HK1178173A - Antibodies with modified affinity to fcrn that promote antigen clearance - Google Patents
Antibodies with modified affinity to fcrn that promote antigen clearance Download PDFInfo
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- HK1178173A HK1178173A HK13104980.9A HK13104980A HK1178173A HK 1178173 A HK1178173 A HK 1178173A HK 13104980 A HK13104980 A HK 13104980A HK 1178173 A HK1178173 A HK 1178173A
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Description
Technical Field
The present invention relates to:
methods for promoting antigen binding molecule-mediated uptake of an antigen by a cell;
a method for increasing the number of antigens to which a single antigen binding molecule can bind;
a method for promoting a decrease in plasma antigen concentration by administering an antigen binding molecule;
methods for improving the pharmacokinetics of antigen binding molecules;
a method for reducing the total antigen concentration or the free antigen concentration of plasma;
an antigen binding molecule that increases the uptake of an antigen by a cell;
an antigen-binding molecule that binds an increased number of antigens;
an antigen binding molecule capable of promoting a reduction in plasma antigen concentration by administration of said molecule;
antigen binding molecules with improved pharmacokinetics;
a pharmaceutical composition comprising said antigen binding molecule;
methods for producing the above, and the like.
Priority
The present invention claims the benefit of japanese patent application No. 2010-079667 filed on 30/2010 and japanese patent application No. 2010-250830 filed on 9/2010, the entire contents of which are incorporated herein by reference.
Background
Antibodies are attracting attention as drugs because they are very stable in plasma with few side effects. Currently, many IgG-type antibody drugs are available on the market, and many are currently under development (NPL 1 and 2). Meanwhile, various technologies suitable for second-generation antibody drugs have been reported, including technologies for improving effector functions, antigen-binding ability, pharmacokinetics, and stability, and technologies for reducing the risk of immunogenicity (NPL 3). Overall, the necessary dosage of antibody drugs is high. This in turn creates problems such as high production costs and difficulty in producing subcutaneous formulations. In theory, the dosage of antibody drugs can be reduced by improving the pharmacokinetics of the antibody or by improving the affinity between the antibody and the antigen.
The literature reports methods for improving antibody pharmacokinetics using artificial substitutions of amino acids in the constant region (NPL 4 and 5). Similarly, affinity maturation is reported as a technique for improving antigen binding ability or antigen neutralizing activity (NPL 6). This technique can improve antigen binding activity by introducing amino acid mutations into the CDR regions of the variable region or such regions. The increased antigen binding capacity can lead to increased in vitro biological activity or to a reduced dose, and also to increased in vivo efficacy (NPL 7).
The neutralizing capacity of an individual antibody molecule depends on its affinity. By increasing the affinity, the antigen can be neutralized by a smaller amount of antibody. Antibody affinity (NPL 6) can be increased in a variety of ways. Furthermore, if the affinity can be made infinite by covalently binding the antibody to the antigen, a single antibody molecule can neutralize one antigen molecule (a bivalent antibody can neutralize two antigen molecules). However, stoichiometric neutralization of one antibody against one antigen (one bivalent antibody against two antigens) is the limit of the existing method, and thus it is impossible to completely neutralize the antigen with an amount of antibody that is less than the amount of antigen. In other words, the affinity-increasing effect has a limit (NPL 9). In order to prolong the duration of the neutralizing effect of neutralizing antibodies, the antibodies must be administered at a higher dose than the amount of antigen produced by the contemporaneous organism. Only with the above improvements in antibody pharmacokinetics or affinity maturation techniques, there is still such a limitation in reducing the required antibody dose. Therefore, in order to maintain antigen neutralization of an antibody for a target time with a smaller amount of antibody than the amount of antigen, a single antibody must neutralize multiple antigens. An antibody that binds to an antigen in a pH-dependent manner has been reported as a novel method for achieving the above object (PTL 1). A pH-dependent antigen-binding antibody that binds strongly to an antigen under neutral conditions in plasma and dissociates from an antigen under acidic conditions in endosomes can dissociate from an antigen in endosomes. When the pH-dependent antigen-binding antibody dissociated from the antigen is recycled to the plasma through FcRn, it can bind to another antigen again. Thus, a single pH-dependent antigen-binding antibody can repeatedly bind to many antigens.
Furthermore, plasma retention of antigen is very short compared to antibodies that are recirculated by FcRn binding. When the antibody having such long-term plasma retention binds to the antigen, the plasma retention time of the antigen-antibody complex is extended as much as the plasma retention time of the antibody. Therefore, the plasma retention of antigen is prolonged by binding to the antibody, and thus the plasma antigen concentration is increased.
IgG antibodies have a longer plasma retention time due to FcRn binding. Binding between IgG and FcRn was only observed under acidic conditions (pH 6.0). In contrast, under neutral conditions (pH7.4), little binding was detected. IgG antibodies are taken up into cells in a non-specific manner. The antibody returns to the cell surface by binding to endosomal FcRn under endosomal acidic conditions, and then dissociates from FcRn under plasma neutral conditions. If FcRn binding is lost under acidic conditions by introducing mutations into the IgG Fc domain, antibodies that recirculate from endosomes to plasma are absent, which significantly reduces antibody residence time in plasma. One reported approach for improving IgG antibody plasma retention is to increase FcRn binding under acidic conditions. Amino acid mutations were introduced into the Fc domain of IgG antibodies to improve FcRn binding under acidic conditions. This increases the efficiency of recirculation from endosomes to plasma, resulting in improved plasma retention. An important requirement for amino acid substitutions is not to increase FcRn binding under neutral conditions. If an IgG antibody binds to FcRn under neutral conditions, the antibody that returns to the cell surface by binding to FcRn under the in vivo acidic conditions does not dissociate from FcRn under plasma neutral conditions. In this case, plasma retention is lost to some extent, as IgG antibodies are not recirculated into the plasma. For example as J Immunol. (2002)169 (9): 5171-80, it is reported that the modified IgG1 antibody, which enables the resulting antibody to bind to mouse FcRn under neutral conditions (pH7.4) by introducing amino acid substitutions, has very poor plasma retention when administered to mice. Furthermore, as in J Immunol. (2009)182 (12): 7663-71; j Biol chem.2007 1 month 19; 282(3): 1709-17; and J immunol.2002, 11 months and 1 days; 169(9): 5171-80, IgG1 antibodies were modified by introducing amino acid substitutions such that the resulting antibodies had improved human FcRn binding under acidic conditions (pH 6.0) while becoming capable of binding human FcRn under neutral conditions (pH 7.4). It was reported that the obtained antibody showed neither improvement nor change in plasma retention when administered to cynomolgus monkeys (cynomolgus monkey). Therefore, antibody engineering techniques for improving antibody function have focused only on improving antibody plasma retention by increasing human FcRn binding under acidic conditions, rather than increasing binding under neutral conditions (pH 7.4). To date, no report has been described describing the advantage of improving human FcRn binding under neutral conditions (pH7.4) by introducing amino acid substitutions into the Fc domain of IgG antibodies. Even if the antigen affinity of the antibody is improved, the elimination of the antigen in the plasma cannot be improved. It is reported that the above-mentioned pH-dependent antigen-binding antibody is more effective as a method for enhancing antigen elimination from plasma than a typical antibody (PTL 1).
Thus, a single pH-dependent antigen-binding antibody binds many antigens and can facilitate elimination of the antigen from plasma, as compared to typical antibodies. Thus, the pH-dependent antigen-binding antibody has an effect that cannot be achieved by typical antibodies. However, to date, there have been no reports on antibody engineering methods for further improving the ability of pH-dependent antigen-binding antibodies to repeatedly bind to antigens and enhancing the effect of antigen elimination from plasma.
The prior art documents relating to the present invention are given below:
reference list
Patent document
[ PTL 1] WO 2009/125825, ANTIGEN-BINDING MOLECULECAPABLE OF BINDING TO TWO OR MORE ANTIGENMOLECULES REPEATEDLY (ANTIGEN-BINDING molecule capable OF REPEATEDLY BINDING TO TWO OR MORE ANTIGEN molecules)
Non-patent document
[ NPL 1] Monoclonal antibodies in the clinic (successful use of Monoclonal antibodies in the clinic), Janic M Reichert, Clark J Rosenssweig, Laura BFaden and Matthew C Dewitz, Nature Biotechnology 23, 1073-
[ NPL 2] Pavlou AK, Belsey MJ., The therapeutic antibodies marketto 2008(2008 therapeutic antibody market), Eur J Pharm Biopharm.2005 month 4; 59(3): 389-96
[ NPL 3] Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies, Mol cells.2005, 8.31 days; 20(1): 17-29 review
[ NPL 4] Hinton PR, Xiong JM, Johnfs MG, Tang MT, Keller S, Tsurushita N., An engineered human IgG 1antibody with a ringer serum half-life engineered human IgG 1antibody, J Immunol.2006, 1 month 1; 176(1): 346-56
[ NPL 5] Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, MedesanC, Ober RJ, Ward ES., Inc. the server persistence of an IgGfragment by random mutagenesis, Nat Biotechnol.1997 for 7 months; 15(7): 637-40
[ NPL 6] Proc Natl Acad Sci U S.2005, 6 months and 14 days; 102(24): 8466-71.Epub, 6.6.6.2005A general method for great improvement of antibody affinity by using combinatorial libraries, Rajpal A, Beyaz N, Haber L, Cappuccil G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R
[ NPL 7] Wu H, Pfar DS, Johnson S, Brewah YA, Woods RM, PatelNK, White WI, Young JF, Kiener PA.development of Motavizumab, and ultra-potential Antibody for the development of the Prevention of Respiratory Syncytial virus infection of the Upper and Lower Respiratory tracts ], J Mol Biol (2007) 368: 652-665
[ NPL 8] Hanson CV, Nishiyama Y, Paul S. catalytic antibodies and their applications, Curr Opin Biotechnol.2005, 12 months; 16(6): 631-6
[ NPL 9] Rathanawami P, Roalstad S, Roskos L, Su QJ, Lackie S, Babcook J. Demonration of an in vivo generated sub-picomolar affinity fully human monoclonal antibody to interleukin-8 (argument for in vivo generated sub-picomolar affinity fully human monoclonal antibody against interleukin-8.) Biochem Biophys Res Commun.2005, 9 months; 334(4): 1004-13
Summary of The Invention
Technical problem
The present invention has been achieved in view of the above circumstances. The object of the present invention is to provide: a method for promoting cellular uptake of an antigen by using an antigen binding molecule, a method for increasing the number of antigens to which a single antigen binding molecule can bind, a method for promoting a decrease in plasma antigen concentration by administering an antigen binding molecule, a method for improving the pharmacokinetics of an antigen binding molecule, an antigen binding molecule that promotes cellular uptake of an antigen, an antigen binding molecule that binds an increased number of antigens, an antigen binding molecule that is capable of promoting a decrease in plasma antigen concentration by administration, an antigen binding molecule with improved pharmacokinetics, a pharmaceutical composition comprising said antigen binding molecule and a method for producing the same.
Solution to the problem
The present inventors have conducted intensive studies on the following methods: methods for promoting uptake of an antigen by a cell via an antigen-binding molecule (a molecule having antigen-binding ability, e.g., a polypeptide), methods for allowing repeated binding of an antigen-binding molecule to an antigen, methods for promoting a decrease in plasma antigen concentration by administering an antigen-binding molecule, and methods for improving plasma retention of an antigen-binding molecule. Thus, the inventors have found that antigen-binding molecules having human FcRn binding capacity at early endosomal pH (early endosomal pH) and human FcRn binding activity at plasma pH higher than that of intact human IgG-type immunoglobulin can facilitate antigen uptake by cells. The present inventors have also found that by using an antigen-binding molecule with weaker antigen-binding activity at early endosomal pH compared to plasma pH, antigen-binding molecule-mediated uptake of antigen by cells can be further increased and the number of antigens that can be bound to a single antigen-binding molecule can be increased; by administering such antigen binding molecules, a reduction in plasma antigen concentration may be facilitated; and can improve the pharmacokinetics of the antigen binding molecule.
In particular, the present invention relates to:
methods for promoting antigen binding molecule-mediated uptake of an antigen by a cell;
a method for increasing the number of antigens to which a single antigen binding molecule can bind;
a method for promoting a decrease in plasma antigen concentration by administering an antigen binding molecule;
methods for improving the pharmacokinetics of antigen binding molecules;
a method for reducing the total antigen concentration or the free antigen concentration of plasma;
an antigen binding molecule that increases the uptake of an antigen by a cell;
an antigen-binding molecule that binds an increased number of antigens;
an antigen binding molecule capable of promoting a reduction in plasma antigen concentration by administration of said molecule;
antigen binding molecules with improved pharmacokinetics;
a pharmaceutical composition comprising said antigen binding molecule;
methods for producing the above, and the like. More specifically, the present invention provides:
[1] an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, having human FcRn binding activity in acidic and neutral pH ranges, wherein human FcRn binding activity is greater than 3.2 micromolar in the neutral pH range;
[2] an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, having human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is 28-fold that of intact human IgG;
[3] An antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, having human FcRn binding activity in a neutral pH range, wherein human FcRn binding activity in the neutral pH range is greater than 2.3 micromolar;
[4] an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, having human FcRn binding activity in a neutral pH range, wherein the human FcRn binding activity in the neutral pH range is 38-fold that of intact human IgG;
[5] [1] to [4], wherein the neutral pH ranges from pH7.0 to 8.0;
[6] an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, wherein the total plasma antigen concentration following administration of the antigen binding molecule to a non-human animal is lower than the total plasma antigen concentration following administration of a reference antigen binding molecule to a non-human animal, the reference antigen binding molecule comprising the same antigen binding domain and a fully human IgG Fc domain as the human FcRn binding domain;
[7] an antigen binding molecule, wherein the plasma antigen concentration following administration of the antigen binding molecule to a non-human animal is lower than the total plasma antigen concentration obtained from a non-human animal to which the antigen binding molecule has not been administered;
[8] An antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, wherein the antigen/antigen binding molecule molar ratio (C) of the antigen binding molecule is calculated as follows:
C＝A/B，
an antigen/antigen binding molecule molar ratio (C') lower than that of an antigen binding molecule comprising the same antigen binding domain and a fully human IgG Fc domain as the human FcRn binding domain calculated as follows;
C′＝A′/B′，
wherein;
a is the total antigen concentration in plasma following administration of the antigen binding molecule to a non-human animal,
b is the plasma concentration of the antigen binding molecule after administration of the antigen binding molecule to a non-human animal,
a' is the total antigen concentration in plasma after administration of the reference antigen binding molecule to a non-human animal,
b' is the plasma concentration of the antigen-binding molecule after administration of the reference antigen-binding molecule to a non-human animal;
[9] [6] to [8], wherein the non-human animal is a human FcRn transgenic mouse;
[10] [6] the antigen binding molecule of any one of [9], wherein the plasma antigen concentration is long-term plasma total antigen concentration;
[11] [6] the antigen binding molecule of any one of [9], wherein the plasma antigen concentration is a short-term plasma total antigen concentration;
[12] an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, having human FcRn binding activity in acidic and neutral pH ranges, wherein the human FcRn binding activity in the neutral pH range is stronger than the binding activity of intact human IgG;
[13] [1] the antigen-binding molecule according to any one of [11], wherein the antigen-binding activity of the antigen-binding domain is lower in an acidic pH range than in a neutral pH range;
[14] [12] or [13], wherein the ratio of antigen binding activity in the acidic pH range and the neutral pH range is at least 2 in terms of values of KD (in the acidic pH range)/KD (in the neutral pH range);
[15] [12] to 14] an antigen binding molecule comprising an amino acid mutation of the antigen binding domain comprising a substitution of at least one amino acid of the antigen binding domain with histidine or an insertion of at least one histidine;
[16] the antigen binding molecule of any one of [12] to [14], wherein the antigen binding domain is obtained from a library of antigen binding domains;
[17] [1] to 16] an antigen binding molecule comprising as a human FcRn binding domain an Fc domain resulting from the substitution of at least one amino acid in the Fc domain of a parent IgG with a different amino acid;
[18] [1] to [17], wherein the human FcRn-binding domain is a human FcRn-binding domain comprising an amino acid sequence that replaces at least one of the following amino acids in an Fc domain selected from a parent IgG with a different amino acid: 237. 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436(EU numbering);
[19] [1] - [18] an antigen binding molecule comprising a human FcRn binding domain comprising an amino acid substitution in the Fc domain of a parent IgG comprising at least one amino acid substitution (EU numbering) selected from the group consisting of:
an amino acid substitution in which Gly at position 237 is substituted into Met;
amino acid substitution of Pro at position 238 to Ala;
an amino acid substitution wherein Ser at position 239 is substituted with Lys;
an amino acid substitution wherein Lys at position 248 is replaced with Ile;
an amino acid substitution at Thr at position 250 with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr;
an amino acid substitution of Met at position 252 with Phe, Trp or Tyr;
an amino acid substitution of Ser at position 254 to Thr;
amino acid substitution of Arg at position 255 with Glu;
an amino acid substitution at position 256 of Thr substituted with Asp, Glu or Gln;
an amino acid substitution at position 257 of Pro substituted with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val;
an amino acid substitution wherein Glu at position 258 is substituted with His;
an amino acid substitution at position 265 wherein Asp is substituted with Ala;
270 Asp substituted with an amino acid Phe;
an amino acid substitution wherein Asn at position 286 is replaced with Ala or Glu;
An amino acid substitution of Thr at position 289 with His;
an amino acid substitution wherein Asn at position 297 is substituted with Ala;
298 amino acid substitutions in which Ser is replaced with Gly;
an amino acid substitution wherein Val at position 303 is substituted with Ala;
an amino acid substitution wherein Val at position 305 is substituted with Ala;
a substitution of Thr at position 307 with an amino acid Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr;
an amino acid substitution of Val at position 308 with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr;
an amino acid substitution of Leu or Val at position 309 with Ala, Asp, Glu, Pro or Arg;
an amino acid substitution in which Gln at position 311 is replaced with Ala, His, or Ile;
an amino acid substitution wherein Asp at position 312 is substituted with Ala or His;
an amino acid substitution wherein Leu at position 314 is substituted with Lys or Arg;
an amino acid substitution wherein Asn at position 315 is substituted with Ala or His;
an amino acid substitution wherein Lys at position 317 is substituted with Ala;
an amino acid substitution wherein Asn at position 325 is replaced with Gly;
an amino acid substitution wherein Ile at position 332 is substituted with Val;
an amino acid substitution wherein Lys at position 334 is substituted with Leu;
An amino acid substitution wherein Lys at position 360 is replaced by His;
an amino acid substitution at position 376 of Asp to Ala;
an amino acid substitution in which Glu at position 380 is substituted with Ala;
an amino acid substitution wherein Glu at position 382 is substituted with Ala;
an amino acid substitution of Asn or Ser at position 384 with Ala;
an amino acid substitution wherein Gly at position 385 is substituted by Asp or His;
an amino acid substitution of Gln at position 386 with Pro;
amino acid substitution of Pro at position 387 with Glu;
an amino acid substitution wherein Asn at position 389 is substituted with Ala or Ser;
an amino acid substitution of Ser at position 424 with Ala;
an amino acid substitution of Met at position 428 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr;
an amino acid substitution of His at position 433 with Lys;
an amino acid substitution in which Asn at position 434 is substituted with Ala, Phe, His, Ser, Trp or Tyr;
and an amino acid substitution of Tyr or Phe at position 436 with His;
[20] [1] to 18] an antigen binding molecule according to any one of claims, wherein the human FcRn binding domain comprises at least one amino acid (EU numbering) selected from the group consisting of the Fc domain of a parent IgG:
Met at amino acid position 237;
ala at amino acid position 238;
lys at amino acid position 239;
an 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, 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;
phe at amino acid position 270;
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;
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 or His at amino acid position 315;
ala at amino acid position 317;
gly at amino acid position 325;
val at amino acid position 332;
a 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 at amino acid position 436;
[21] [18] to [20], wherein the parent IgG is selected from IgG obtained from a non-human animal;
[22] [18] to 20], wherein the parent IgG is a human IgG;
[23] [1] to [22] which have an antagonistic activity;
[24] [1] to 23] which bind to a membrane antigen or a soluble antigen;
[25] The antigen binding molecule of any one of [1] to [24], wherein the antigen binding domain comprises an artificial ligand that binds to a receptor;
[26] the antigen binding molecule of any one of [1] to [24], wherein the antigen binding domain comprises an artificial receptor that binds to a ligand;
[27] [1] to [24] which is an antibody;
[28] the antigen binding molecule of [27], wherein the antibody is selected from a chimeric antibody, a humanized antibody or a human antibody;
[29] a pharmaceutical composition comprising the antigen binding molecule of any one of [1] to [28 ];
[30] a method for promoting antigen binding molecule-mediated uptake of an antigen by a cell by increasing its human FcRn binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;
[31] a method for promoting antigen binding molecule mediated uptake of a cell to an antigen by increasing its human FcRn binding activity in the neutral pH range and decreasing its antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;
[32] A method for increasing the number of antigens to which a single antigen binding molecule can bind by increasing its human FcRn binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;
[33] a method for increasing the number of antigens to which a single antigen binding molecule can bind by increasing its human FcRn binding activity in the neutral pH range and decreasing its antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;
[34] a method for increasing the ability of an antigen binding molecule to eliminate antigen from plasma by increasing its human FcRn binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;
[35] a method for increasing the ability of an antigen binding molecule to eliminate antigen from plasma by increasing its human FcRn binding activity in the neutral pH range and decreasing its antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;
[36] A method for improving the pharmacokinetics of an antigen binding molecule by increasing its human FcRn binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain, and has human FcRn binding activity in the acidic pH range;
[37] a method for improving the pharmacokinetics of an antigen binding molecule by increasing its human FcRn binding activity in the neutral pH range and decreasing its antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain, and has human FcRn binding activity in the acidic pH range;
[38] a method for promoting intracellular dissociation of an antigen binding molecule from an antigen to which the antigen binding molecule binds extracellularly, by increasing its human FcRn binding activity in the neutral pH range and decreasing its antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;
[39] A method for promoting the extracellular release of an antigen binding molecule in antigen-bound form taken up into a cell in antigen-free form by increasing its human FcRn binding activity in the neutral pH range and decreasing its antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;
[40] a method for reducing the total plasma antigen concentration or the free plasma antigen concentration in plasma by increasing its human FcRn binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;
[41] a method for reducing the concentration of total plasma antigens or free plasma antigens in plasma by increasing their human FcRn binding activity in the neutral pH range and reducing their antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range;
[42] [30] the process according to any one of [41], wherein the acidic pH is in the range of pH 5.5 to pH6.5 and the neutral pH is in the range of pH 7.0 to pH 8.0;
[43] [30] to 41], wherein the increase in human FcRn binding activity in the neutral pH range is an increase caused by substituting at least one amino acid in a parent IgG Fc domain of the human FcRn binding domain with a different amino acid;
[44] [30] to 41], wherein the increase in binding activity of human FcRn in the neutral pH range is an increase caused by substituting a different amino acid for at least one of the following amino acids in a parent IgG Fc domain selected from the group consisting of human FcRn binding domains: 237. 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436(EU numbering);
[45] the method of any one of [31], [33], [35], [37] - [39] and [41], wherein the antigen-binding activity of the antigen-binding molecule in the acidic pH range is reduced to less than the antigen-binding activity in the neutral pH range by substituting at least one amino acid of the antigen-binding molecule with histidine or inserting at least one histidine;
[46] The method of any one of [31], [33], [35], [37] - [39] and [41], wherein the antigen-binding domain is obtained from a library of antigen-binding domains;
[47] the method of any one of [31], [33], [35], [37] - [39] and [41], wherein the decrease in antigen-binding activity is represented by an increase in the value of KD (in the acidic pH range)/KD (in the neutral pH range), relative to the ratio of antigen-binding activity in the acidic pH range and the neutral pH range prior to histidine substitution or insertion;
[48] a method for producing an antigen binding molecule, the method comprising the steps of:
(a) selecting an antigen binding molecule having a human FcRn binding activity of greater than 3.2 micromolar over a neutral pH range obtained by altering at least one amino acid in the human FcRn binding domain of the antigen binding molecule;
(b) obtaining a gene encoding an antigen binding molecule, wherein the human FcRn binding domain prepared in (a) is linked to an antigen binding domain; and
(c) generating an antigen binding molecule using the gene prepared in (b);
[49] a method for producing an antigen binding molecule, the method comprising the steps of:
(a) selecting an antigen binding molecule having greater human FcRn binding activity in the neutral pH range than before altering at least one amino acid in the human FcRn binding domain of an antigen binding molecule having human FcRn binding activity in the acidic pH range;
(b) Altering at least one amino acid in the antigen binding domain of the antigen binding molecule and selecting an antigen binding molecule having a stronger antigen binding activity in the neutral pH range than in the acidic pH range;
(c) obtaining a gene encoding an antigen binding molecule, wherein the human FcRn binding domain prepared in (a) and (b) is linked to an antigen binding domain; and
(d) generating an antigen binding molecule using the gene prepared in (c); and
[50] a method for producing an antigen binding molecule, the method comprising the steps of:
(a) selecting an antigen binding molecule having greater human FcRn binding activity in the neutral pH range than before altering at least one amino acid in the human FcRn binding domain of an antigen binding molecule having human FcRn binding activity in the acidic pH range;
(b) selecting an antigen binding molecule having a greater antigen binding activity in the neutral pH range than in the acidic pH range;
(c) obtaining a gene encoding an antigen binding molecule, wherein the human FcRn binding domain prepared in (a) and (b) is linked to an antigen binding domain; and
(d) generating an antigen binding molecule using the gene prepared in (c);
[51] an antigen-binding molecule produced by the production method of any one of [48] to [50 ];
[52] A method for screening for antigen binding molecules, the method comprising the steps of:
(a) selecting an antigen binding molecule having a human FcRn binding activity of greater than 3.2 micromolar over a neutral pH range obtained by altering at least one amino acid in the human FcRn binding domain of the antigen binding molecule;
(b) obtaining a gene encoding an antigen binding molecule, wherein the human FcRn binding domain prepared in (a) is linked to an antigen binding domain; and
(c) generating an antigen binding molecule using the gene prepared in (b);
[53] a method for screening for antigen binding molecules, the method comprising the steps of:
(a) selecting an antigen binding molecule having greater human FcRn binding activity in the neutral pH range than before altering at least one amino acid in the human FcRn binding domain of an antigen binding molecule having human FcRn binding activity in the acidic pH range;
(b) altering at least one amino acid in the antigen binding domain of the antigen binding molecule and selecting an antigen binding molecule having a stronger antigen binding activity in the neutral pH range than in the acidic pH range;
(c) obtaining a gene encoding an antigen binding molecule, wherein the human FcRn binding domain prepared in (a) and (b) is linked to an antigen binding domain; and
(d) Generating an antigen binding molecule using the gene prepared in (c);
[54] a method for screening for antigen binding molecules, the method comprising the steps of:
(a) selecting an antigen binding molecule having greater human FcRn binding activity in the neutral pH range than before altering at least one amino acid in the human FcRn binding domain of an antigen binding molecule having human FcRn binding activity in the acidic pH range;
(b) selecting an antigen binding molecule having a greater antigen binding activity in the neutral pH range than in the acidic pH range;
(c) obtaining a gene encoding an antigen binding molecule, wherein the human FcRn binding domain prepared in (a) and (b) is linked to an antigen binding domain; and
(d) generating an antigen binding molecule using the gene prepared in (c);
[55] the method of any one of [30] to [54], wherein the antigen binding domain comprises an artificial ligand that binds to a receptor;
[56] the method of any one of [30] to [54], wherein the antigen binding domain comprises an artificial receptor that binds to a ligand; and
[57] [30] the method of any one of [54] to [30], wherein the antigen-binding molecule is an antibody.
Advantageous effects of the invention
The present invention provides:
methods for promoting antigen binding molecule-mediated uptake of an antigen by a cell; a method for increasing the number of antigens to which a single antigen binding molecule can bind; and methods for promoting a decrease in plasma antigen concentration by administering an antigen binding molecule. When antigen-binding molecule-mediated uptake of an antigen by a cell is promoted, a decrease in plasma antigen concentration can be promoted by administering such antigen-binding molecules, and the pharmacokinetics of the antigen-binding molecules can be improved to increase the number of antigens to which a single antigen-binding molecule can bind. Thus, the antigen binding molecules can produce superior in vivo effects than ordinary antigen binding molecules.
Brief Description of Drawings
FIG. 1 shows in a graph the time course of plasma concentrations of the soluble form of human IL-6 receptor after administration of an anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 276), wherein the plasma concentrations of the soluble form of human IL-6 receptor are constant (steady state infusion model).
FIG. 2 is a schematic showing dissociation of IgG antibody molecules from soluble antigen in endosomes resulting in enhanced antigen elimination, resulting in a new round of binding to another antigen.
Figure 3 shows in a graph the time course of plasma antibody concentrations in human FcRn transgenic mice.
Figure 4 shows in a graph the time course of plasma concentrations of the soluble form of human IL-6 receptor in human FcRn transgenic mice.
FIG. 5 is a graph showing the time course of plasma antibody concentration in normal mice.
FIG. 6 shows in a graph the time course of the plasma concentration of the soluble form of human IL-6 receptor in normal mice.
FIG. 7 shows in a graph the time course of plasma concentrations of the unbound soluble form of human IL-6 receptor in normal mice.
Figure 8 shows in a graph the time course of plasma concentrations of the soluble form of human IL-6 receptor in human FcRn transgenic mice.
FIG. 9 shows in a graph the time course of plasma concentrations of the soluble form of human IL-6 receptor after administration of Fv4-IgG1-F14 at low doses (0.01mg/kg) or 1 mg/kg.
FIG. 10 is a graph showing the time course of plasma antibody concentration after administration of Fv4-IgG1-F14 at a low dose (0.01mg/kg) or 1 mg/kg.
FIG. 11 is a graph showing the time course of plasma concentration of the soluble form of human IL-6 receptor after administration of an anti-human IL-6 receptor antibody to normal mice in which the plasma concentration of the soluble form of human IL-6 receptor is constant.
FIG. 12 is a graph showing the time course of plasma antibody concentration following co-injection of hsIL-6R and anti-human IL-6 receptor antibody into human FcRn transgenic mice (strain 276).
FIG. 13 is a graph showing the time course of plasma concentrations of soluble forms of human IL-6 receptor following co-injection of hsIL-6R and anti-human IL-6 receptor antibody into human FcRn transgenic mice (strain 276).
FIG. 14 depicts the relationship between binding affinity of Fc variants to human FcRn at pH 7.0 and plasma hsIL-6R concentrations at day 1 after co-injection of hsIL-6R and anti-human IL-6 receptor antibody into human FcRn transgenic mice (line 276).
FIG. 15 depicts the relationship between binding affinity of Fc variants to human FcRn at pH 7.0 and plasma antibody concentrations at day 1 following co-injection of hsIL-6R and anti-human IL-6 receptor antibody to human FcRn transgenic mice (line 276).
FIG. 16 depicts the time course of antigen/antibody molar ratio (C value) after co-injection of hsIL-6R and anti-human IL-6 receptor antibody into human FcRn transgenic mice (strain 276).
Figure 17 depicts the relationship between binding affinity of Fc variants to human FcRn at pH 7.0 and the time course of antigen/antibody molar ratio (C-value) at day 1 after co-injection of hsIL-6R and anti-human IL-6 receptor antibody to human FcRn transgenic mice (line 276).
FIG. 18 shows in a graph the time course of plasma concentrations of hsIL-6R after administration of Fv4-IgG1-F14 to human FcRn transgenic mice (strain 276) at low doses (0.01 or 0.2mg/kg) or 1mg/kg (where plasma concentrations of hsIL-6R are constant) (steady state infusion model).
FIG. 19 depicts the time course of plasma hsIL-6R concentrations in human FcRn transgenic mouse lines 276 and 32 following co-injection of hsIL-6R and anti-human IL-6 receptor antibody to human FcRn transgenic mice (lines 276 and 32).
FIG. 20 depicts the time course of plasma antibody concentrations in human FcRn transgenic mouse lines 276 and 32 following co-injection of hsIL-6R and anti-human IL-6 receptor antibody to human FcRn transgenic mice (lines 276 and 32).
FIG. 21 is a graph showing the time course of plasma concentrations of hsIL-6R after administration of anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 32) in which plasma concentrations of hsIL-6R are constant (steady state infusion model).
FIG. 22 is a graph showing the time course of antibody plasma concentrations following administration of anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 32) in which plasma concentrations of hsIL-6R are constant (steady state infusion model).
FIG. 23 depicts the time course of antigen/antibody molar ratio (C value) after administration of anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 32) in which plasma concentrations of hsIL-6R were constant (steady state infusion model).
FIG. 24 depicts the relationship between binding affinity of Fc variants to human FcRn at pH7.0 and the antigen/antibody molar ratio (C-value) on day 1 after administration of anti-human IL-6 receptor antibody to human FcRn transgenic mice (strain 32) in which plasma concentrations of hsIL-6R were constant (steady state infusion model).
Figure 25 in the figure shows the time course of plasma antibody concentrations after administration of anti-human IL-6 receptor antibodies with Fc variants of F11, F39, F48 and F264 to human FcRn transgenic mice (strain 32) in which plasma concentrations of hsIL-6R are constant (steady state infusion model).
FIG. 26 shows in a graph the time course of plasma concentrations of hsIL-6R after administration of anti-human IL-6 receptor antibodies with Fc variants of F11, F39, F48, and F264 to human FcRn transgenic mice (strain 32) in which the plasma concentration of hsIL-6R is constant (steady state infusion model).
FIG. 27 shows in a graph the time course of plasma antibody concentrations after administration of anti-human IL-6 receptor antibodies with Fc variants of F157, F196 and F262 to human FcRn transgenic mice (strain 32) in which plasma concentrations of hsIL-6R were constant (steady state infusion model).
FIG. 28 shows in a graph the time course of plasma concentrations of hsIL-6R after administration of anti-human IL-6 receptor antibodies with Fc variants of F157, F196 and F262 to human FcRn transgenic mice (strain 32) in which plasma concentrations of hsIL-6R are constant (steady state infusion model).
FIG. 29 depicts a pharmacokinetic model for in silico studies (in silico study) of conventional and antigenemic antibodies.
FIG. 30 is a graph showing the time course of plasma concentrations of human IL-6 after co-injection of human IL-6 and an anti-human IL-6 antibody to normal mice.
FIG. 31 is a graph showing the time course of the plasma concentration of the antibody after co-injection of human IL-6 and anti-human IL-6 antibody to normal mice.
FIG. 32 shows a sensorgram for human IgA binding to CD89-Fc fusion protein using Biacore at pH 7.4 and pH 6.0.
FIG. 33 is a graph showing the time course of plasma concentrations of human IgA after co-injection of human IgA and CD89-Fc fusion protein to normal mice.
FIG. 34 is a graph showing the time course of antibody plasma concentrations following co-injection of human IgA and CD89-Fc fusion proteins to normal mice.
FIG. 35 is a graph showing the plasma concentration of soluble human Plexin A1 7 hours after co-injection of soluble human Plexin A1 and anti-human Plexin A1 antibodies in normal mice.
Description of the embodiments
The present invention provides methods for promoting antigen binding molecule-mediated uptake of an antigen by a cell. More specifically, the present invention provides methods for promoting cellular uptake of an antigen by an antigen binding molecule having human FcRn binding activity in the acidic pH range, based on increasing human FcRn binding activity of the antigen binding molecule in the neutral pH range. The invention also provides methods of increasing cellular uptake of an antigen by an antigen binding molecule having human FcRn binding activity in the acidic pH range based on altering at least one amino acid in the human FcRn binding domain of the antigen binding molecule.
The invention also provides a method of promoting uptake of an antigen by a cell by an antigen binding molecule having human FcRn binding activity in the acidic pH range, the method being based on the use of a human FcRn binding domain comprising an amino acid sequence with a substitution of at least one of the following amino acids in a parent IgGFc domain selected from the group consisting of a human FcRn binding domain comprising the Fc domain of a parent IgG with a different amino acid: 237. 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436(EU numbering).
The present invention also provides a method for promoting antigen-binding molecule-mediated antigen uptake by cells by decreasing the antigen-binding activity (binding capacity) of the above antigen-binding molecule in an acidic pH range to less than its antigen-binding activity in a neutral pH range; and this facilitates antigen uptake by the cells. The present invention also provides a method for promoting antigen-binding molecule mediated uptake of an antigen by a cell, said method being based on altering at least one amino acid in the antigen-binding domain of the above antigen-binding molecule, which promotes antigen uptake by the cell. The invention also provides a method for promoting antigen-binding molecule-mediated antigen uptake by cells based on the substitution of at least one amino acid with histidine or the insertion of at least one histidine into the antigen-binding domain of the above antigen-binding molecule, which promotes antigen uptake by cells.
Herein, "uptake of antigen by a cell" mediated by an antigen binding molecule means uptake of antigen into a cell by endocytosis. Meanwhile, in the present context, "promoting the uptake of cells" means increasing the intracellular uptake rate of an antigen-binding molecule that binds to an antigen in plasma, and/or reducing the amount of the taken antigen that is recirculated to plasma. This means that the rate of uptake into cells is enhanced compared to the antigen binding molecule prior to increasing the human FcRn binding activity of the antigen binding molecule in the neutral pH range, or prior to increasing the human FcRn binding activity of the antigen binding molecule in the acidic pH range and decreasing the antigen binding activity (binding capacity) of the antigen binding molecule in the acidic pH range to less than its antigen binding activity in the neutral pH range. Preferably, the rate is improved compared to intact human IgG, more preferably compared to intact human IgG. Thus, in the present invention, whether an antigen binding molecule promotes antigen uptake by a cell can be assessed based on an increase in the rate of antigen uptake by the cell. The rate of uptake of an antigen by a cell can be calculated, for example, by monitoring the decrease in antigen concentration over time in a culture medium containing cells expressing human FcRn, or monitoring the amount of antigen taken up by cells expressing human FcRn over time, after addition of the antigen and antigen binding molecule to the culture medium. For example, the present methods of promoting the rate of cellular uptake of an antigen mediated by an antigen binding molecule can be used to increase the rate of antigen elimination from plasma by administering an antigen binding molecule. Thus, whether to promote antigen-binding molecule-mediated uptake of an antigen by cells can be assessed by testing whether the rate of antigen elimination from plasma is increased or whether the total antigen concentration in plasma is decreased, for example, by administering the antigen-binding molecule.
Herein, "total plasma antigen concentration" means the sum of the concentration of antigen bound by the antigen binding molecule and the concentration of unbound antigen, i.e., "plasma free antigen concentration", which is the concentration of antigen unbound by the antigen binding molecule. Various methods of measuring "total antigen concentration in plasma" or "free antigen concentration in plasma" are well known in the art as described below.
As used herein, "fully human IgG" means unmodified human IgG and is not limited to a particular class of IgG. This means that human IgG1, IgG2, IgG3 or IgG4 can be used as "fully human IgG" as long as it can bind to human FcRn in the acidic pH range. Preferably, the "fully human IgG" may be human IgG 1.
The invention also provides methods for increasing the number of antigens to which a single antigen binding molecule can bind. More specifically, the present invention provides methods for increasing the number of antigens to which a single antigen-binding molecule having human FcRn binding activity can bind in the acidic pH range by increasing the human FcRn binding activity of the antigen-binding molecule in the neutral pH range. The invention also provides methods of increasing the number of antigens to which a single antigen binding molecule having human FcRn binding activity can bind in the acidic pH range by altering at least one amino acid in the human FcRn binding domain of the antigen binding molecule.
The present invention also provides a method of increasing the number of antigens to which a single antigen binding molecule having human FcRn binding activity in the acidic pH range may bind, by using a human FcRn binding domain comprising an amino acid sequence in which at least one amino acid selected from the following position amino acids in a parent IgG Fc domain of a human FcRn binding domain comprising a parent IgG Fc domain is substituted with a different amino acid: 237. 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436(EU numbering).
As used herein, "parent IgG" means an unmodified IgG that is subsequently modified to produce a variant, so long as the modified variant of the parent IgG can bind to human FcRn at acidic pH ranges (thus, under acidic conditions, the parent IgG does not necessarily require binding activity to human FcRn). The parent IgG may be a naturally occurring IgG, or a variant or engineered form of a naturally occurring IgG. A parent IgG may refer to the polypeptide itself, a composition comprising the parent IgG, or an amino acid sequence encoding the parent IgG. It should be noted that parent IgG "includes known commercially available recombinantly produced IgG as outlined below. The source of the "parent IgG" is not limited and may be obtained from any organism other than a human animal or human. Preferably the organism is selected from the group consisting of mouse, rat, guinea pig, hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel and non-human primate. In another embodiment, the "parent IgG" may also be obtained from a cynomolgus monkey (cynomologous), marmoset monkey, macaque, chimpanzee or human. Preferably, the "parent IgG" is obtained from human IgG1, but is not limited to a particular class of IgG. This means that human IgG1, IgG2, IgG3 or IgG4 can suitably be used as "parent IgG". Similarly, any class or subclass of IgG of any of the organisms described above may be preferably used as a "parent IgG". Examples of variants or engineered forms of naturally occurring IgG are described in Curr Opin biotechnol.2009, 12 months; 20(6): 685-91; curr Opin immunol.2008, 8 months; 20(4): 460 to 70 parts; protein Eng Des Sel.2010 for 4 months; 23(4): 195- > 202; WO2009/086320, WO 2008/092117, WO 2007/041635 and WO 2006/105338, but are not limited thereto.
In addition, the present invention provides a method of increasing the number of antigens to which a single antigen binding molecule can bind by decreasing the antigen binding activity (binding capacity) of the above-described antigen binding molecule, which has an increased number of antigen binding events, in the acidic pH range to less than its antigen binding activity in the neutral pH range. The invention also provides a method of increasing the number of antigens to which a single antigen binding molecule can bind by altering at least one amino acid of the antigen binding domain of the above-described antigen binding molecule, which has an increased number of antigen binding events. The present invention also provides methods of increasing the number of antigens to which a single antigen-binding molecule can bind by substituting at least one amino acid with histidine or inserting at least one histidine into an antigen-binding domain having an antigen-binding molecule as described above, which has an increased number of antigen-binding events.
Herein, "the number of antigens to which a single antigen binding molecule can bind" means the number of antigens to which a single antigen binding molecule can bind until the molecule is eliminated by degradation. Herein, "increasing the number of antigens to which a single antigen binding molecule can bind" means an increase in the number of cycles achieved until the antigen binding molecule is eliminated by degradation, wherein each cycle consists of: the antigen binds to the antigen-binding molecule in plasma, the antigen-binding molecule bound to the antigen is taken up intracellularly, dissociated from the antigen in an endosome, and then the antigen-binding molecule is returned to the plasma. This means that the number of cycles is increased compared to the antigen binding molecule prior to increasing the human FcRn binding activity of the antigen binding molecule in the neutral pH range, or prior to increasing the human FcRn binding activity of the antigen binding molecule in the acidic pH range and decreasing the antigen binding activity (binding capacity) of the antigen binding molecule in the acidic pH range to less than its antigen binding activity in the neutral pH range. Thus, whether the number of cycles is increased can be evaluated by testing whether the above-mentioned "intracellular uptake is promoted" or whether the following "pharmacokinetics is improved".
The invention also provides methods for promoting intracellular dissociation of an antigen from an antigen-binding molecule that is extracellularly bound to the antigen. More specifically, the present invention provides a method of promoting intracellular dissociation of an antigen from an antigen-binding molecule that is extracellularly bound to the antigen, by: an antigen binding molecule having human FcRn binding activity in the acidic pH range has increased human FcRn binding activity in the neutral pH range and decreased antigen binding activity in the acidic pH range to less than the neutral pH range. The invention also provides a method for promoting intracellular dissociation of an antigen from an antigen binding molecule that binds to the antigen extracellularly, based on altering at least one amino acid of the antigen binding domain of the antigen binding molecule and simultaneously altering at least one amino acid of the human FcRn binding domain of the antigen binding molecule having human FcRn binding activity in the acidic pH range. The present invention also provides a method for promoting intracellular dissociation of an antigen from an antigen-binding molecule that is extracellularly bound to the antigen, the method performed by: by substituting at least one amino acid with histidine or inserting at least one histidine into the antigen binding domain of the antigen binding molecule, while substituting at least one of the following amino acids in the parent IgG Fc domain selected from the human FcRn binding domain with a different amino acid: 237. 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436(EU numbering).
In the present invention, an antigen can be dissociated from an antigen-binding molecule at any position within a cell; however, the preferred site of dissociation is the early endosome. Herein, "to dissociate intracellularly from the antigen-binding molecule the antigen bound to the antigen-binding molecule extracellularly" does not necessarily mean that all the antigen taken into the cell by binding to the antigen-binding molecule dissociates intracellularly from the antigen-binding molecule. Thus, it is acceptable that the proportion of antigen that is dissociated from the antigen binding molecule within the cell is increased compared to before decreasing the antigen binding activity of the antigen binding molecule in the acidic pH range to less than the antigen binding activity in the neutral pH range while increasing the human FcRn binding activity in the neutral pH range. Such methods for facilitating intracellular dissociation of antigen from an antigen-binding molecule that is extracellularly bound to the antigen are synonymous with the following methods: a method of conferring to an antigen-binding molecule the property of promoting intracellular dissociation of an antigen from the antigen-binding molecule by promoting uptake of the antigen-binding molecule bound to the antigen.
The invention also provides methods for promoting extracellular release of antigen-free antigen binding molecules that are taken up into cells in an antigen-bound form. More specifically, the present invention provides a method for promoting the extracellular release of an antigen-free antigen-binding molecule, which is taken up into a cell in an antigen-bound form, by: by increasing the human FcRn binding activity of an antigen binding molecule having human FcRn binding activity in the acidic pH range in the neutral pH range and decreasing its antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range. The invention also provides a method for promoting the extracellular release of an antigen-free antigen binding molecule which is taken up into a cell in an antigen-bound form, based on altering at least one amino acid in the antigen binding molecule and simultaneously altering at least one amino acid in the human FcRn binding domain. The present invention also provides a method for promoting extracellular release of an antigen-free antigen-binding molecule taken up into a cell in an antigen-bound form, the method performed by: by substituting at least one amino acid with histidine or inserting at least one histidine into the antigen binding molecule, while substituting at least one amino acid selected from the following position amino acids in the parent IgG Fc domain of the human FcRn binding domain with a different amino acid: 237. 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436(EU numbering).
Herein, "the antigen-binding molecule that is taken up into the cell in an antigen-bound form without containing the antigen is released extracellularly" does not necessarily mean that all the antigen-binding molecule that is taken up into the cell and bound to the antigen is released extracellularly in an antigen-free form. It is acceptable that the proportion of antigen binding molecule released extracellularly in an antigen-free form is increased compared to before decreasing the antigen binding activity of the antigen binding molecule in the acidic pH range to less than the antigen binding activity in the neutral pH range and increasing the human FcRn binding activity in the neutral pH range. The antigen binding molecules released outside the cells preferably retain antigen binding activity. Such methods for promoting extracellular release of antigen-free antigen-binding molecules that are taken up into cells in antigen-bound form are synonymous with the following methods: a method of conferring to an antigen-binding molecule the property of promoting extracellular release of an antigen-free antigen-binding molecule that is taken up into a cell in an antigen-bound form by promoting uptake of the antigen-binding molecule into the cell.
The invention also provides methods for increasing the ability to eliminate plasma antigens by administering antigen binding molecules. In the present invention, "a method for increasing the ability to eliminate plasma antigens" is synonymous with "a method for increasing the ability of an antigen-binding molecule to eliminate antigens from plasma". More specifically, the present invention provides methods for increasing the ability of an antigen binding molecule having human FcRn binding activity in the acidic pH range to eliminate plasma antigens by increasing the human FcRn binding activity of the antigen binding molecule in the neutral pH range. The invention also provides a method for increasing the ability to eliminate plasma antigens from an antigen binding molecule having human FcRn binding activity in the acidic pH range, said method being based on altering at least one amino acid in the human FcRn binding domain of the antigen binding molecule.
The present invention also provides a method for increasing the ability of an antigen binding molecule having human FcRn binding activity in the acidic pH range to eliminate plasma antigens by using a human FcRn binding domain comprising an amino acid sequence with a substitution of at least one of the following amino acids selected from the following positions in the parent IgG Fc domain of a human FcRn binding domain comprising the Fc domain of a parent IgG with a different amino acid: 237. 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436(EU numbering).
The present invention also provides a method for increasing the ability of an antigen binding molecule to eliminate a plasma antigen, the method performed by: the antigen binding activity of the above antigen binding molecule in an acidic pH range, which is improved by reducing the ability to eliminate plasma antigens, as compared to the antigen binding activity in a neutral pH range. The present invention also provides a method for increasing the ability of an antigen binding molecule to eliminate a plasma antigen, the method performed by: at least one amino acid in the antigen binding domain of the above antigen binding molecules that is improved by altering its ability to eliminate plasma antigens. The present invention also provides a method for increasing the ability to eliminate plasma antigens by administering an antigen binding molecule, said method performed by: an antigen binding domain of the above antigen binding molecule improved by the ability to eliminate plasma antigens by substituting at least one amino acid with histidine or inserting at least one histidine.
Herein, "the ability to eliminate plasma antigens" means the ability to eliminate antigens from plasma when the antigen binding molecule is administered or secreted in vivo. Thus, by "an increase in the ability of an antigen binding molecule to eliminate plasma antigens" is meant herein an increased rate of elimination of antigens from plasma when the antigen binding molecule is administered compared to prior to increasing human FcRn binding activity of the antigen binding molecule in the neutral pH range or prior to increasing human FcRn binding activity while simultaneously decreasing its antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range. The increase in the activity of an antigen-binding molecule to eliminate antigen from plasma can be assessed, for example, by administering soluble antigen and antigen-binding molecule in vivo and measuring the concentration of soluble antigen in plasma after administration. An increased ability of an antigen-binding molecule to eliminate plasma antigens is assumed when the concentration of soluble antigens in plasma is reduced after administration of soluble antigens and antigen-binding molecules, either by increasing the human FcRn binding activity of the antigen-binding molecule in the neutral pH range, or by increasing its human FcRn binding activity while reducing its antigen-binding activity in the acidic pH range to less than the antigen-binding activity in the neutral pH range. The soluble antigen may be in the form of antigen bound by the antigen binding molecule or antigen unbound by the antigen binding molecule, the concentrations of which may be determined as "concentration of antigen bound by the antigen binding molecule in plasma" and "concentration of antigen unbound by the antigen binding molecule in plasma", respectively (the latter being synonymous with "concentration of free antigen in plasma"). Since "total plasma antigen concentration" means the sum of the concentration of antigen bound to the antigen-binding molecule and the concentration of unbound antigen or "plasma free antigen", the latter being the concentration of antigen unbound to the antigen-binding molecule, the concentration of soluble antigen can be determined as "total plasma antigen concentration". Various methods for measuring "total antigen concentration in plasma" or "free antigen concentration in plasma" are well known in the art as described below.
The invention also provides methods for improving the pharmacokinetics of antigen binding molecules. More specifically, the present invention provides methods for improving the pharmacokinetics of antigen binding molecules having human FcRn binding activity in the acidic pH range by increasing human FcRn binding activity of antigen binding molecules in the neutral pH range. Furthermore, the present invention provides methods for improving the pharmacokinetics of antigen binding molecules having human FcRn binding activity in the acidic pH range by altering at least one amino acid in the human FcRn binding domain of the antigen binding molecule.
The present invention also provides a method for improving the pharmacokinetics of an antigen binding molecule having human FcRn binding activity in the acidic pH range by using a human FcRn binding domain comprising an amino acid sequence with a substitution of at least one of the following amino acids selected from the amino acids at the following positions in the parent IgG Fc domain of a human FcRn binding domain comprising an Fc domain of an IgG with a different amino acid: 237. 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436(EU numbering).
Furthermore, the present invention provides a method for improving the pharmacokinetics of an antigen binding molecule by: the antigen binding activity of the above antigen binding molecules in the acidic pH range, which is improved by reducing the pharmacokinetics, to less than its antigen binding activity in the neutral pH range. The present invention also provides a method for improving the pharmacokinetics of an antigen binding molecule having human FcRn binding activity in the acidic pH range by: at least one amino acid in the antigen binding domain of the above antigen binding molecule that is improved by altering pharmacokinetics. The present invention also provides a method for improving pharmacokinetics by: an antigen binding domain of the above antigen binding molecule having improved pharmacokinetics by substituting at least one amino acid with histidine or inserting at least one histidine into the antigen binding domain.
Herein, "improvement of pharmacokinetics" and "superior pharmacokinetics" may be described in another way as "improvement of plasma (blood) retention", "superior plasma (blood) retention" and "prolonged plasma (blood) retention". These terms are synonymous.
In this context, "improvement of pharmacokinetics" refers not only to an extension of time after administration of the antigen binding molecule to a human or non-human animal, e.g., mouse, rat, monkey, rabbit, and dog, until elimination from plasma (e.g., until the antigen binding molecule degrades intracellularly, etc., and cannot return to plasma), but also to an extension of plasma retention of the antigen binding molecule in a form that allows antigen binding (e.g., in an antigen-free form of the antigen binding molecule) during the period of administration to elimination due to degradation. Intact human IgG can bind to FcRn in non-human animals. For example, it may be preferable to administer it using mice to confirm the properties of the antigen-binding molecules of the invention, since intact human IgG binds to mouse FcRn more strongly than to human FcRn (Int Immunol.2001, 12 months; 13 (12): 1551-9). As another example, it may also be preferred to use a mouse in which the native FcRn gene is disrupted and which carries the transgene of the human FcRn gene to be expressed (MethodsMol biol. 2010; 602: 93-104), which is administered to confirm the properties of the antigen binding molecules of the invention described herein below. In particular, "improvement of pharmacokinetics" also includes an extended period of time until elimination by degradation of the antigen binding molecule not bound to the antigen (the antigen-free form of the antigen binding molecule). If the antigen binding molecule has bound to the antigen, the antigen binding molecule in the plasma cannot bind to the new antigen. Thus, the longer the antigen binding molecule does not bind to an antigen, the longer it can bind to a new antigen (the higher the chance of binding to another antigen). This can shorten the time for the antigen to free from the antigen binding molecule and prolong the time for the antigen to bind to the antigen binding molecule in vivo. Antigen elimination from plasma can be accelerated by administering the antigen binding molecule, increasing the plasma concentration of the antigen-free form of the antigen binding molecule and prolonging the time that the antigen binds to the antigen binding molecule. Specifically, herein, "improvement of the pharmacokinetics of the antigen binding molecule" includes improvement of pharmacokinetic parameters of antigen-free form of the antigen binding molecule (any of prolonged plasma half-life, prolonged mean plasma residence time, and impaired plasma clearance), prolonged time of antigen binding to the antigen binding molecule, and accelerated antigen elimination mediated by the antigen binding molecule in plasma after administration of the antigen binding molecule. The improvement in the pharmacokinetics of the antigen binding molecule can be assessed by determining any of the following parameters: plasma half-life, mean plasma residence time and plasma clearance of the antigen-binding molecule or antigen-free form thereof ("pharmaceuticals: Enshu-niyoru Rikai (outstanding) Nanzando). For example, the plasma concentration of the antigen-binding molecule or antigen-free form thereof is determined after administration of the antigen-binding molecule to a mouse, rat, monkey, rabbit, dog, or human. Then, each parameter was measured. The pharmacokinetics of the antigen binding molecules are believed to be improved when the plasma half-life or mean plasma residence time is extended. The parameters can be determined by methods known to those skilled in the art. Appropriate evaluation of the parameters can be carried out, for example, by non-compartmental analysis using the pharmacokinetic analysis software winnonlin (pharsight) according to the manual of the attached instructions. Can be prepared by methods known to those skilled in the art, for example, using Clin Pharmacol.2008, month 4; 48(4): 406-17, the plasma concentration of antigen-binding molecules without antigen is determined.
Herein, "improvement of pharmacokinetics" also includes an increase in the time that an antigen binds to an antigen binding molecule after administration of the antigen binding molecule. Whether the time for the antigen to bind to the antigen-binding molecule is prolonged after administration of the antigen-binding molecule can be evaluated by measuring the plasma concentration of free antigen. Prolongation can be determined by the plasma concentration of free antigen measured or the time required to increase the ratio of free antigen concentration to total antigen depth.
The concentration of the compound can be determined by methods known to those skilled in the art, for example by Pharm res.2006, month 1; 23(1): 95-103 to determine the plasma concentration or the ratio of the concentration of free antigen to the total concentration of free antigen not bound to the antigen binding molecule. Alternatively, when an antigen has a specific function in vivo, whether the antigen binds to an antigen binding molecule (antagonist molecule) that neutralizes the function of the antigen can be evaluated by testing whether the function of the antigen is neutralized. Whether antigen function is neutralized can be assessed by measuring in vivo markers that reflect antigen function. Whether an antigen binds to an antigen binding molecule (agonist) that activates the function of the antigen can be assessed by measuring in vivo markers that reflect the function of the antigen.
The determination of the plasma concentration of free antigen and the ratio of the amount of free antigen in plasma to the total amount of antigen in plasma, the in vivo marker determination and such measurements are not particularly limited; however, it is preferred that the assay is performed after a certain time has elapsed after administration of the antigen binding molecule. In the present invention, the time after administration of the antigen-binding molecule is not particularly limited; one skilled in the art can determine the appropriate time based on the nature of the antigen binding molecule administered, and the like. Such times include, for example, 1 day after administration of the antigen binding molecule, 3 days after administration of the antigen binding molecule, 7 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. Herein, "plasma antigen concentration" means "total plasma antigen concentration" which is the sum of the antigen bound to the antigen-binding molecule and the antigen unbound, or "free plasma antigen concentration" which is the antigen concentration to which the antigen-binding molecule is unbound.
By administering the antigen binding molecule of the 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, 1,000-fold, or even more, compared to administering a reference antigen binding molecule comprising an intact human IgG Fc domain as a human FcRn binding domain, or compared to when the antigen binding domain molecule of the invention is not administered.
The molar ratio of antigen/antigen binding molecule can be calculated as follows:
a value: molar concentration of antigen at each time point
B value: molar concentration of antigen binding molecule at each time point
C value: molar concentration of antigen/molar concentration of antigen-binding molecule (antigen/molar ratio of antigen-binding molecule) at each time point
C＝A/B。
A smaller C value indicates a higher antigen elimination efficiency per antigen-binding molecule, and a higher C value indicates a lower antigen elimination efficiency per antigen-binding molecule.
The antigen/antigen binding molecule molar ratio can be calculated as described above.
Administration of the antigen binding molecules of the invention may reduce the antigen/antigen binding molecule molar ratio by a factor of 2, 5, 10, 20, 50, 100, 200, 500, 1,000 or even more compared to a reference antigen binding molecule comprising a fully human IgG Fc domain as human FcRn binding domain.
Herein, intact human IgG1, IgG2, IgG3 or IgG4 is preferably used as an intact human IgG for the purpose of comparing a reference intact human IgG to an antigen binding molecule for its human FcRn binding activity or in vivo activity. Preferably, a reference antigen binding molecule comprising the same antigen binding domain as the antigen binding molecule of interest and a fully human IgG Fc domain as the human FcRn binding domain may be suitably used. More preferably, intact human IgG1 is used, with the aim of comparing a reference intact human IgG to the antigen binding molecule for its human FcRn binding activity or in vivo activity.
The reduction in total antigen concentration or antigen/antibody molar ratio in plasma can be assessed as described in examples 6, 8 and 13. More specifically, human FcRn transgenic mouse strain 32 or strain 276(Jackson Laboratories, Methods Mol biol. 2010; 602: 93-104) was used and when the antigen binding molecule did not cross-react with the mouse counterpart antigen, it could be evaluated by an antigen-antibody co-injection model or a steady state antigen infusion model. When the antigen binding molecule is cross-reactive with the mouse counterpart, it can be evaluated by simply injecting it into human FcRn transgenic mouse strain 32 or strain 276(Jackson Laboratories). In the co-injection model, a mixture of an antigen binding molecule and an antigen is administered to mice. In a steady state antigen infusion model, an infusion pump containing an antigen solution is implanted into mice to achieve a constant plasma antigen concentration, and then the mice are injected with an antigen binding molecule. The test antigen binding molecules were administered at the same dose. Plasma total antigen concentration, plasma free antigen concentration and plasma antigen-binding molecule concentration are measured at appropriate time points using methods known to those skilled in the art.
The total or free antigen concentration in plasma and the antigen/antigen-binding molecule molar ratio can be measured at 2, 4, 7, 14, 28, 56 or 84 days after administration to evaluate the long-term effects of the invention. In other words, the long-term plasma antigen concentration is determined by measuring the total or free antigen concentration in plasma and the antigen/antigen-binding molecule molar ratio 2, 4, 7, 14, 28, 56, or 84 days after administration of the antigen-binding molecule to evaluate the properties of the antigen-binding molecules of the invention. Whether a reduction in plasma antigen concentration or antigen/antigen-binding molecule molar ratio is achieved by the antigen-binding molecules of the invention can be determined by assessing the reduction at any one or more of the above time points.
The total or free antigen concentration in plasma and the antigen/antigen-binding molecule molar ratio can be measured 15 minutes, 1, 2, 4, 8, 12 or 24 hours after administration to evaluate the short-term effects of the invention. In other words, short term plasma antigen concentrations are determined by measuring the total or free antigen concentration and the antigen/antigen binding molecule molar ratio in plasma 15 minutes, 1, 2, 4, 8, 12 or 24 hours after administration of the antigen binding molecule to assess the properties of the antigen binding molecules of the invention.
The route of administration of the antigen binding molecules of the present invention may be selected from intradermal, intravenous, intravitreal, subcutaneous, intraperitoneal, parenteral, and intramuscular injections.
In the present invention, an improvement in pharmacokinetics in humans is preferred. If plasma retention in humans is difficult to determine, it can be predicted from plasma retention in mice (e.g., normal mice, transgenic mice expressing human antigens, transgenic mice expressing human FcRn) or monkeys (e.g., cynomolgus monkeys).
In this context, an acidic pH range generally means a pH of 4.0 to 6.5. The acidic pH range is preferably a range expressed by any pH value within the range of pH 5.5 to pH 6.5, preferably selected from the group consisting of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 and 6.5, particularly preferably pH 5.8 to pH6.0, which is close to the pH of early endosomes in vivo. Meanwhile, the neutral pH range herein generally means pH 6.7 to pH 10.0. The neutral pH range is preferably a range expressed by any pH value within the range of pH 7.0 to pH 8.0, 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, particularly preferably pH 7.4, which is close to the in vivo plasma (blood) pH. If it is difficult to assess the binding affinity between the human FcRn binding domain and human FcRn due to its low affinity at pH 7.4, pH 7.0 may be used instead of pH 7.4. As for the temperature used for the assay conditions, the binding affinity between the human FcRn binding domain and human FcRn can be assessed at any temperature from 10 ℃ to 50 ℃. Temperatures between 15 ℃ and 40 ℃ are preferably used to determine the binding affinity between the human FcRn binding domain and human FcRn. More preferably also any temperature between 20 ℃ and 35 ℃, such as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 ℃, is used to determine the binding affinity between the human FcRn binding domain and human FcRn. The 25 ℃ temperature described in example 5 is an example of an embodiment of the present invention.
Thus, herein "reducing the antigen binding activity of an antigen binding molecule in the acidic pH range to less than the antigen binding activity in the neutral pH range" means that the antigen binding activity of the antigen binding molecule is reduced at pH 4.0 to pH 6.5 compared to its antigen binding activity at pH 6.7 to pH 10.0. Preferably the above expression means that the antigen binding activity of the antigen binding molecule is reduced at pH 5.5 to pH 6.5 compared to its antigen binding activity at pH 7.0 to pH 8.0, more preferably it means that its antigen binding activity at early endosomal pH is reduced compared to its antigen binding activity at plasma pH in vivo. Specifically, the antigen binding activity of the antigen binding molecule is reduced at pH 5.8 to pH 6.0 compared to the antigen binding activity of the antigen binding molecule at pH 7.4.
Meanwhile, the expression "reducing the antigen binding activity of the antigen binding molecule in the acidic pH range to less than the antigen binding activity in the neutral pH range" herein may also be expressed as "increasing the antigen binding activity of the antigen binding molecule in the neutral pH range to more than the antigen binding activity in the acidic pH range". In particular, in the present invention, the ratio of the antigen binding activity of the antigen binding molecule between acidic and neutral pH ranges can be increased. For example, in the following embodiments, the value of KD (pH 5.8)/KD (pH 7.4) is increased. The ratio of antigen binding activity of the antigen binding molecule between the acidic and neutral pH ranges can be increased by, for example, decreasing its antigen binding activity in the acidic pH range, increasing its antigen binding activity in the neutral pH range, or both.
Herein, the expression "the antigen binding activity in the acidic pH range is decreased as compared to the antigen binding activity in the neutral pH range" is sometimes replaced with "the antigen binding activity in the acidic pH range is decreased to be less than the antigen binding activity in the neutral pH range".
In this context, human FcRn binding activity in the acidic pH range means human FcRn binding activity at pH 4.0-pH6.5, preferably at pH 5.5-pH 6.5, particularly preferably at pH 5.8-pH 6.0, comparable to early in vivo endosomal pH. Meanwhile, herein, human FcRn binding activity in the neutral pH range means human FcRn binding activity at pH 6.7-pH 10.0, preferably human FcRn binding activity at pH7.0-pH 8.0, particularly preferably human FcRn binding activity at pH 7.4, pH 7.4 being comparable to plasma pH in vivo.
The antigen binding molecules of the invention have a human FcRn binding domain. The human FcRn binding domain is not particularly limited as long as the antigen binding molecule has human FcRn binding activity in acidic and neutral pH ranges. Alternatively, the domain may have direct or indirect human FcRn binding activity. Such domains include, for example, immunoglobulins of the IgG type, albumin domain 3, anti-human FcRn antibodies, anti-human FcRn peptides, and Fc domains of anti-human FcRn scaffold molecules, all of which have direct binding activity to human FcRn; and molecules that bind IgG or albumin, which have indirect binding activity to human FcRn. Such preferred domains of the invention have human FcRn binding activity in the acidic and neutral pH ranges. The domains can be used without any modification, as long as they already have human FcRn binding activity in the acidic and neutral pH ranges. If the domain has only weak or no human FcRn binding activity in the acidic and/or neutral pH range, it may be made human FcRn binding activity by altering the amino acids in the antigen binding molecule. However, it is preferred to have human FcRn binding activity in the acidic and/or neutral pH range by altering the amino acids of the human FcRn binding domain. Alternatively, amino acids in domains that already have human FcRn binding activity in acidic and/or neutral pH ranges may be altered to increase human FcRn binding activity. The desired amino acid changes in the human FcRn binding domain can be selected by comparing the binding activity of human FcRn in the acidic and/or neutral pH range before and after the amino acid change.
Preferred human FcRn binding domains are those regions that bind directly to human FcRn. Such preferred human FcRn binding regions include, for example, antibody Fc domains. Meanwhile, a region capable of binding to a polypeptide such as albumin or IgG (which has human FcRn binding activity) can indirectly bind to human FcRn through albumin, IgG, or the like. Thus, such human FcRn binding regions of the invention may be regions that bind to polypeptides having human FcRn binding activity.
The antigen-binding molecules of the present invention are not particularly limited as long as they include an antigen-binding domain having a binding activity specific to a target antigen. Such preferred antigen binding domains include, for example, domains having an antigen binding region of an antibody. The antigen binding region of an antibody comprises, for example, CDRs and variable regions. When the antigen binding region of an antibody is a CDR, it may contain all 6 CDRs, or 1, 2 or more CDRs, of the whole antibody. When CDRs are contained as binding regions of antibodies, they may comprise amino acid deletions, substitutions, additions and/or insertions, or may be part of a CDR.
In another aspect, the antigen binding molecules used in the methods of the invention comprise antigen binding molecules having antagonistic activity (antagonistic antigen binding molecules), antigen binding molecules having agonistic activity (agonistic antigen binding molecules), and molecules having cytotoxicity. In a preferred embodiment, the antigen binding molecule comprises an antagonistic antigen binding molecule, in particular an antagonistic antigen binding molecule recognizing an antigen (e.g. a receptor or a cytokine).
In the present invention, the antigen-binding molecule of interest is not particularly limited and may be any antigen-binding molecule. The antigen binding molecules of the present invention preferably comprise both an antigen binding activity (antigen binding domain) and a human FcRn binding domain. In particular, preferred antigen binding molecules of the invention comprise a domain that binds to human FcRn. Antigen binding molecules that include both an antigen binding domain and a human FcRn binding domain include, for example, antibodies. Preferred antibodies in the context of the present invention include, for example, IgG antibodies. When the antibody to be used is an IgG antibody, the type of IgG is not limited; IgG belonging to any isotype (subclass) such as IgG1, IgG2, IgG3 or IgG4 can be used. In addition, the antigen binding molecules of the present invention may include antibody constant regions, and amino acid mutations may be introduced into the constant regions. Amino acid mutations to be introduced include, for example, amino acid mutations that potentiate or attenuate binding to the Fc γ receptor (Proc Natl Acad Sci U S A.2006, 3/14/2006; 103 (11): 4005-10), but are not limited to these examples. Alternatively, pH-dependent binding can also be altered by selecting appropriate constant regions, such as that of IgG 2.
When the antigen binding molecule of interest of the present invention is an antibody, it may be an antibody derived from any animal, such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody or a camel antibody. Further, the antibody may be an altered antibody, such as a chimeric antibody, and in particular, an altered antibody comprising amino acid substitutions in the sequence of a humanized antibody, or the like. Antibodies also include bispecific antibodies, antibody modification products linked to a variety of molecules, and polypeptides including antibody fragments.
"chimeric antibody" is an antibody prepared by combining sequences derived from different animals. Specifically, chimeric antibodies include, for example, antibodies having variable (V) regions from the heavy and light chains of a mouse antibody and constant (C) regions from the heavy and light chains of a human antibody.
A "humanized antibody", also known as an engineered human antibody, is an antibody in which Complementarity Determining Regions (CDRs) of an antibody derived from a non-human mammal (e.g., a mouse) are grafted into the CDRs of a human antibody. Methods for identifying CDRs are known (Kabat et al, Sequence of proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al, Nature (1989) 342: 877). General genetic recombination techniques suitable for this purpose are also known (see European patent applications EP 125023 and WO 96/02576).
Bispecific antibodies refer to antibodies having variable regions that recognize different epitopes in the same antibody molecule. Bispecific antibodies can be antibodies that recognize two or more different antigens, or antibodies that recognize two or more different epitopes on the same antigen.
In addition, polypeptides comprising antibody fragments include, for example, Fab fragments, F (ab') 2 fragments, scFvs (Nat Biotechnol.2005, 9 months; 23 (9): 1126-36), domain antibodies (dAbs) (WO2004/058821, WO 2003/002609), scFv-Fc (WO 2005/037989), dAb-Fc, and Fc fusion proteins. When the molecule includes an Fc domain, the Fc domain may function as a human FcRn binding domain. Alternatively, FcRn binding domains may be fused to these molecules.
Furthermore, antigen binding molecules suitable for the present invention may be antibody-like molecules. Antibody-like molecules (scaffold molecules, peptide molecules) are molecules that can show function by binding to a target molecule (Current Opinion in Biotechnology (2006) 17: 653-; Current Opinion in Biotechnology (2007) 18: 1-10; Current Opinion in Structural Biology (1997) 7: 463-; Protein Science (2006) 15: 14-27) including, for example, DARPins (WO 2002/020565), affibodies (WO 1995/001937), Avimers (WO 2004/044011; WO 2005/040229) and Adnectins (WO 2002/032925). If these antibody-like molecules can bind to the target molecule in a pH-dependent manner and/or have human FcRn binding activity in the neutral pH range, cellular uptake of the antigen can be facilitated by the antigen-binding molecule, reduction of plasma antigen concentration can be facilitated by administration of the antigen-binding molecule, and the pharmacokinetics of the antigen-binding molecule is improved, increasing the number of antigens to which a single antigen-binding molecule can bind.
Furthermore, the antigen binding molecule may be a protein resulting from fusion between a human FcRn binding domain and a receptor protein (including a ligand) that binds to a target, including, for example, TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, and CTLA4-Fc fusion protein (Nat Med.2003, 1/9 (1): 47-52; BioDrugs. (2006)20 (3): 151-60). If these receptor-human FcRn binding domain fusion proteins bind to a target molecule (including a ligand) in a pH-dependent manner and/or have human FcRn binding activity in the neutral pH range, cellular uptake of antigen can be facilitated by the antigen binding molecule, reduction of plasma antigen concentration can be facilitated by administration of the antigen binding molecule, and the pharmacokinetics of the antigen binding molecule is improved, and the number of antigens to which a single antigen binding molecule can bind is increased. The receptor protein is suitably designed and modified so as to include a domain of the receptor protein that binds to a target (including a ligand). As mentioned hereinabove including examples of TNFR-Fc fusion proteins, IL1R-Fc fusion proteins, VEGFR-Fc fusion proteins and CTLA4-Fc fusion proteins, it is preferred in the present invention to use soluble receptor molecules which comprise the extracellular domain of the receptor protein required for binding to the target (including the ligand). These designed and modified receptor molecules are referred to herein as artificial receptors. Methods employed to design and modify receptor molecules to construct artificial receptor molecules are known in the art.
Furthermore, the antigen binding molecule may be a fusion protein in which an artificial ligand protein that binds to a target and has a neutralizing effect is fused to a human FcRn binding domain, and the artificial ligand protein includes, for example, mutant IL-6(EMBO J.1994, 12, 15; 13 (24): 5863-70). If such artificial ligand fusion proteins can bind to a target molecule in a pH-dependent manner and/or have human FcRn binding activity in the neutral pH range, cellular uptake of the antigen can be facilitated by the antigen binding molecule, reduction of plasma antigen concentration can be facilitated by administration of the antigen binding molecule, and the pharmacokinetics of the antigen binding molecule can be improved, and the number of antigens to which a single antigen binding molecule can bind can be increased.
Furthermore, the antibody of the present invention may comprise a modified sugar chain. Antibodies having modified sugar chains include, for example, an antibody having modified glycosylation (WO 99/54342), an antibody lacking fucose added to a sugar chain (WO 00/61739, WO 02/31140, WO 2006/067847, WO2006/067913), and an antibody having a sugar chain with bisecting GlcNAc (WO 02/79255).
The conditions used for the determination of antigen binding or human FcRn binding activity other than pH can be appropriately selected by those skilled in the art, and the conditions are not particularly limited. For example, the activity can be determined by using MES buffer at 37 ℃ as described in WO 2009/125825. In another embodiment, the activity can be measured using a sodium phosphate buffer at 25 ℃ as described in example 4 or 5. Meanwhile, the antigen binding activity of the antigen binding molecule and the human FcRn binding activity can be measured by a method known to those skilled in the art, for example, using biacore (ge healthcare), etc. If the antigen is a soluble antigen, the activity of the antigen-binding molecule in binding to the soluble antigen can be determined by applying the antigen as an analyte to a chip on which the antigen-binding molecule is immobilized. Alternatively, if the antigen is a membrane-type antigen, the activity of binding of the antigen-binding molecule to the membrane-type antigen can be measured by adding the antigen-binding molecule as an analyte to an antigen-immobilized chip. The human FcRn binding activity of an antigen binding molecule can be determined by adding the human FcRn or antigen binding molecule as an analyte to a chip on which the antigen binding molecule or human FcRn is immobilized, respectively.
In the present invention, the ratio between the antigen binding activity in the acidic pH range and the antigen binding activity in the neutral pH range is not particularly limited as long as the antigen binding activity in the acidic pH range is lower than the antigen binding activity in the neutral pH range. However, the value of KD (pH5.8)/KD (pH 7.4), which is the ratio of the dissociation constants (KD) of the antigen at pH5.8 and pH 7.4, is preferably 2 or more, more preferably 10 or more, and still more preferably 40 or more. The upper limit of the KD (pH5.8)/KD (pH 7.4) value is not particularly limited and may be any value, for example 400, 1,000 or 10,000, as long as it can be produced by techniques known to those skilled in the art.
If the antigen is a soluble antigen, the value of the antigen binding activity can be expressed as the dissociation constant (KD). On the other hand, if the antigen is a membrane-type antigen, the activity can be expressed by an apparent dissociation constant (apparent KD). Dissociation constants (KD) and apparent dissociation constants (apparent KD) can be determined by methods known to those skilled in the art using, for example, biacore (ge healthcare), Scatchard curves, flow cytometry, and the like.
In the present invention, other parameters indicating the ratio of antigen binding activity between acidic and neutral pH ranges include, for example, the dissociation rate constant k d. If the dissociation rate constant (k) is usedd) And not the dissociation constant (KD) as a parameter representing the ratio of binding activities, then kd(in the acidic pH range)/kd(in the neutral pH range) which is the k of the antigen in the acidic and neutral pH rangesd(dissociation rate constant) is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, still more preferably 30 or more. k is a radical ofd(in the acidic pH range)/kdThe upper limit of the value (in the neutral pH range) is not particularly limited and may be anyAs long as it can be produced using techniques known to those skilled in the art, e.g., 50, 100, or 200.
If the antigen is a soluble antigen, the value of the antigen-binding activity can be determined using the dissociation rate constant (k)d) And (4) showing. Alternatively, if the antigen is a membrane-type antigen, the C value may be the apparent kd(apparent dissociation rate constant) representation. The dissociation rate constant (k) can be determined by methods known to those skilled in the art, for example, using Biacore (GE healthcare), flow cytometry, and the liked) And apparent dissociation rate constant (apparent k)d)。
In the present invention, when the antigen binding activity of the antigen binding molecule is measured at different pH, it is preferable that the measurement conditions other than pH are constant.
The method for reducing (attenuating) the antigen binding activity of the antigen binding molecule in the acidic pH range to less than the antigen binding activity in the neutral pH range (the method for imparting pH-dependent binding ability) is not particularly limited and can be achieved by any method. Specifically, as described in WO 2009/125825, the method includes a method for reducing (attenuating) an antigen binding activity in an acidic pH range to less than an antigen binding activity in a neutral pH range, for example, by substituting histidine for an amino acid in the antigen binding molecule or inserting histidine into the antigen binding molecule. It is known that an antibody pH-dependent antigen-binding activity can be imparted by substituting histidine for an amino acid in the antibody (FEBS Letter (1992)309 (1): 85-88). Such histidine mutation (substitution) or insertion site is not particularly limited; histidine may be substituted for amino acids at any position or inserted at any position. Preferred sites for histidine mutations (substitutions) or insertions include, for example, regions in which the mutation or insertion has an effect on the antigen binding activity of the antigen binding molecule. Such regions include sites where the mutation or insertion reduces (attenuates) the antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range (KD (in the acidic pH range)/KD (in the neutral pH range) value increase) as compared to prior to the mutation or insertion. If the antigen binding molecule is an antibody, such regions include, for example, antibody variable regions and CDRs. The number of histidine mutations or insertions to be introduced (effected) can be appropriately determined by the person skilled in the art. Histidine substitutions may be introduced at a single unique site or at two or more sites. Alternatively, histidine may be inserted at a single unique site or at two or more sites. In addition, in addition to the histidine mutation, a mutation other than the histidine mutation (a mutation (deletion, addition, insertion and/or substitution) of an amino acid other than histidine) may be introduced. Alternatively, histidine mutations may be combined with histidine insertions. Such histidine substitutions or insertions can be achieved by random methods, such as histidine scanning, which is performed by using histidine rather than alanine in alanine scanning as known to those skilled in the art. Antigen binding molecules having a KD (in the acidic pH range)/KD (in the neutral pH range) value greater than before the introduction of the mutation can then be selected from a library of antigen binding molecules introduced with random histidine mutations or insertions.
If histidine is substituted for an amino acid in the antigen-binding molecule or histidine is inserted into the antigen-binding molecule, the antigen-binding activity of the antigen-binding molecule in the neutral pH range after the histidine substitution or insertion is preferably equal to the antigen-binding activity of the antigen-binding molecule in the neutral pH range before the histidine substitution or insertion, but there is no particular limitation thereto. Herein, "the antigen binding activity of the antigen binding molecule in the neutral pH range after histidine substitution or insertion is equal to the antigen binding activity of the antigen binding molecule in the neutral pH range before histidine substitution or insertion" means that the antigen binding molecule retains 10% or more, preferably 50% or more, more preferably 80% or more, still more preferably 90% or more of the antigen binding activity of the antigen binding molecule before histidine substitution or insertion after histidine substitution or insertion. If the antigen binding activity of the antigen binding molecule is reduced due to a histidine substitution or insertion, the antigen binding activity may be adjusted by substituting, deleting, adding and/or inserting one or more amino acids of the antigen binding molecule to the antigen binding molecule such that the antigen binding activity becomes equal to the antigen binding activity prior to the histidine substitution or insertion. The invention also encompasses antigen binding molecules whose binding activity is equalized by substitution, deletion, addition and/or insertion of one or more amino acids of the antigen binding molecule after histidine substitution or insertion.
Other methods for reducing (attenuating) the antigen binding activity of an antigen binding molecule in the acidic pH range to less than the antigen binding activity in the neutral pH range include methods of substituting or inserting unnatural amino acids into antigen binding molecules with unnatural amino acids. It is known that pKa can be adjusted manually by using unnatural Amino Acids (Angew. Chem. int. Ed.2005, 44, 34; Chem Soc Rev.2004, 9/10/33 (7): 422-30; Amino Acids (1999)16 (3-4): 345-79). Therefore, in the present invention, an unnatural amino acid may be used in place of histidine as described above. The site of introducing the unnatural amino acid is not particularly limited, and substitution or insertion of the unnatural amino acid can be performed at any site. Preferred sites for non-natural amino acid substitutions or insertions include, for example, regions in which a substitution or insertion has an effect on the antigen-binding activity of the antigen-binding molecule. For example, if the antigen binding molecule is an antibody, such regions include antibody variable regions and Complementarity Determining Regions (CDRs). Meanwhile, the number of unnatural amino acids to be introduced is not particularly limited; substitutions with unnatural amino acids can be made at a single unique site or at two or more sites. Alternatively, the unnatural amino acid can be inserted at a single site only or at two or more sites. In addition, other amino acids may be deleted, added, inserted, and/or substituted in addition to the substitution or insertion of the unnatural amino acid. Furthermore, substitution and/or insertion of unnatural amino acids can be performed in combination with the histidine substitution and/or insertion described above. Any unnatural amino acid can be used in the invention. Unnatural amino acids known to those skilled in the art can be used.
In the present invention, if the antigen binding molecule is an antibody, possible sites for histidine or unnatural amino acid substitutions include, for example, CDR sequences and sequences responsible for the CDR structure of the antibody, including, for example, the sites described in WO 2009/125825. Amino acid positions are labeled according to Kabat numbering (Kabat EA et al (1991) Sequences of Proteins of immunologicalcatest, NIH).
When referring to residues of the variable domain (roughly residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al, Sequences of immunological interest. 5 th edition. Public Health Service, National institutes of Health, Bethesda, Md. (1991)). When referring to residues of the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., EU index as reported by Kabat et al, supra). "EU index according to Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise indicated herein, reference to residue numbering of antibody variable domains means residue numbering by the Kabat numbering system. Unless otherwise stated herein, reference to residue numbering of antibody constant domains means residue numbering by the EU numbering system (see, e.g., WO 2006073941).
Heavy chain: h27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b and H102
Light chain: l24, L27, L28, L32, L53, L54, L56, L90, L92 and L94
Of these sites of alteration, H32, H61, L53, L90 and L94 are assumed to be the most common sites of alteration.
If the antigen is an IL-6 receptor (e.g., human IL-6 receptor), preferred sites of alteration include the following. However, the site of alteration is not particularly limited herein.
Heavy chain: h27, H31, H32, H35, H50, H58, H61, H62, H63, H64, H65, H100b and H102
Light chain: l24, L27, L28, L32, L53, L56, L90, L92 and L94
In particular, preferred combinations of histidine or unnatural amino acid substitution sites include, for example, combinations of H27, H31, and H35; a combination of H27, H31, H32, H35, H58, H62, and H102; a combination of L32 and L53; and a combination of L28, L32, and L53. In addition, preferred combinations of substitution sites in the heavy and light chains include, for example, combinations of H27, H31, L32, and L53.
In these sites, histidine or an unnatural amino acid is substituted at only a single site or at more sites.
Meanwhile, when the antigen-binding molecule is a substance having an antibody constant region, a method for reducing (attenuating) the antigen-binding activity of the antigen-binding molecule in an acidic pH range to less than the antigen-binding activity in a neutral pH range includes, for example, a method for changing an amino acid in an antibody constant region. In particular, such methods include, for example, methods for substituting the constant regions (SEQ ID NOS: 11, 12, 13 and 14) described in WO 2009/125825. Meanwhile, methods for changing the constant region of an antibody include, for example, methods for evaluating various constant region isotypes (IgG1, IgG2, IgG3, and IgG4) and selecting an isotype that reduces antigen binding activity in an acidic pH range (increases the off-rate in an acidic pH range). Such methods also include methods for reducing antigen binding activity (increasing off-rate in acidic pH range) in acidic pH range by introducing amino acid substitutions into the amino acid sequence of the wild-type isotype (amino acid sequence of wild-type IgG1, IgG2, IgG3 or IgG 4). The hinge region sequence in the antibody constant region is quite different between isotypes (IgG1, IgG2, IgG3, and IgG4), and the difference in the hinge region amino acid sequence has a great influence on antigen-binding activity. Therefore, depending on the type of antigen or epitope, an appropriate isotype can be selected to reduce antigen binding activity (increase off-rate in acidic pH range) in acidic pH range. In addition, since the difference in amino acid sequence of the hinge region has a great influence on antigen-binding activity, it is considered that a preferred amino acid substitution site in the amino acid sequence of the wild type isoform is within the hinge region.
When the antigen-binding activity of the antigen-binding molecule in the acidic pH range is reduced (attenuated) to be less than the antigen-binding activity in the neutral pH range (when the value of KD (in the acidic pH range)/KD (in the neutral pH range) is increased) by the above-mentioned method or the like, it is generally preferable that the value of KD (in the acidic pH range)/KD (in the neutral pH range) is two or more times, preferably 5 or more times, more preferably 10 or more times, as compared with the original antibody, but it is not particularly limited herein.
The above-described method can be employed to produce an antigen-binding molecule whose antigen-binding activity in the acidic pH range is reduced (attenuated) to less than the antigen-binding activity in the neutral pH range (antigen-binding molecule bound in a pH-dependent manner) by amino acid substitution or insertion of an antigen-binding molecule not having such properties. Other methods include methods for directly obtaining antigen binding molecules having the above properties. For example, pH-dependent antigen binding can be used as an indicator of antibodies obtained by immunizing an animal (mouse, rat, hamster, rabbit, human immunoglobulin transgenic mouse, human immunoglobulin transgenic rat, human immunoglobulin transgenic rabbit, llama (llamas), camel, etc.) with an antigen, and antibodies having desired properties of interest can be selected by screening. Antibodies can be produced by hybridoma technology or B-cell cloning technology (Proc Natl Acad Sci U A.1996, 7/23; 93 (15): 7843-8; J immunological methods.2006, 10/20/2006; 316 (1-2): 133-43; Journal of the Association for Laboratory Automation; WO 2004/106377, WO 2008/045140 and WO2009/113742), which are methods known to those skilled in the art, but are not limited thereto. Alternatively, antibodies with the properties of interest can be directly selected by screening using pH-dependent antigen binding as an indicator to provide a library of antigen binding domains in vitro. Such libraries include human naive libraries, non-human animal and human immune libraries, semi-synthetic libraries and synthetic libraries, which are known to those skilled in the art (Methods Mol Biol. 2002; 178: 87-100; J Immunol Methods.2004, 6 months; 289 (1-2): 65-80; and ExpertOpin Biol. Ther.2007, 5 months; 7 (5): 763-79), but are not limited thereto. However, the method is not particularly limited to these examples.
The present invention utilizes the pH difference as an environmental difference between plasma and endosomes to achieve differential binding affinity of antigen binding molecules to antigen in plasma and endosomes (strong binding in plasma and weak binding in endosomes). Since the environmental differences between plasma and endosomes are not limited to differences in pH, other factors whose concentrations differ within plasma and endosomes can be used instead of the pH-dependent binding properties for antigen binding molecules to antigen. Such factors can also be used to generate antibodies that bind to the antigen in plasma but dissociate the antigen in vivo. Thus, the invention also encompasses antigen binding molecules comprising an antigen binding domain and a human FcRn binding domain, which have human FcRn binding activity in the acidic and neutral pH range and lower antigen binding activity in vivo than in plasma, wherein human FcRn binding activity in plasma is stronger than the FcRn binding activity of intact human IgG.
The method for increasing the human FcRn binding activity of the human FcRn binding domain of the antigen binding molecule of the present invention in the neutral pH range is not particularly limited and may be increased by any method. In particular, when the Fc domain of an immunoglobulin of the IgG class is used as a human FcRn binding domain, human FcRn binding activity in the neutral pH range can be increased by changing the amino acids thereof. The Fc domain of such preferred IgG-type immunoglobulins to be altered include, for example, the Fc domain of parent human IgG (IgG1, IgG2, IgG3 or IgG4 and engineered variants thereof). Amino acids at any position may be changed to other amino acids as long as human FcRn binding activity in the neutral pH range is conferred or enhanced. When the antigen binding molecule has a human IgG1 Fc domain as the human FcRn binding domain, the molecule has alterations that enhance binding to human FcRn in the neutral pH range, preferably compared to that of the parent human IgG 1. Amino acids that can effect such changes include, for example, amino acids at the following positions: 221-. More specifically, such amino acid changes include, for example, those listed in table 1. Human FcRn binding of the Fc domain of an immunoglobulin of the IgG class in the neutral pH range can be increased by using the above modifications.
Furthermore, the changes that enhance binding to human FcRn in the acidic pH range compared to parental IgG are shown as examples in table 2. When appropriate changes that also enhance binding to human FcRn in the neutral pH range are selected from the above changes, they may be suitable for use in the present invention. Also, the combination of changes that potentiate binding of Fv4-IgG1 to human FcRn under acidic conditions are shown in tables 6-1 and 6-2. Particularly preferred amino acids to be altered in the Fc domain of the parental IgG include, for example, the amino acids at the following positions: 237. 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436(EU numbering). The human FcRn binding activity of the antigen binding molecule in the neutral pH range may be increased by substituting at least one amino acid selected from the above amino acids with a different amino acid.
Particularly preferred alterations include, for example, the parent IgG Fc domain:
an amino acid substitution in which Gly at position 237 is substituted into Met;
amino acid substitution of Pro at position 238 to Ala;
An amino acid substitution wherein Ser at position 239 is substituted with Lys;
an amino acid substitution wherein Lys at position 248 is replaced with Ile;
an amino acid substitution at Thr at position 250 with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr;
an amino acid substitution of Met at position 252 with Phe, Trp or Tyr;
an amino acid substitution of Ser at position 254 to Thr;
amino acid substitution of Arg at position 255 with Glu;
an amino acid substitution at position 256 of Thr substituted with Asp, Glu or Gln;
an amino acid substitution at position 257 of Pro substituted with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val;
an amino acid substitution wherein Glu at position 258 is substituted with His;
an amino acid substitution at position 265 wherein Asp is substituted with Ala;
270 Asp substituted with an amino acid Phe;
an amino acid substitution wherein Asn at position 286 is replaced with Ala or Glu;
an amino acid substitution of Thr at position 289 with His;
an amino acid substitution wherein Asn at position 297 is substituted with Ala;
298 amino acid substitutions in which Ser is replaced with Gly;
an amino acid substitution wherein Val at position 303 is substituted with Ala;
an amino acid substitution wherein Val at position 305 is substituted with Ala;
a substitution of Thr at position 307 with an amino acid Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr;
An amino acid substitution of Val at position 308 with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr;
an amino acid substitution of Leu or Val at position 309 with Ala, Asp, Glu, Pro or Arg;
an amino acid substitution in which Gln at position 311 is replaced with Ala, His, or Ile;
an amino acid substitution wherein Asp at position 312 is substituted with Ala or His;
an amino acid substitution wherein Leu at position 314 is substituted with Lys or Arg;
an amino acid substitution wherein Asn at position 315 is substituted with Ala or His;
an amino acid substitution wherein Lys at position 317 is substituted with Ala;
an amino acid substitution wherein Asn at position 325 is replaced with Gly;
an amino acid substitution wherein Ile at position 332 is substituted with Val;
an amino acid substitution wherein Lys at position 334 is substituted with Leu;
an amino acid substitution wherein Lys at position 360 is replaced by His;
an amino acid substitution at position 376 of Asp to Ala;
an amino acid substitution in which Glu at position 380 is substituted with Ala;
an amino acid substitution wherein Glu at position 382 is substituted with Ala;
an amino acid substitution of Asn or Ser at position 384 with Ala;
an amino acid substitution wherein Gly at position 385 is substituted by Asp or His;
an amino acid substitution of Gln at position 386 with Pro;
amino acid substitution of Pro at position 387 with Glu;
An amino acid substitution wherein Asn at position 389 is substituted with Ala or Ser;
an amino acid substitution of Ser at position 424 with Ala;
an amino acid substitution of Met at position 428 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr;
an amino acid substitution of His at position 433 with Lys;
an amino acid substitution in which Asn at position 434 is substituted with Ala, Phe, His, Ser, Trp or Tyr;
and amino acid substitution of Tyr or Phe at position 436 with His (EU numbering). Meanwhile, the number of amino acids to be changed is not particularly limited; the amino acids may be changed at a single unique site or at two or more sites. Combinations of two or more amino acid changes include, for example, those listed in table 3. Also, the combination of changes that enhance binding to human FcRn in the acidic pH range compared to parental IgG are shown in tables 4-1 to 4-5. When suitable combinations of alterations that also potentiate binding to human FcRn in the neutral pH range are selected from the above, they may be suitable for use in the present invention. Furthermore, the combination of changes that potentiate binding of Fv4-IgG1 to human FcRn under neutral conditions are shown in tables 6-1 and 6-2.
The symbol "^" in the table indicates that an amino acid is inserted after the numbering shown by EU numbering. For example, 281S means that S is inserted between 281 and 282 bits numbered by EU.
[ Table 1]
| Position of | Amino acid changes |
| 256 | P |
| 280 | K |
| 339 | T |
| 385 | H |
| 428 | L |
| 434 | W，Y，F，A，H |
[ Table 2]
[ Table 3]
| Combinations of amino acid alterations |
| M252Y/S254T/T256E |
| M252Y/S254T/T256E/H433K/N434F/Y436H |
| H433K/N434F/Y436H |
| T307A/E380A |
| T307A/E380A/N434H |
| T307A/E380A/N434A |
| N434H/N315H |
| N434H/T289H |
| N434H/T370A/E380A |
| T250Q/M428L |
| T250Q/N434A |
| M252W/N434A |
| M252Y/N434A |
| T256A/N434A |
| T256D/N434A |
| T256E/N434A |
| T256S/N434A |
| P257I/Q311I |
| T307A/N434A |
| T307E/N434A |
| T307Q/N434A |
| V308P/N434A |
| L309G/N434A |
| Q311H/N434A |
| Q311R/N434A |
| N315D/N434A |
| A378V/N434A |
| E380S/N434A |
| E382V/N434A |
| S424E/N434A |
| M428L/N434A |
| N434A/Y436I |
| T437Q/N434A |
| T437R/N434A |
[ Table 4-1]
| Combinations of amino acid alterations |
| L234I/L235D |
| G236A/V308F/I332E |
| G236R/L328R |
| G236A/I332E/N434S |
| S239E/V264I/A330Y/I332E |
| S239E/V264I/I332E |
| S239E/V264I/S298A/A330Y/I332E |
| S239D/D265H/N297D/I332E |
| S239D/E272Y/I332E |
| S239D/E272S/I332E |
| S239D/E272I/I332E |
| S239D/N297D/I332E |
| S239D/K326T/I332E |
| S239Q/I332Q |
| S239Q/I332N |
| S239D/I332D |
| S239D/I332E |
| S239Q/I332E |
| S239E/I332E |
| F241W/F243W |
| F241Y/F243Y/V262T/V264T |
| F241W/F243W/V262A/V264A |
| F241L/V262I |
| F243L/V262I/V264W |
| F243L/K288D/R292P/Y300L/V305I/P396L/H435K |
| F243L/K288D/R292P/Y300L/H435K |
| F243L/R292P/Y300L/V305I/P396L/H435K |
| P245G/V308F |
| T250I/V259I/V308F |
| T250I/V308F |
| T250I/V308F/N434S |
| T250Q/V308F/M428L |
| T250Q/M428L |
| L251I/N434S |
| L251N/N434S |
| L251F/N434S |
| L251V/N434S |
| L251M/N434S |
| T252L/T254S/T256F |
| M252Y/S254T/T256E/N434M |
| M252Y/S254T/T256E/M428L/N434S |
| M252Y/S254T/T256E |
| M252Y/S254T/T256E/V308F |
| M252Y/S254T/T256E/N434S |
| M252Y/S254T/T256E/N434A |
| M252Y/S254T/T256E/M428L |
| M252Y/S254T/T256E/T307Q |
| M252F/T256D |
| M252Y/T256Q |
| M252Y/P257L |
| M252Y/P257N |
| M252Y/V259I |
| M252Y/V279Q |
| M252Y/V308P/N434Y |
| M252Q/V308F |
| M252Y/V308F |
Table 4-2 is a continuation of Table 4-1.
[ tables 4-2]
| M252Q/V308F/N434S |
| M252Y/V308F/M428L |
| M252Y/V308F/N434M |
| M252Y/V308F/N434S |
| M252Y/Y319I |
| M252Q/M428L/N434S |
| M252Y/M428L |
| M252Y/N434M |
| M252Y/N434S |
| M252Y/N434A |
| M252Y/N434Y |
| S254T/V308F |
| R255H/N434A |
| R255Q/N434S |
| R255H/N434S |
| T256V/V308F |
| T256P/Q311I |
| T256P/I332E |
| T256P/I332E/S440Y |
| T256P/E430Q |
| T256P/N434H |
| T256E/N434Y |
| T256P/S440Y |
| P257Y/V279Q |
| P257L/V279E |
| P257N/V279Q |
| P257N/V279E |
| P257N/V279Y |
| P257L/V279Q |
| P257N/^281S |
| P257L/^281S |
| P257N/V284E |
| P257N/L306Y |
| P257L/V308Y |
| P257L/V308F |
| P257N/V308Y |
| P257I/Q311I/N434H |
| P257L/Q311V |
| P257L/G385N |
| P257L/M428L |
| P257I/E430Q |
| P257I/N434H |
| P257L/N434Y |
| E258H/N434A |
| E258H/N434H |
| V259I/T307Q/V308F |
| V259I/V308F |
| V259I/V308F/Y319L |
| V259I/V308F/Y319I |
| V259A/V308F |
| V259I/V308F/N434M |
| V259I/V308F/N434S |
| V259I/V308F/M428L/N434S |
| V259I/V308F/M428L |
| V259I/Y319I |
| V259I/Y319I/N434S |
| V259I/M428L |
| V259I/M428L/N434S |
| V259I/N434S |
Table 4-3 is a continuation of Table 4-2.
[ tables 4 to 3]
| V259I/N434Y |
| V264I/A330L/I332E |
| V264I/I332E |
| D265F/N297E/I332E |
| S267L/A327S |
| E272R/V279L |
| V279E/V284E |
| V279Q/L306Y |
| V279Y/V308F |
| V279Q/V308F |
| V279Q/G385H |
| ^281S/V308Y |
| ^281S/V308F |
| ^281S/N434Y |
| E283F/V284E |
| V284E/V308F |
| V284E/G385H |
| K288A/N434A |
| K288D/H435K |
| K288V/H435D |
| T289H/N434A |
| T289H/N434H |
| L306I/V308F |
| T307P/V308F |
| T307Q/V308F/N434S |
| T307Q/V308F/Y319L |
| T307S/V308F |
| T307Q/V308F |
| T307A/E310A/N434A |
| T307Q/E380A/N434A |
| T307Q/M428L |
| T307Q/N434M |
| T307I/N434S |
| T307V/N434S |
| T307Q/N434S |
| T307Q/N434Y |
| V308T/L309P/Q311S |
| V308F/L309Y |
| V308F/Q311V |
| V308F/Y319F |
| V308F/Y319I/N434M |
| V308F/Y319I |
| V308F/Y319L |
| V308F/Y319I/M428L |
| V308F/Y319I/M428L/N434S |
| V308F/Y319L/N434S |
| V308F/I332E |
| V308F/G385H |
| V308F/M428L/N434M |
| V308F/M428L |
| V308F/M428L/N434S |
| V308P/N434Y |
| V308F/N434M |
| V308F/N434S |
| V308F/N434Y |
| Q311G/N434S |
| Q311D/N434S |
| Q311E/N434S |
| Q311N/N434S |
Tables 4-4 are continuation tables of tables 4-3.
[ tables 4 to 4]
| Q311Y/N434S |
| Q311F/N434S |
| Q311W/N434S |
| Q311A/N434S |
| Q311K/N434S |
| Q311T/N434S |
| Q311R/N434S |
| Q311L/N434S |
| Q311M/N434S |
| Q311V/N434S |
| Q311I/N434S |
| Q311A/N434Y |
| D312H/N434A |
| D312H/N434H |
| L314Q/N434S |
| L314V/N434S |
| L314M/N434S |
| L314F/N434S |
| L314I/N434S |
| N315H/N434A |
| N315H/N434H |
| Y319I/V308F |
| Y319I/M428L |
| Y319I/M428L/N434S |
| Y319I/N434M |
| Y319I/N434S |
| L328H/I332E |
| L328N/I332E |
| L328E/I332E |
| L328I/I332E |
| L328Q/I332E |
| L328D/I332E |
| L328R/M428L/N434S |
| A330L/I332E |
| A330Y/I332E |
| I332E/D376V |
| I332E/N434S |
| P343R/E345D |
| D376V/E430Q |
| D376V/E430R |
| D376V/N434H |
| E380A/N434A |
| G385R/Q386T/P387R/N389P |
| G385D/Q386P/N389S |
| N414F/Y416H |
| M428L/N434M |
| M428L/N434S |
| M428L/N434A |
| M428L/N434Y |
| H429N/N434S |
| E430D/N434S |
| E430T/N434S |
| E430S/N434S |
| E430A/N434S |
| E430F/N434S |
| E430Q/N434S |
| E430L/N434S |
| E430I/N434S |
| A431T/N434S |
Tables 4-5 are continuation tables of tables 4-4.
[ tables 4 to 5]
| A431S/N434S |
| A431G/N434S |
| A431V/N434S |
| A431N/N434S |
| A431F/N434S |
| A431H/N434S |
| L432F/N434S |
| L432N/N434S |
| L432Q/N434S |
| L432H/N434S |
| L432G/N434S |
| L432I/N434S |
| L432V/N434S |
| L432A/N434S |
| H433K/N434F |
| H433L/N434S |
| H433M/N434S |
| H433A/N434S |
| H433V/N434S |
| H433K/N434S |
| H433S/N434S |
| H433P/N434S |
| N434S/M428L |
| N434S/Y436D |
| N434S/Y436Q |
| N434S/Y436M |
| N434S/Y436G |
| N434S/Y436E |
| N434S/Y436F |
| N434S/Y436T |
| N434S/Y436R |
| N434S/Y436S |
| N434S/Y436H |
| N434S/Y436K |
| N434S/Y436L |
| N434S/Y436V |
| N434S/Y436W |
| N434S/Y436I |
| N434S/T437I |
Such amino acid changes can be suitably introduced using known methods. For example, alterations in the Fc domain of intact human IgG1 are described in Drug meta dispos.2007, month 1.35 (1): 86-94 parts of; int immunol.2006, 12 month and 18 days, (12): 1759 to 69; j Biol chem.2001, 3/2/276 (9): 6591 and 604; j Biol Chem. (2007)282 (3): 1709-17; JImmunol. (2002)169 (9): 5171-80; j Immunol. (2009)182 (12): 7663-71; molecular Cell, Vol.7, 867 and 877, month 4 of 2001; nat biotechnol.1997, 7 month 15, (7): 637-40; nat biotechnol.2005, month 10, 23, (10): 1283-8; proc Natl Acad Sci U S.2006, 12/5/103 (49): 18709-14; EP2154157, US 20070141052, WO 2000/042072, WO 2002/060919, WO2006/020114, WO 2006/031370, WO 2010/033279, WO 2006/053301 and WO 2009/086320.
According to Journal of Immunology (2009) 182: 7663-7671 the human FcRn binding activity of intact human IgG1 is KD 1.7 micromolar (microM) in the acidic pH range (pH 6.0), whereas almost no activity is detectable in the neutral pH range. Thus, in a preferred embodiment, the antigen binding molecules used in the methods of the invention include those that have human FcRn binding activity in the acidic pH range of KD 20 micromolar or greater, and that have the same or greater human FcRn binding activity as intact human IgG in the neutral pH range. In a more preferred embodiment, the antigen binding molecule comprises an antigen binding molecule that has a binding activity for human FcRn of KD 2.0 micromolar or more in the acidic pH range and KD 40 micromolar or more in the neutral pH range. In an even more preferred embodiment, the antigen binding molecule comprises an antigen binding molecule having a binding activity for human FcRn in the acidic pH range of KD 0.5 micromolar or more and a binding activity for human FcRn in the neutral pH range of KD 15 micromolar or more. By Journal of immunology (2009) 182: 7663-7671 (by immobilizing antigen binding molecules on a chip and loading human FcRn as an analyte) to determine the KD values.
The dissociation constant (KD) can be used as a value for human FcRn binding activity. However, the human FcRn binding activity of intact human IgG has almost no human FcRn binding activity in the neutral pH range (pH 7.4), and thus it is difficult to calculate the activity as KD. The method for assessing whether the human FcRn binding activity at pH 7.4 is higher than that of intact human IgG comprises an assessment method of comparing the intensity of Biacore reactions after loading the analyte at the same concentration. Specifically, when the reaction at pH 7.4 after loading human FcRn to the chip immobilized with the antigen-binding molecule is stronger than the reaction at pH 7.4 after loading human FcRn to the chip immobilized with complete human IgG, the human FcRn-binding activity of the antigen-binding molecule is regarded as higher than that of complete human IgG at pH 7.4.
A pH of 7.0 may also be used as the neutral pH range. Using pH 7.0 as neutral pH may promote a weak interaction between human FcRn and FcRn binding domains. As for the temperature used for the determination conditions, the binding affinity can be evaluated at any temperature of 10 ℃ to 50 ℃. Temperatures between 15 ℃ and 40 ℃ are preferably used to determine the binding affinity between the human FcRn binding domain and human FcRn. More preferably also any temperature between 20 ℃ and 35 ℃, such as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 ℃, is used to determine the binding affinity between the human FcRn binding domain and human FcRn. The 25 ℃ temperature described in example 5 is an example of an embodiment of the present invention. In a preferred embodiment, the interaction between human FcRn and FcRn binding domain may be measured at pH 7.0 and at 25 ℃ as described in example 5. The binding affinity of an antigen binding molecule to human FcRn can be measured by Biacore as described in example 5.
In a more preferred embodiment, the antigen binding molecules of the invention have human FcRn binding activity at pH 7.0 and at 25 ℃ which is stronger than the binding activity of intact human IgG. In a more preferred embodiment, the human FcRn binding activity at pH 7.0 and at 25 ℃ is 28-fold greater than that of intact human IgG or greater than KD 3.2 micromolar. In a more preferred embodiment, the human FcRn binding activity at pH 7.0 and at 25 ℃ is 38 times greater than that of intact human IgG or greater than KD 2.3 micromolar.
As intact human IgG, preferably intact human IgG1, IgG2, IgG3 or IgG4 is used, a reference intact human IgG aimed at comparing to antigen binding molecules for its human FcRn binding activity or in vivo activity. Preferably, a reference antigen binding molecule comprising the same antigen binding domain as the antigen binding molecule of interest and a fully human IgG Fc domain as the human FcRn binding domain may suitably be used. More preferably, intact human IgG1 is used, a reference intact human IgG aimed at comparing antigen binding molecules against their human FcRn binding activity or in vivo activity.
More specifically, the antigen binding molecules described herein that have a long-term effect on plasma antigen-depleting activity have human FcRn binding activity at pH 7.0 and at 25 ℃ in the range of 28-fold to 440-fold for full human IgG1 or a KD in the range of 3.0 micromolar to 0.2 micromolar. The long-term plasma antigen concentration is determined by measuring the total or free antigen concentration and the antigen/antigen-binding molecule molar ratio in plasma 2, 4, 7, 14, 28, 56, or 84 days after administration of the antigen-binding molecule to evaluate the long-term effect of the antigen-binding molecules of the invention on the activity of eliminating plasma antigens. Whether a reduction in plasma antigen concentration or antigen/antigen-binding molecule molar ratio is achieved by the antigen-binding molecules of the invention can be determined by assessing the reduction at any one or more of the above time points.
Even more specifically, the antigen binding molecules of the invention with short-term effects on the activity to deplete plasma antigens have human FcRn binding activity at pH 7.0 and at 25 ℃, which is 440-fold stronger than that of intact human IgG or KD stronger than 0.2 micromolar. Short-term plasma antigen concentrations are determined by measuring the total or free antigen concentration and the antigen/antigen-binding molecule molar ratio in plasma 15 minutes, 1, 2, 4, 8, 12 or 24 hours after administration of the antigen-binding molecule to evaluate the short-term effect of the antigen-binding molecules of the invention on the activity of eliminating plasma antigens.
The methods of the invention are applicable to any antigen binding molecule, regardless of the type of target antigen.
In the method of the present invention, the antigen recognized by the antigen binding molecule (e.g., target antibody) is not particularly limited. Such target antibodies may recognize any antigen. Antibodies whose pharmacokinetics are improved by the methods of the present invention include, for example, receptor proteins (membrane-bound receptor and soluble receptor), antibodies that recognize membrane antigens (e.g., cell surface markers), and antibodies that recognize soluble antigens (e.g., cytokines). Specific examples of antigens recognized by antibodies whose pharmacokinetics are improved by the method of the present invention include, for example: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE-2, activin A, activin AB, activin B, activin C, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS4, ADAMTS5, addressin, adiponectin, ADP ribosylcyclase-1, aIA, AGE, ALCAM, ALK-1, ALK-7, allergen, alpha 1-antitrypsin (antitrypsin antagonist), alpha 1-trrypsin, alpha-V-beta-synuclein, amyloid-beta-alpha-beta-synuclein, amyloid-beta-synuclein, and alpha-beta, Amyloid immunoglobulin heavy chain variable region, amyloid immunoglobulin light chain variable region, androgen, ANG, angiotensinogen, angiopoietin ligand-2, anti-Id, antithrombin III, anthrax, APAF-1, APE, APJ, apo A1, apo serum amyloid A, Apo-SAA, APP, APRIL, AR, ARC, ART, Artemin, ASPARTIC, atrial natriuretic factor, atrial natriuretic peptide A, atrial natriuretic peptide B, atrial natriuretic peptide C, av/B3 integrin, Axl, B7-1, B7-2, B7-H, lactamase, BACE-1, Bacillus anthracis (Bacillus anthracis) protective antigen, Bad, BAFF-R, Bag-1, BAK, Bax, BCA-1, GF, Bcl, BCMA, BDNF, B-ECs, beta-2-microglobulin, beta-SAA, beta-2-D, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BB lymphocyte stimulating factor (BIyS), BMP-2(BMP-2a), BMP-3 (osteogenic protein), BMP-4(BMP-2B), BMP-5, BMP-6(Vgr-1), BMP-7(OP-1), BMP-8(BMP-8a), BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3), BMPs, BOK, bombesin, bone derived neurotrophic factor, bovine growth hormone, BPDE-DNA, BRK-2, BTC, B lymphocyte cell adhesion molecule, C10, C1-inhibitor, C1q, C3, C3a, C4, C5, C5a (5 a), CA125, CAD-8, cadherin-3, Calcitonin, cAMP, carbonic anhydrase-IX, carcinoembryonic antigen (CEA), carcinoma-associated antigen, cardiac dystrophin-1, 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, CCL1/I-309, CCL 11/Eotaxin (Eotaxin), CCL12/MCP-5, CCL13/MCP-4, CCL14/HCC-1, CCL15/HCC-2, CCL16/HCC-4, CCL17/TARC, CCL18/PARC, CCL19/ELC, CCL2/MCP-1, CCL 20/3-alpha, MIP, CCL/SLC, CCL/MDC, CCL/MPIF-1, CCL/eotaxin-2, CCL/TECK, CCL/eotaxin-3, CCL/CTACK, CCL/MEC, CCL/M1-alpha, CCL/LD-78-beta, CCL/MIP-1-beta, CCL/RANTES, CCL/C, CCL/MCP-3, CCL/MCP-2, CCL/10/MTP-1-gamma, CCR, CD105, CD11, CD123, CD137, CD138, CD140, CD146, CD147, CD148, CD152, CD164, CD27, CD27, CCL/MPIF-1-alpha, CCL/alpha, CCL3, CCL/CTC/MCR-1-alpha, CCL/, CD3, CD30, CD30L, CD32, CD33(p67 protein), CD34, CD37, CD38, CD3E, CD4, CD40, CD40 40, CD49 40, CD66 40, CD40 (B40-1), CD40, CD105, CD158 40, CEA, ACACCEM 40, CFTR, cGMP, CGRP receptor, CINC, CKb 40-1, claudin 18, CLC, Clostridium botulinum toxin (Clostridium botulinum toxin), Clostridium difficile (Clostridium difficile) toxin, Clostridium difficile 3-CtC, CTC-40, CTC-3-C, CTC-40, CTC-C-3-C, CTC-C3, CTC-C, CTC-C3, CTC-C, CTC 3-C, CT, CXCL10, CXCL11/I-TAC, CXCL 12/SDF-1-alpha/beta, CXCL13/BCA-1, CXCL14/BRAK, CXCL15/Lungkine, CXCL16, CXCL16, CXCL 2/Gro-beta, CXCL 3/Gro-gamma, CXCL3, CXCL4/PF4, CXCL5/ENA-78, CXCL 2/GCP-2, CXCL7/NAP-2, CXCL8/IL-8, CXCL9/Mig, CXCLlO/IP-10, CXCR1, CXCR2, CXCR3, CXCR4, cystatin C, cytokeratin tumor-associated antigen, DAN, DCC, DKR 4, DC-SIGN, accelerated factor, delta-like protein ligand, IGF 1-alpha-beta-peptidyl-1, IGF-peptidyl-1-peptidyl-digoxido-1, CXCL-alpha-beta-peptidyl-1, CXCL-alpha-peptidyl-1, CXCL-alpha-beta-peptidyl-1, CXCL, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EGF-like domain containing protein 7, elastase, elastin, EMA, EMMPRIN, ENA-78, endosialin, endothelin receptor, endotoxin, enkephalinase, eNOS, Eot, eosinophil-selective chemokine-2, eotaxin, EpCAM, Ephrin B2/EphB4, Epha2 tyrosine kinase receptor, Epidermal Growth Factor Receptor (EGFR), ErbB2 receptor, Erb 3 tyrosine kinase receptor, ERCC, Erythropoietin (EPO), erythropoietin receptor, EPO-selectin, ET-1, Exodus-2, RSV F protein, F366323, F11, F12, F13F 2, F-Xa factor, factor Ia, EPO, Factor VII, factor VIII, factor VIIIc, Fas, Fc α R, Fc ε RI, Fc γ IIb, Fc γ RI, Fc γ RIIa, Fc γ RIIIa, Fc γ RIIIb, FcRn, FEN-1, ferritin, FGF-19, FGF-2 receptor, FGF-3, FGF-8, FGF-acidic, FGF-basic, FGFR-3, fibrin, Fibroblast Activation Protein (FAP), fibroblast growth factor-10, fibronectin, FL, FLIP, Flt-3, FLT3 ligand, folate receptor, Follicle Stimulating Hormone (FSH), fractal chemokine (CX3C), free heavy chain, free light chain, FZD1, FZD10, F2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD3, GCG 250, CSF 7376, GD 250, GD2, GD 42, GD-42 GD-G3884, GD-3 receptor, GDF, GDF-1, GDF-15(MIC-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, GDNF, gelsolin, GFAP, GF-CSF, GFR-alpha 1, GFR-alpha 2, GFR-alpha 3, GF-beta 1, gH envelope glycoprotein, GITR, glucagon receptor, glucagon-like peptide 1 receptor, Glut 4, glutamate carboxypeptidase II, glycoprotein hormone receptor, glycoprotein IIb/IIIa (GP IIb/IIIa), phosphatidylinositosan-3, GM-receptor, and, GP130, GP140, GP72, granulocyte-CSF (G-CSF), GRO/MGSA, growth hormone releasing factor, GRO- β, GRO- γ, H.pylori (H.pylori) hapten (NP-cap or NIP-cap), HB-EGF, HCC 1, HCMV gB envelope glycoprotein, HCMV UL, Hematopoietic Growth Factor (HGF), Hep B GP120, heparanase, heparin cofactor II, hepatic growth factor, Bacillus anthracis protective antigen, hepatitis C virus E2 glycoprotein, hepatitis E, Hepcidin, Her1, Her2/neu (ErbB-2), Her3(ErbB-3), Her4(ErbB-4), Herpes Simplex Virus (HSV) gB glycoprotein, HGF, HGFA, high molecular weight melanoma associated antigen (HMW-MAA), HIV envelope proteins such as GP120, HIV MIB 3, HLA-HA, HLA-gB glycoprotein, HLA-EGF, HCF, and HIV, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human cardiac myosin, Human Cytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin, human tissue plasminogen activator (t-PA), Huntingtin (Huntingtin), HVEM, IAP, ICAM-1, ICAM-3, ICE, ICOS, IFN- α, IFN- β, IFN- γ, IgA receptor, IgE, IGF binding protein, IGF-1R, IGF-2, IGFBP, IGFR, IL-1, IL-10 receptor, IL-11 receptor, IL-12 receptor, IL-13 receptor, IL-15 receptor, IL-16 receptor, IL-17, IL-17 receptor, IL-18(IGIF), IL-18 receptor, IL-1 alpha, IL-1 beta, IL-1 receptor, IL-2 receptor, IL-20 receptor, IL-21 receptor, IL-23 receptor, IL-2 receptor, IL-3 receptor, IL-31 receptor, IL-3 receptor, IL-4 receptor IL-5, IL-5 receptor, IL-6 receptor, IL-7 receptor, IL-8 receptor, IL-9 receptor, immunoglobulin immune complex, immunoglobulin, INF-alpha, INF-alpha receptor, INF-beta receptor, INF-gamma receptor, IFN type I receptor, influenza virus (influenza), inhibin alpha, inhibin beta, iNOS, insulin A-chain, insulin B-chain, insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein, integrin alpha 2, integrin alpha 3, integrin alpha 4/beta 1, integrin alpha-V/beta-3, integrin alpha-V/beta-6, integrin alpha 4/beta 7, integrin alpha 5/beta 1, integrin alpha 5/beta 3, integrin alpha 5/beta 6, integrin alpha-delta (alpha V), Integrin alpha-theta, integrin beta 1, integrin beta 2, integrin beta 3(GPIIb-IIIa), IP-10, I-TAC, JE, kalliklein, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, kallistatin, KC, KDR, Keratinocyte Growth Factor (KGF), keratinocyte growth factor-2 (KGF-2), KGF, killer cell immunoglobulin-like receptor, Kit Ligand (KL), Kit tyrosine kinase, laminin 5, LAPP (pancreatic amyloid peptide, pancreatic islet amyloid polypeptide), LAP (TGF-1), latency-related peptide, latent TGF-1, TGF-1bp1, LBP, LDGF, LDL receptors, LECT2, Lefty, leptin, Luteinizing Hormone (LH), Lewis-Y antigen (Lewis-Y antigen), Lewis-Y associated antigen, LFA-1, LFA-3 receptor, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surfactant, luteinizing hormone, lymphotactin, lymphotoxin beta receptor, Lysosphingolipid receptor (Lysosphingolipid receptor), Mac-1, macrophage-CSF (M-CSF), MAdCAM, MAG, MCMAP 2, MARC, maspin, MCAM, MDCK-2, MCP-1, MCP-2, MCP-3, MCP-4, MCAF-F, MCAA, 67 MDC (69a.a.), megsin, Mer, MET tyrosine kinase receptor family, metalloprotease, membrane glycoprotein OX2, Mesothelin, MGDF receptor, MGMT, MHC (HLA-DR), microbial protein, MIF, MIG, MIP-1 alpha, MIP-1 beta, MIP-3 alpha, MIP-3 beta, MIP-4, MK, MMAC1, 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, monocyte chemotactic protein (monoclonal protein), monocyte colony inhibitory factor, mouse gonadotropin-related peptide, MPP, Mpo, MSK, IF, MUC-16, MUC18, mucin (Mud), Mueller-associated substance, Mu-associated substance, Munk, MuC-16, MuC18, Mu-associated substance, Mu-I, and Mu-1 alpha, MIP-3 alpha, MIP, MMAC-1 alpha, MMAC, Myeloid progenitor inhibitory factor-1 (MPIF-I), NAIP, Nanobody (Nanobody), NAP-2, NCA 90, NCAD, N-cadherin, NCAM, enkephalinase (Neprilysin), a neural cell adhesion molecule, a neural serine protease inhibitor (nerosepin), Neuronal Growth Factor (NGF), neurotrophin-3, neurotrophin-4, neurotrophin-6, neuropilin 1, Neurturin, NGF-beta, NGFR, NKG20, N-methionyl human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, nonstructural protein type 3 of hepatitis C virus (NS3), NOS, NnG, NRG-3, NT-3, NT-4, NTN, OB, OGG1, oncostatin M, OP-2, OPG, OPN, OSM receptor, bone inducing factor, osteopontin, and so, OX40L, OX40R, oxidized LDL, P150, P95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PCSK9, PDGF receptor, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-D, PDK-1, PECAM, PEDF, PEM, PF-4, PGE, PGF, PGI2, PGJ2, PIGF, PIN, PLA2, placental growth factor, placental alkaline phosphatase (PLAP), placental lactogen, plasminogen activator inhibitor-1, platelet growth factor, plgR, PLP, polyethylene glycol chains of different sizes (e.g., PEG-20, PEG-30, PEG40), PP14, prokallikrein, prion proteins, procalcitonin, apoprotein 1, proinsulin, PCL, proprotein convertase 9, pinorexin, PSMA-specific membrane antigen (PSMA), prostate antigen A), and the like, Protein C, protein D, protein S, protein Z, PS, PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PTN, P-selectin glycoprotein ligand-1, R51, RAGE, RANK, RANKL, RANTES, relaxin A-chain, relaxin B-chain, renin, Respiratory Syncytial Virus (RSV) F, Ret, reticululon 4, rheumatoid factor, RLIP76, RPA2, RPK-1, RSK, RSV Fgp, S100, RON-8, SCF/KL, SCGF, Sclerostin, SDF-1, SDF1 alpha, SDF1 beta, SERINE, serum amyloid P, serum albumin, sFRP-3, Shh, shiga-like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, DF, SMOH, SPA, SMRC, SMAP 1-1, Stat-phosphate receptor, Stat-AP-1, Stat-like kinase, Stat-like toxin II, S-1, S-8, SCF, S-1, S-, Superoxide dismutase, cohesin-1, TACE, TACI, TAG-72 (tumor associated glycoprotein-72), TARC, TB, TCA-3, T-cell receptor alpha/beta, TdT, TECK, TEM1, TEM5, TEM7, TEM8, tenascin, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF-alpha, TGF-beta Pan-specific, TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta R1(ALK-5), TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, TGF-I, thrombin, Thrombopoietin (TPO), thymic stromal lymphopoietin (lymphoprotein) receptor, thymic Ck-1, Thyroid Stimulating Hormone (TSH), thyroxine binding globulin, and thyroxine-binding globulin, Tie, TIMP, TIQ, tissue factor protease inhibitor, tissue factor protein, TMEFF, Tmpo, TMPRSS, TNF receptor II, TNF- α, TNF- β 2, TNFa, TNF-RI, TNF-RII, TNFRSF10 (TRAIL Apo-2/DR), TNFRSF10 (TRAIL DR/KILLER/TRICK-2A/TRICK-B), TNFRSF10 (TRAR/LIT/TRID), TNFRSF10 (TRAIL DcR/TRUNDD), TNFRSF11 (RANODF R/TRANCER), TNFRSF11 (OPGOCIF/TR), TNFRSF (TWEAK R FN), TNFRSF12, TNFRSF13 (TACI), TNFRSF13 (BAFF), TNFRSF (HVTREMAF/HTR/LIGHT/TNFRR/TNFRSF 75), TNFRSF (TNFRSF) and TNFRSTRATFRSF (TNFRSF) 13 (TNFRSTRATFRSF) and TNFRSF (TNFRSF) 1/DRST-R), TNFRFR (TNFRFR, TNFRSF) and TNFRSF (TNFRSF) as, TNFRSF1B (TNFRII CD120b/p75-80), TNFRSF21(DR6), TNFRSF22(DcTRAIL R22 TNFRH 22), TNFRSF22 (DR3Apo-3/LARD/TR-3/TRAMP/WSL-1), TNFRSF22 (TNFRH 22), TNFRSF22 (LTbR TNF RIII/TNFRR), TNFRSF22 (TNFR72 ACT 22/TXGP 22R), TNFRSF22 (CD 22 p 22), TNFRSF22 (Fas Apo-1/APT 22/CD 22), TNFRSF6 22 (DcR 22M 22/TR 22), TNFRSF22 (CD 22), TNFRSF 36137 (TNFRSF 36137 CD/ILBB/ILA), TNFRSF22 (TNFRSF 22/TRAIL 22), TNFRSF22 (TNFRSF 22/TRAP 22), TNFRSF 22/22 (TNFRSF 22), TNFRSF22 (TNFRSF 22/TRASF 22) ligand (TNFRSF 22) TNFRSF22, TNFRSF 3633/TRAP 22, TNFRSF 3633 (TNFRSF 22, TN, TNFSF15(TL1A/VEGI), TNFSF18(GITR ligand AITR ligand/TL 6), TNFSF1A (TNF-aConnectin/DIF/TNFSF 2), TNFSF1B (TNF-b LTa/TNFSF1), TNFSF3(LTbTNFC/p33), TNFSF4(OX40 ligand gp34/TXGP 34), TNFSF 34 (CD 34 ligand CD154/gp 34/HIGM 34/TRAP), TNFSF 34 (Fas ligand Apo-1 ligand/APT 34 ligand), TNFSF 34 (CD 34 ligand CD 34), TNFSF 34 (CD 34 ligand CD 137), TNF-alpha, TNF-beta, TNIL-I, toxin metabolites, transmembrane TP-1, TRAIL-TRAIL, Tpo, TRAIL, TNSF 72, TNSF 36137, TGF-beta, TGF-glycoprotein, TGF-beta, TGF-converting glycoprotein, TGF-beta, TGF-TGF, Trk, TROP-2, trophoblast glycoprotein, TSG, TSLP, Tumor Necrosis Factor (TNF), tumor associated antigen CA125, tumor associated antigen expressing Lewis Y-associated carbohydrate, TWEAK, TXB2, Ung, uPAR-1, urokinase, VAP-1, Vascular Endothelial Growth Factor (VEGF), vaspin, VCAM-1, VECAD, VE-cadherin-2, VEFGR-1(flt-1), VEFGR-2, VEGF receptor (VEGFR), VEGFR-3(flt-4), VEGI, VIM, viral antigen, VitB12 receptor, vitronectin receptor, VLA-1, VLA-4, VNR integrin, Victorial Wilrand factor (vWF), WIF-1, WNT1, WNT10, WNT B, WNT 6346T 84, WNT-84, WNT 8225, WNT-9/8511, WNT-3, WNT-6, 3611, 369, 367, 3648, 369, 368, 369, 3611, 368, 3611, 3, 3611, 3, WNT8B, WNT9A, WNT9B, XCL1, XCL 2/SCM-1-. beta., XCL 1/lymphotactin, XCR1, XEDAR, XIAP, and XPD.
The antigen binding molecules of the present invention are capable of reducing the total plasma antigen concentration of the above antigens. The antigen binding molecules of the present invention are also capable of eliminating plasma viruses, bacteria and fungi by binding to their structural components. In particular, the F protein of RSV, staphylococcal lipoteichoic acid, Clostridium difficile toxin, Shiga-like toxin II, Bacillus anthracis protective antigen and hepatitis C virus E2 glycoprotein may be used as structural components of viruses, bacteria and fungi.
Although the method of the present invention is not limited to any particular theory, it may be explained as follows, due to the promotion of uptake of antigen binding molecules into cells and increased elimination of antigen from plasma: reducing (attenuating) the antigen binding capacity of the antigen binding molecule in the acidic pH range to less than the antigen binding activity in the neutral pH range and/or increasing (increasing) the relationship between the human FcRn binding activity in the neutral pH range and the increase in the number of antigens to which a single antigen binding molecule can bind.
For example, if the antigen binding molecule is an antibody that binds to a membrane antigen, the antibody administered to the body binds to the antigen and is then taken up into the endosome of the cell by internalization with the antigen, during which time the antibody remains bound to the antigen. The antibody then migrates to the lysosome, during which time the antibody remains bound to the antigen, along with which it is degraded by the lysosome. Internalization-mediated self-plasma abrogation is referred to as antigen-dependent abrogation, and such abrogation has been reported in numerous antibody molecules (drug discov today.2006, month 1; 11 (1-2): 81-8). When a single molecule of an IgG antibody binds to an antigen in a bivalent manner, the single antibody molecule is internalized, during which time the antibody remains bound to both antigen molecules and degrades in lysosomes. Thus, in the case of a typical antibody, one IgG antibody molecule cannot bind to 3 or more antigen molecules. For example, a single IgG antibody molecule with neutralizing activity is unable to neutralize 3 or more antigen molecules.
The relatively prolonged retention (slow elimination) of IgG molecules in plasma is caused by the action of a human FcRn called the salvage receptor (salvaging receptor) for IgG molecules. When taken up into the endosome by pinocytosis, IgG molecules bind to human FcRn expressed in the endosome under the acidic conditions of the endosome. Although IgG molecules that do not bind to human FcRn migrate into lysosomes where they degrade, IgG molecules that bind to human FcRn migrate to the cell surface and return to the plasma again by dissociating from human FcRn under the neutral conditions of the plasma.
Alternatively, where the antigen binding molecule is an antibody that binds to a soluble antigen, then the antibody administered to the body binds to the antigen and is then taken up into the cells, during which time the antibody remains bound to the antigen. Many antibodies taken up into cells are released extracellularly via FcRn. However, since the antibody is released outside the cell and the antibody remains bound to the antigen, the antibody can no longer bind to the antigen. Thus, similar to antibodies that bind membrane antigens, in the case of typical antibodies, one IgG antibody molecule cannot bind 3 or more antigen molecules.
A pH-dependent antigen-binding antibody that binds strongly to an antigen under neutral conditions of plasma but dissociates from the antigen under acidic conditions of endosomes (an antibody that binds under neutral conditions but dissociates under acidic conditions) can dissociate from the antigen in endosomes. Such pH-dependent antigen-binding antibodies can re-bind antigen when recirculated to plasma via FcRn following antigen dissociation; thus, each antibody can repeatedly bind to many antigens. Furthermore, the antigen bound to the antigen binding molecule dissociates in the endosomes without recycling into the plasma. This facilitates antigen-binding molecule mediated uptake of the antigen by the cell. Thus, administration of antigen binding molecules can promote antigen elimination, thus reducing plasma antigen concentrations.
Antigen-binding molecule-mediated uptake of an antigen by cells can be further facilitated by having the antibody, which binds to the antigen in a pH-dependent manner (binding under neutral conditions but dissociating under acidic conditions), have human FcRn binding activity under neutral conditions (pH 7.4). Thus, administration of antigen binding molecules can promote antigen elimination, thus reducing plasma antigen concentrations. Typically, both antibodies and antigen-antibody complexes are taken up into cells by non-specific endocytosis and then transported to the cell surface by binding to FcRn under the acidic conditions of endosomes. Antibodies and antigen-antibody complexes are recycled to plasma by dissociation from FcRn under neutral conditions on the cell surface. Thus, when an antibody that exhibits sufficient pH dependence in antigen binding (binding under neutral conditions but dissociating under acidic conditions) binds to an antigen in plasma and then dissociates from the bound antigen in endosomes, it is assumed that the rate of antigen elimination is equal to the rate at which the antigen is taken up into cells by nonspecific endocytosis. On the other hand, when the pH dependence is insufficient, antigens that do not dissociate in endosomes are also recycled into the plasma. Meanwhile, when the pH dependence is sufficient, the rate-determining step in antigen elimination is taken into cells by nonspecific endocytosis. It is hypothesized that some FcRn is located at the cell surface because FcRn transports antibodies from endosomes to the cell surface.
The present inventors postulate that immunoglobulins of the IgG type, which are one of the antigen binding molecules, generally have little FcRn binding capacity in the neutral pH range, but those exhibiting FcRn binding capacity in the neutral pH range can bind to FcRn on the cell surface and thus be taken up into the cell in an FcRn-dependent manner by binding to the cell surface FcRn. FcRn-mediated incorporation into cells occurs at a faster rate than uptake into cells by nonspecific endocytosis. Thus, the rate of antigen elimination is further accelerated by conferring FcRn binding capacity in the neutral pH range. In particular, antigen binding molecules with FcRn binding capacity in the neutral pH range transport antigen to cells faster than typical (fully human) IgG-type immunoglobulins, and then the antigen binding molecules dissociate from the antigen in the endosome. The antigen binding molecule is recycled to the cell surface or plasma and binds to another antigen and is taken up into the cell via FcRn. The rate of elimination of antigen from plasma can be increased by further increasing the rate of circulation by improving FcRn binding in the neutral pH range. In addition, efficiency can be further improved by reducing the antigen binding activity of the antigen binding molecule in the acidic pH range to less than the binding activity in the neutral pH range. Furthermore, it is postulated that the number of antigens to which a single antigen binding molecule can bind increases with the number of cycles that a single antigen binding molecule can achieve. The antigen binding molecules of the present invention comprise an antigen binding domain and an FcRn binding domain. Because the FcRn binding domain does not affect antigen binding, or in view of the above mechanisms, it is expected that antigen-binding molecule-mediated uptake of antigen by cells can be facilitated irrespective of the antigen type, thus increasing the antigen elimination rate by decreasing the antigen binding activity (binding capacity) of the antigen-binding molecule in the acidic pH range to less than the antigen binding activity in the neutral pH range and/or increasing its FcRn binding activity at plasma pH.
< substance used as antigen-binding molecule >
Furthermore, the present invention provides antigen binding molecules having human FcRn binding activity in the acidic and neutral pH ranges and having antigen binding activity in the acidic pH range that is lower than antigen binding activity in the neutral pH range. Specific examples of antigen binding molecules include those having human FcRn binding activity at pH 5.8-pH6.0 and pH 7.4, which are assumed to be the in vivo pH of early endosomes and plasma, respectively, and whose antigen binding activity at pH 5.8 is lower than that at pH 7.4. An antigen binding molecule having an antigen binding activity at pH 5.8 that is lower than the antigen binding activity at pH 7.4 may also be referred to as an antigen binding molecule having an antigen binding activity at pH 7.4 that is stronger than the antigen binding activity at pH 5.8.
The antigen binding molecules of the invention having human FcRn binding activity in the acidic and neutral pH ranges are preferably antigen binding molecules which also have human FcRn binding activity in the acidic pH range and stronger human FcRn binding activity than intact human IgG in the neutral pH range. The binding activity ratio is not limited as long as its human FcRn binding activity is even slightly stronger at pH 7.4.
According to Journal of Immunology (2009) 182: 7663-7671 the binding activity of human FcRn of intact human IgG1 was KD 1.7 micromolar in the acidic pH range (pH 6.0), whereas almost no activity was detectable in the neutral pH range. Thus, in a preferred embodiment, the antigen binding molecules of the invention having human FcRn binding activity in the acidic and neutral pH ranges comprise antigen binding molecules having human FcRn binding activity with KD 20 micromolar or greater in the acidic pH range which is equal to or greater than the human FcRn binding activity of intact human IgG in the neutral pH range. In a more preferred embodiment, the antigen binding molecules of the invention comprise antigen binding molecules having a human FcRn binding activity of KD 2.0 micromolar or more in the acidic pH range and KD 40 micromolar or more in the neutral pH range. In an even more preferred embodiment, the antigen binding molecules of the invention comprise antigen binding molecules having a human FcRn binding activity of KD 0.5 micromolar or stronger in the acidic pH range and KD15 micromolar or stronger in the neutral pH range. The above KD value is determined by Journal of immunology (2009) 182: 7663-.
The present invention provides an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, which has human FcRn binding activity in the acidic and neutral pH ranges, wherein the antigen binding activity of human FcRn in the acidic pH range is stronger than KD 3.2 micromolar below that in the neutral pH range. The invention also provides an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, which has human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is 28-fold that of intact human IgG. The antigen binding molecules of the invention have human FcRn binding activity at pH 7.0 and at 25 ℃, which is stronger than that of intact human IgG. In a more preferred embodiment, the human FcRn binding activity at pH 7.0 and 25 ℃ is 28-fold greater than that of intact human IgG or greater than KD 3.2 micromolar.
The present invention provides an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, which has human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is greater than KD 2.3 micromolar. The invention also provides an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, which has human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is 38-fold that of intact human IgG.
In this context, an acidic pH range generally means a pH of 4.0 to 6.5. The acidic pH range is preferably a range expressed by any pH value within the range of pH 5.5 to pH 6.5, preferably selected from the group consisting of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 and 6.5, particularly preferably pH 5.8 to pH6.0, which is close to the pH of early endosomes in vivo. Meanwhile, the neutral pH range herein generally means pH 6.7 to pH 10.0. The neutral pH range is preferably a range expressed by any pH value within the range of pH 7.0 to pH 8.0, 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, particularly preferably pH 7.4, which is close to the in vivo plasma (blood) pH. If it is difficult to assess the binding affinity between the human FcRn binding domain and human FcRn due to its low affinity at pH 7.4, pH 7.0 may be used instead of pH 7.4. As for the temperature used for the assay conditions, the binding affinity between the human FcRn binding domain and human FcRn can be assessed at any temperature from 10 ℃ to 50 ℃. Temperatures between 15 ℃ and 40 ℃ are preferably used to determine the binding affinity between the human FcRn binding domain and human FcRn. More preferably also any temperature between 20 ℃ and 35 ℃, such as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 ℃, is used to determine the binding affinity between the human FcRn binding domain and human FcRn. The 25 ℃ temperature described in example 5 is an example of an embodiment of the present invention.
In a more preferred embodiment, the human FcRn binding activity is 38 times greater than that of intact human IgG or greater than KD 2.3 micromolar at pH 7.0 and at 25 ℃. Intact human IgG1, IgG2, IgG3 or IgG4 are used as intact human IgG, reference intact human IgG for comparison with antigen binding molecules for their human FcRn binding activity. More preferably, intact human IgG1 is used, a reference intact human IgG aimed at comparing the antigen binding molecule against its human FcRn binding activity.
The invention provides an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, wherein the total plasma antigen concentration following administration of the antigen binding molecule to a non-human animal is lower than the total plasma antigen concentration following administration of a reference antigen binding molecule to a non-human animal.
The invention also provides antigen binding molecules wherein the plasma antigen concentration following administration of the antigen binding molecule to a non-human animal is lower than the total plasma antigen concentration obtained without administration of the antigen binding molecule to a non-human animal.
By administering the antigen binding molecule of the 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 and 1,000 fold or even higher compared to administration of a reference antigen binding molecule comprising an intact human IgG Fc domain as human FcRn binding domain, or compared to administration without the antigen binding domain molecule of the invention.
In another embodiment, the invention provides an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, wherein the antigen/antigen binding molecule molar ratio of the antigen binding molecule is calculated as follows:
C＝A/B，
an antigen/antigen binding molecule molar ratio (C') lower than that of an antigen binding molecule comprising the same antigen binding domain and a fully human IgG Fc domain as the human FcRn binding domain calculated as follows;
C′＝A′/B′，
wherein;
a is the total antigen concentration in plasma following administration of the antigen binding molecule to a non-human animal,
b is the plasma concentration of the antigen binding molecule after administration of the antigen binding molecule to a non-human animal,
a' is the total antigen concentration in plasma after administration of the reference antigen binding molecule to a non-human animal,
b' is the plasma concentration of the antigen-binding molecule after administration of the reference antigen-binding molecule to a non-human animal.
By administering the antigen binding molecule of the invention, the antigen/antigen binding molecule molar ratio can be reduced by up to 2 fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold, 200 fold, 500 fold and 1,000 fold or even higher compared to administering an antigen binding molecule comprising a fully human IgG Fc domain as human FcRn binding domain.
The reduction in total antigen concentration or antigen/antibody molar ratio in plasma can be assessed as described in examples 6, 8 and 13. More specifically, human FcRn transgenic mouse strain 32 or strain 276(Jackson Laboratories, Methods Mol Biol. (2010) 602: 93-104.) was used and when the antigen binding molecule did not cross-react with the mouse counterpart antigen, it could be evaluated by an antigen-antibody co-injection model or a steady state antigen infusion model. When the antigen binding molecule is cross-reactive with the mouse counterpart, it can be evaluated by injecting it only into human FcRn transgenic mouse strain 32 or strain 276(Jackson Laboratories). In the co-injection model, a mixture of an antigen binding molecule and an antigen is administered to mice. In a steady-state antigen infusion model, an infusion pump containing an antigen solution is implanted into mice to achieve a constant plasma antigen concentration, and then the mice are injected with an antigen binding molecule. The test antigen binding molecules were administered at the same dose. Plasma total antigen concentration, plasma free antigen concentration and plasma antigen-binding molecule concentration are measured at appropriate time points using methods known to those skilled in the art.
The route of administration of the antigen binding molecules of the present invention may be selected from intradermal, intravenous, intravitreal, subcutaneous, intraperitoneal, parenteral, and intramuscular injections.
More specifically, the antigen binding molecules described herein that have a long-term effect on plasma antigen-depleting activity have human FcRn binding activity at pH 7.0 and 25 ℃ in the range of 28-fold to 440-fold for full human IgG1 or a KD in the range of 3.0 micromolar to 0.2 micromolar. The long-term plasma antigen concentration is determined by measuring the total or free antigen concentration and the antigen/antigen-binding molecule molar ratio in plasma 2, 4, 7, 14, 28, 56, or 84 days after administration of the antigen-binding molecule to evaluate the long-term effect of the antigen-binding molecules of the invention on the activity of eliminating plasma antigens. Whether a reduction in plasma antigen concentration or antigen/antigen-binding molecule molar ratio is achieved by the antigen-binding molecules of the invention can be determined by assessing the reduction at any one or more of the above time points.
Even more specifically, the antigen binding molecules of the invention with short term effects on the elimination of plasma antigen activity have human FcRn binding activity at pH 7.0 and at 25 ℃ that is 440-fold or more than 0.2 micromolar that of intact human IgG. Short-term plasma antigen concentrations are determined by measuring the total or free antigen concentration and the antigen/antigen-binding molecule molar ratio in plasma 15 minutes, 1, 2, 4, 8, 12 or 24 hours after administration of the antigen-binding molecule to evaluate the short-term effect of the antigen-binding molecules of the invention on the activity of eliminating plasma antigens.
Further, in the antigen-binding molecule of the present invention, which has antigen-binding activity in the acidic pH range lower than that in the neutral pH range, the binding activity ratio thereof is not limited as long as the antigen-binding activity in the acidic pH range is lower than that in the neutral pH range. Antigen binding molecules are acceptable if the antigen binding activity in the acidic pH range is even slightly lower. In a preferred embodiment, the antigen binding molecules of the invention comprise antigen binding molecules that have 2-fold or greater antigen binding activity at pH7.4 than at pH 5.8. In a more preferred embodiment, the antigen binding molecules of the invention include antigen binding molecules that have 10-fold or greater antigen binding activity at pH7.4 as compared to pH 5.8. In an even more preferred embodiment, the antigen binding molecules of the invention comprise antigen binding molecules that have 40-fold or greater antigen binding activity at pH7.4 as compared to pH 5.8.
In particular, the antigen binding molecules of the present invention include embodiments as described in, for example, WO 2009/125825. More specifically, in a preferred embodiment, the antigen binding activity of the antigen binding molecules of the invention is lower at pH5.8 than at pH7.4, wherein the value of KD (pH5.8)/KD (pH7.4), which is the ratio of the KD of the antigen at pH5.8 to the KD at pH7.4, is preferably 2 or more, more preferably 10 or more, still more preferably 40 or more. The upper limit of the KD (pH5.8)/KD (pH7.4) value is not particularly limited and may be any value, for example 400, 1,000 or 10,000, as long as it can be produced by techniques known to those skilled in the art.
In another preferred embodiment, the antigen binding molecule of the invention having an antigen binding activity at pH 5.8 which is lower than the antigen binding activity at pH7.4 has kd(pH5.8)/kd(pH7.4) (which is the k of the antigen at pH 5.8dAnd k of antigen at pH7.4dIs 2 or more, more preferably 5 or more, even more preferably 10 or more, still more preferably 30 or more. k is a radical ofd(pH5.8)/kdThe upper limit of the value (ph7.4) is not particularly limited and may be any value, for example 50, 100 or 200, as long as it can be produced by techniques known to those skilled in the art.
The conditions for measuring the antigen binding activity and the human FcRn binding activity other than pH can be appropriately selected by those skilled in the art, and the conditions are not particularly limited; however, the measurement can be carried out under conditions such as MES buffer and 37 ℃ described in examples. Furthermore, the antigen binding activity of the antigen binding molecule can be determined by methods known to those skilled in the art, using, for example, Biacore T100(GEHealthcare) and the like as described in the examples.
The antigen binding molecules of the present invention facilitate antigen uptake by cells. In vivo the molecule readily dissociates from the antigen and is then released extracellularly by binding to human FcRn. It is postulated that the antigen binding molecules of the present invention readily re-bind to antigens in plasma. Thus, for example, where the antigen binding molecule of the invention is a neutralizing antigen binding molecule, a reduction in plasma antigen concentration may be facilitated by administration of the molecule. Thus, an antigen binding molecule having human FcRn binding activity in the acidic pH range has lower antigen binding activity in the acidic pH range than in the neutral pH range; an antigen binding molecule with human FcRn binding activity in the neutral pH range may be an antigen binding molecule with superior pharmacokinetics and which can bind more antigen per molecule.
In preferred embodiments, such antigen binding molecules having human FcRn binding activity in the acidic and neutral pH ranges include antigen binding molecules comprising a human FcRn binding domain having the ability to bind directly or indirectly to human FcRn. This domain can be used as such if it already has human FcRn binding capacity in the acidic and neutral pH ranges. Alternatively, even if the domain has human FcRn binding activity in the acidic pH range but exhibits only weak or non-human FcRn binding activity in the neutral pH range, it may be used after changing the amino acids of the domain to human FcRn binding activity in the neutral pH range. Alternatively, human FcRn binding activity can be increased by altering amino acids in domains that already have human FcRn binding capacity in the acidic and neutral pH ranges. Such antigen binding molecules include, for example, those having an amino acid sequence of an IgG Fc domain that contains at least one amino acid change. The amino acid change is not particularly limited; and can be altered at any site, provided that the binding activity of human FcRn in the neutral pH range is stronger than the binding activity of human FcRn prior to the alteration.
Specifically, amino acid changes that result in human FcRn binding activity in acidic and neutral pH ranges include, for example, amino acid changes at the following positions in the parent IgG Fc domain described above: 221-. More specifically, amino acid changes include changes at the positions shown in Table 1, Table 2, Table 6-1, Table 6-2, and Table 9 (by EU numbering). Preferred antigen binding molecules include antigen binding molecules comprising an amino acid sequence resulting from a change in at least one amino acid at a position selected from the group consisting of: 237. 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (numbered EU).
In preferred embodiments, such amino acid changes include:
an amino acid substitution in which Gly at position 237 is substituted into Met;
amino acid substitution of Pro at position 238 to Ala;
an amino acid substitution wherein Ser at position 239 is substituted with Lys;
an amino acid substitution wherein Lys at position 248 is replaced with Ile;
an amino acid substitution at Thr at position 250 with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr;
an amino acid substitution of Met at position 252 with Phe, Trp or Tyr;
an amino acid substitution of Ser at position 254 to Thr;
amino acid substitution of Arg at position 255 with Glu;
an amino acid substitution at position 256 of Thr substituted with Asp, Glu or Gln;
an amino acid substitution at position 257 of Pro substituted with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val;
an amino acid substitution wherein Glu at position 258 is substituted with His;
an amino acid substitution at position 265 wherein Asp is substituted with Ala;
270 Asp substituted with an amino acid Phe;
an amino acid substitution wherein Asn at position 286 is replaced with Ala or Glu;
an amino acid substitution of Thr at position 289 with His;
an amino acid substitution wherein Asn at position 297 is substituted with Ala;
298 amino acid substitutions in which Ser is replaced with Gly;
An amino acid substitution wherein Val at position 303 is substituted with Ala;
an amino acid substitution wherein Val at position 305 is substituted with Ala;
a substitution of Thr at position 307 with an amino acid Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr;
an amino acid substitution of Val at position 308 with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr;
an amino acid substitution of Leu or Val at position 309 with Ala, Asp, Glu, Pro or Arg;
an amino acid substitution in which Gln at position 311 is replaced with Ala, His, or Ile;
an amino acid substitution wherein Asp at position 312 is substituted with Ala or His;
an amino acid substitution wherein Leu at position 314 is substituted with Lys or Arg;
an amino acid substitution wherein Asn at position 315 is substituted with Ala or His;
an amino acid substitution wherein Lys at position 317 is substituted with Ala;
an amino acid substitution wherein Asn at position 325 is replaced with Gly;
an amino acid substitution wherein Ile at position 332 is substituted with Val;
an amino acid substitution wherein Lys at position 334 is substituted with Leu;
a substitution of Lys at position 360 to His amino acid;
an amino acid substitution at position 376 of Asp to Ala;
an amino acid substitution in which Glu at position 380 is substituted with Ala;
An amino acid substitution wherein Glu at position 382 is substituted with Ala;
an amino acid substitution of Asn or Ser at position 384 with Ala;
an amino acid substitution wherein Gly at position 385 is substituted by Asp or His;
an amino acid substitution of Gln at position 386 with Pro;
amino acid substitution of Pro at position 387 with Glu;
an amino acid substitution wherein Asn at position 389 is substituted with Ala or Ser;
an amino acid substitution of Ser at position 424 with Ala;
an amino acid substitution of Met at position 428 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr;
an amino acid substitution of His at position 433 with Lys;
an amino acid substitution in which Asn at position 434 is substituted with Ala, Phe, His, Ser, Trp or Tyr;
and an amino acid substitution of Tyr or Phe at position 436 with His (numbering according to EU).
The number of amino acids to be changed is not particularly limited; the amino acids may be changed at only a single site or at two or more sites. Combinations of two or more amino acid changes include, for example, those listed in Table 3, tables 4-1 to 4-5, Table 6-1, Table 6-2, and Table 9.
Meanwhile, domains that already have human FcRn binding ability in acidic and neutral pH ranges include, for example, a human FcRn binding domain comprising at least one amino acid selected from the following in the parent IgG Fc domain:
Met at amino acid position 237;
ala at amino acid position 238;
lys at amino acid position 239;
an 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, 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;
phe at amino acid position 270;
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;
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 or His at amino acid position 315;
ala at amino acid position 317;
gly at amino acid position 325;
val at amino acid position 332;
a 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 at amino acid position 436 (EU numbering).
The amino acid at one site or the amino acids at two or more sites may have these amino acids. Combinations of amino acids at two or more positions include, for example, those listed in Table 3, tables 4-1 to 4-5, Table 6-1, Table 6-2, and Table 9.
Alternatively, in a preferred embodiment, an antigen binding molecule having an antigen binding activity in the acidic pH range that is lower than the antigen binding activity in the neutral pH range comprises an antigen binding molecule wherein at least one amino acid of the antigen binding molecule is replaced with a histidine or a non-natural amino acid, or wherein at least one histidine or non-natural amino acid has been inserted. The site to which histidine or an unnatural amino acid mutation is introduced is not particularly limited, and may be any site as long as the antigen-binding activity in the acidic pH range is weaker than the antigen-binding activity in the neutral pH range (KD (in the acidic pH range)/KD (in the neutral pH range) value is larger than that before the substitution, or k is larger d(in the acidic pH range)/kdThe (in the neutral pH range) values are large). Where the antigen binding molecule is an antibody, examples include the variable regions and CDRs of the antibody. The number of amino acids substituted with histidine or an unnatural amino acid and the number of amino acids to be inserted can be determined appropriately by those skilled in the art. One amino acid may be substituted with histidine or a non-natural amino acid, or one amino acid may be inserted, or two or more amino acids may be substituted with histidine or a non-natural amino acid, or two or more amino acids may be inserted. In addition, in addition to histidine or unnatural amino acid substitution or insertion of histidine or unnatural amino acid, deletion, addition, insertion and/or substitution of such other amino acids may be performed simultaneously. Substitutions of histidine or unnatural amino acids or insertions of histidine or unnatural amino acids can be made randomly, e.g., by histidine scanningThe histidine scan method uses histidine instead of alanine in the alanine scan as known to those skilled in the art. KD (pH5.8)/KD (pH7.4) or k compared to before mutationd(pH5.8)/kd(pH7.4) the increased antigen binding molecule may be selected from antigen binding molecules into which histidine or unnatural amino acid mutations are randomly introduced.
Preferred antigen binding molecules having an antigen binding activity in the acidic pH range which is lower than the antigen binding activity in the neutral pH range, mutated to histidine or to an unnatural amino acid, include, for example, antigen binding activity at pH 7.4 of the antigen binding molecule after mutation to histidine or to an unnatural amino acid which is equivalent to antigen binding activity at pH 7.4 prior to mutation to histidine or to an unnatural amino acid. In the present invention, "the antigen binding activity of the antigen binding molecule after histidine or unnatural amino acid mutation is equivalent to the antigen binding activity of the antigen binding molecule before histidine or unnatural amino acid mutation" means that if the antigen binding activity of the antigen binding molecule before histidine or unnatural amino acid mutation is set to 100%, the antigen binding activity of the antigen binding molecule after histidine or unnatural amino acid mutation is at least 10% or more, preferably 50% or more, more preferably 80% or more, still more preferably 90% or more. The antigen binding activity at pH 7.4 after histidine or unnatural amino acid mutation can be stronger than the antigen binding activity at pH 7.4 prior to histidine or unnatural amino acid mutation. If the antigen binding activity of the antigen binding molecule is reduced due to histidine or unnatural amino acid substitutions or insertions, the antigen binding activity can be modulated by introducing one or more amino acid substitutions, deletions, additions and/or insertions into the antigen binding molecule such that the antigen binding activity becomes comparable to the antigen binding activity prior to the histidine substitution or insertion. The invention also includes such antigen binding molecules that have comparable binding activity due to substitution, deletion, addition and/or insertion of one or more amino acids after histidine substitution or insertion.
Furthermore, when the antigen binding molecule is a substance comprising an antibody constant region, in another preferred embodiment of the antigen binding molecule, which has a lower antigen binding activity at pH5.8 than at pH7.4, the invention comprises a method for altering the antibody constant region comprised in the antigen binding molecule. Specific examples of the constant region of the antibody after modification include the constant regions described in examples of WO2009/125825 (SEQ ID NOS: 11, 12, 13 and 14).
When the antigen-binding activity of the antigen-binding substance at pH5.8 is decreased (when KD (pH5.8)/KD (pH7.4) value is increased) as compared to that at pH7.4 by the above-mentioned method and such method, it is generally preferable that KD (pH5.8)/KD (pH7.4) value is 2-fold or more, more preferably 5-fold or more, even more preferably 10-fold or more with respect to the original antibody, but there is no particular limitation thereto.
In addition, the present invention provides antigen binding molecules that replace at least one amino acid at one of the following positions with a histidine or an unnatural amino acid. Amino acid positions are indicated by Kabat numbering (Kabat EA et al (1991) Sequences of Proteins of Immunological Interest, NIH).
Heavy chain: h27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b and H102
Light chain: l24, L27, L28, L32, L53, L54, L56, L90, L92 and L94
Of these sites of alteration, H32, H61, L53, L90 and L94 are assumed to be the most common sites of alteration.
In particular, preferred combinations of histidine or unnatural amino acid substitution sites include, for example, combinations of H27, H31, and H35; a combination of H27, H31, H32, H35, H58, H62, and H102; a combination of L32 and L53; and a combination of L28, L32, and L53. In addition, preferred combinations of substitution sites in the heavy and light chains include, for example, combinations of H27, H31, L32, and L53.
The antigen binding molecules of the invention may have other properties, for example may be agonistic or antagonistic antigen binding molecules, as long as they have an antigen binding activity in the acidic pH range which is lower than the antigen binding activity in the neutral pH range, and have human FcRn binding activity in the acidic and neutral pH ranges. Preferred antigen binding molecules of the invention include, for example, antagonistic antigen binding molecules. Such antagonistic antigen binding molecules are typically antigen binding molecules that inhibit receptor-mediated intracellular signal transduction by blocking the binding between a ligand (agonist) and the receptor.
Also, the antigen binding molecules of the present invention may recognize any antigen. Specifically, antigens recognized by the antigen-binding molecules of the present invention include, for example, the above-described receptor proteins (membrane-bound receptor and soluble receptor), membrane antigens (e.g., cell surface markers), and soluble antigens (e.g., cytokines). Such antigens include, for example, the antigens described above.
In a preferred embodiment, the antigen binding molecules of the invention comprise immunoglobulins of the IgG class (IgG antibodies) with an antigen binding domain and a human FcRn binding domain. When an IgG antibody is used as the antigen-binding molecule, the type is not limited; it is possible to use IgG1, IgG2, IgG3, IgG4, and the like.
The source of the antigen binding molecule of the present invention is not particularly limited and may be any source. It is possible to use, for example, mouse antibodies, human antibodies, rat antibodies, rabbit antibodies, goat antibodies, camel antibodies, and other antibodies. Furthermore, the antibody may be, for example, the above-mentioned chimeric antibody, particularly an altered antibody having amino acid sequence substitutions, such as a humanized antibody. The antibody may also be the bispecific antibody described above, antibody modification products to which various molecules are linked, polypeptides including antibody fragments, and antibodies having modified sugar chains.
Bispecific antibodies refer to antibodies having variable regions that recognize different epitopes in the same antibody molecule. A bispecific or multispecific antibody may be an antibody that recognizes two or more different antigens, or an antibody that recognizes two or more different epitopes on the same antigen.
In addition, polypeptides comprising antibody fragments include, for example, Fab fragments, F (ab') 2 fragments, scFvs (Nat Biotechnol.2005, 9 months; 23 (9): 1126-36), domain antibodies (dAbs) (WO2004/058821, WO 2003/002609), scFv-Fc (WO 2005/037989), dAb-Fc, and Fc fusion proteins. When the molecule includes an Fc domain, the Fc domain may function as a human FcRn binding domain. Alternatively, the FcRn binding domain may be fused to these molecules.
Furthermore, antigen binding molecules suitable for use in the present invention may be antibody-like molecules. Antibody-like molecules (scaffold molecules, peptide molecules) are molecules that show function by binding to a target molecule (Current Opinion in Biotechnology (2006) 17: 653-; Current Opinion in Biotechnology (2007) 18: 1-10; Current Opinion in Structural Biology (1997) 7: 463-; Protein Science (2006) 15: 14-27) including, for example, DARPins (WO 2002/020565), avidin (WO 1995/001937), Avimer (WO 2004/044011; WO 2005/040229) and Adnectin (WO 2002/032925). If these antibody-like molecules can bind to the target molecule in a pH-dependent manner and/or have human FcRn binding activity in the neutral pH range, antigen uptake by the cells can be facilitated by the antigen-binding molecule, reduction of plasma antigen concentration can be facilitated by administration of the antigen-binding molecule, the pharmacokinetics of the antigen-binding molecule is improved, and the number of antigens to which a single antigen-binding molecule can bind is increased.
Furthermore, the antigen binding molecule may be a protein resulting from fusion between a human FcRn binding domain and a receptor protein that binds to a target (including a ligand), including, for example, TNFR-Fc fusion proteins, IL1R-Fc fusion proteins, VEGFR-Fc fusion proteins, and CTLA4-Fc fusion proteins (Nat Med.2003, 1/9 (1): 47-52; BioDrugs. (2006)20 (3): 151-60). If these receptor-human FcRn binding domain fusion proteins bind to a target molecule (including a ligand) in a pH-dependent manner and/or have human FcRn binding activity in the neutral pH range, antigen uptake by cells can be facilitated by the antigen binding molecule, a decrease in plasma antigen concentration can be facilitated by administration of the antigen binding molecule, and the pharmacokinetics of the antigen binding molecule is improved, increasing the number of antigens to which a single antigen binding molecule can bind. The receptor protein is suitably designed and modified so as to include a domain of the receptor protein that binds to a target (including a ligand). As with the examples provided above including TNFR-Fc fusion proteins, IL1R-Fc fusion proteins, VEGFR-Fc fusion proteins, and CTLA4-Fc fusion proteins, soluble receptor molecules comprising the extracellular domains of these receptor proteins necessary for binding to these targets (including ligands) are preferred for use in the present invention. These designed and modified receptor molecules are referred to herein as artificial receptors. Methods for designing and modifying receptor molecules to construct artificial receptor molecules are known in the art.
Furthermore, the antigen binding molecule may be a fusion protein in which an artificial ligand protein that binds to the target and has a neutralizing effect is fused to the human FcRn binding domain, including, for example, mutant IL-6(EMBO J.1994, 12/15; 13 (24): 5863-70). If such artificial ligand fusion proteins can bind to a target molecule in a pH-dependent manner and/or have human FcRn binding activity in the neutral pH range, antigen uptake by cells can be facilitated by the antigen binding molecule, reduction of plasma antigen concentration can be facilitated by administration of the antigen binding molecule, the pharmacokinetics of the antigen binding molecule is improved, and the number of antigens to which a single antigen binding molecule can bind is increased.
Furthermore, the antibody of the present invention may comprise a modified sugar chain. Antibodies having modified sugar chains include, for example, an antibody having modified glycosylation (WO 99/54342), an antibody lacking fucose added to a sugar chain (WO 00/61739, WO 02/31140, WO 2006/067847, WO 2006/067913), and an antibody having a sugar chain with bisecting GlcNAc (WO 02/79255).
The conditions other than pH for the antigen binding or human FcRn binding activity assay may be appropriately selected by those skilled in the art, and are not particularly limited. For example, the activity can be determined as described in WO2009/125825 using MES buffer at 37 ℃. Meanwhile, the antigen binding activity of the antigen binding molecule and the human FcRn binding activity can be measured by a method known to those skilled in the art, for example, using biacore (gehealthcare) and the like. When the antigen is a soluble antigen, the activity of binding of the antigen-binding molecule to the soluble antigen can be measured by adding the antigen as an analyte to a chip on which the antigen-binding molecule is immobilized. Alternatively, when the antigen is a membrane-type antigen, the activity of binding of the antigen-binding molecule to the membrane-type antigen can be measured by adding the antigen-binding molecule as an analyte to an antigen-immobilized chip. The human FcRn binding activity of an antigen binding molecule can be determined by adding the human FcRn or antigen binding molecule as an analyte to a chip on which the antigen binding molecule or human FcRn is immobilized, respectively.
The production of chimeric antibodies is known. In the case of a human-mouse chimeric antibody, for example, a DNA encoding a V region of an antibody may be linked to a DNA encoding a C region of a human antibody; this can be inserted into an expression vector and introduced into a host to produce a chimeric antibody.
A "humanized antibody" is also referred to as a reshaped human antibody, and is an antibody in which Complementarity Determining Regions (CDRs) of a non-human mammal (e.g., a mouse) are grafted into CDRs of a human antibody. Methods for identifying CDRs are known (Kabat et al, Sequence of Proteins of immunological interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al, Nature (1989) 342: 877). General genetic recombination techniques suitable for this purpose are also known (see European patent applications EP 125023 and WO 96/02576). Humanized antibodies can be produced by known methods, for example, the CDRs of a mouse antibody can be determined, and DNA encoding the antibody in which the CDRs are linked to the Framework Regions (FRs) of a human antibody can be obtained. Humanized antibodies can then be generated using systems employing conventional expression vectors. Such DNA can be synthesized by PCR using several oligonucleotides prepared to have a portion overlapping with the terminal regions of both CDR and FR as primers (see the method described in WO 98/13388). The human antibody FRs linked by CDRs are selected so that the CDRs form a suitable antigen-binding site. If desired, the amino acids in the FRs of the antibody variable regions can be changed so that the CDRs of the reshaped human antibody form a suitable antigen-binding site (Sato et al, Cancer Res. (1993) 53: 10.01-6). Amino acid residues in the modified FR include a moiety that directly binds to the antigen by non-covalent bonds (Amit et al, Science (1986) 233: 747-53), a moiety that affects or has an effect on the CDR structure (Chothia et al, J.mol.biol. (1987) 196: 901-17), and a moiety that participates in VH-VL interactions (EP 239400).
When the antigen binding molecules of the present invention are chimeric or humanized antibodies, the C region of these antibodies is preferably derived from a human antibody. For example, C-. gamma.1, C-. gamma.2, C-. gamma.3 and C-. gamma.4 can be used for the H chain, while C-. kappa.and C-. lambda.can be used for the L chain. In addition, amino acid mutations can be introduced into the human antibody C region to increase or decrease binding to Fc-gamma receptors or to improve antibody stability or productivity, if desired. The chimeric antibody of the present invention preferably comprises a variable region derived from an antibody of a non-human mammal and a constant region derived from a human antibody. Meanwhile, the humanized antibody preferably comprises CDRs of an antibody derived from a non-human mammal and FRs and C regions derived from a human antibody. The human antibody-derived constant domain preferably comprises a human FcRn binding domain. Such antibodies include, for example, IgG (IgG1, IgG2, IgG3, and IgG 4). The constant region of the humanized antibody used in the present invention may be of any isotype. The constant region derived from human IgG1 is preferably used, but not limited thereto. The FR derived from a human antibody for use in a humanized antibody is also not particularly limited and may be derived from an antibody of any isotype.
The variable and constant regions of the chimeric and humanized antibodies of the present invention can be altered by deletion, substitution, insertion and/or addition, etc., as long as the binding specificity of the original antibody is exhibited.
Because of the reduced immunogenicity in humans, it is believed that chimeric and humanized antibodies using human-derived sequences are beneficial when administered to humans for therapeutic purposes and the like.
Such antigen binding molecules of the invention may be obtained by any method. For example, an antigen binding molecule that originally does not have human FcRn binding activity in the acidic pH and neutral pH ranges, an antigen binding molecule that has stronger antigen binding activity in the acidic pH range than in the neutral pH range, or an antigen binding molecule that has comparable antigen binding activity in the acidic and neutral pH ranges can be artificially altered to an antigen binding molecule having a desired activity by the above-described amino acid alteration or the like. Alternatively, an antibody having a desired activity can be selected by screening from a plurality of antibodies obtained from an antibody library or hybridoma described below.
When changing the amino acids in the antigen binding molecule, it is possible to use the known sequence of the amino acid sequence of the antigen binding molecule prior to the change or the amino acid sequence of the antigen binding molecule newly identified by methods known to those skilled in the art. For example, if the antigen binding molecule is an antibody, it may be obtained from an antibody library or by cloning antibody coding genes from a hybridoma that produces a monoclonal antibody.
As for antibody libraries, many antibody libraries are known, and methods for producing antibody libraries are also known; thus, an antibody library can be appropriately obtained by those skilled in the art. For example, with regard to phage libraries, reference may be made to, for example, Clackson et al, Nature (1991) 352: 624-8; marks et al, j.mol.biol. (1991) 222: 581-97; waterhouses et al, Nucleic Acids Res (1993) 21: 2265-6; griffiths et al, EMBO J. (1994) 13: 324.0-60; vaughan et al, Nature Biotechnology (1996) 14: 309-14; and Japanese patent Kohyo publication No. (JP-A) H20-504970 (corresponding to unexamined Japanese national phase publication No. of non-Japanese international application). Furthermore, it is possible to use known methods such as a method using eukaryotic cells as a library (WO 95/15393) and a ribosome display method. In addition, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, phage display methods can be used to express the variable regions of human antibodies as single chain antibodies (scfvs) on the phage surface and select phage that bind antigen. Genetic analysis of the selected phage allows determination of the DNA sequence encoding the variable region of the human antibody that binds the antigen. Once the DNA sequence of the scFv that binds to the antigen is determined, human antibodies can be obtained by generating an appropriate expression vector based on these sequences. These methods are well known and can be found in WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388.
As for the method for obtaining a gene encoding an antibody from a hybridoma, basically known techniques can be employed, which comprise using a desired antigen or a cell expressing a desired antigen as an sensitizing antigen, immunizing according to a conventional immunization method using these, fusing the resulting immune cell with a known parent cell by a conventional cell fusion method, screening cells producing a monoclonal antibody (hybridoma) by a conventional screening method, synthesizing a cDNA for an antibody variable region (V region) from the mRNA of the resulting hybridoma using a reverse transcriptase, and ligating it with a DNA encoding a desired antibody constant region (C region).
More specifically, the sensitizing antigen from which the above-mentioned antigen-binding molecule genes encoding the H chain and the L chain are obtained may include, for example, both a complete antigen having immunogenicity and an incomplete antigen (including a hapten and the like) having no immunogenicity; however they are not limited to these examples. For example, it is possible to use whole proteins and partial peptides of the target protein. Further, it is known that substances including polysaccharides, nucleic acids, lipids, and the like may be antigens. Accordingly, the antigen of the antigen binding molecule of the present invention is not particularly limited. The antigen may be prepared by methods known to those skilled in the art, for example by baculovirus-based methods (e.g. WO 98/46777) and the like. Hybridomas can be produced, for example, by the method of Milstein et al (G.Kohler and C.Milstein, Methods Enzymol. (1981) 73: 3-46), and the like. If the immunogenicity of the antigen is low, immunization is performed after linking the antigen to a macromolecule having immunogenicity (e.g., albumin). Alternatively, if necessary, the antigen can be converted to a soluble antigen by linking the antigen to other molecules. If a transmembrane molecule such as a membrane antigen (e.g., receptor) is used as the antigen, a portion of the extracellular region of the membrane antigen can be used as the fragment, or a cell expressing the transmembrane molecule on its cell surface can be used as the immunogen.
Cells producing the antigen binding molecule can be obtained by immunizing an animal with the appropriate sensitizing antigen described above. Alternatively, cells producing the antigen binding molecule can be prepared by in vitro immunization of lymphocytes that produce the antigen binding molecule. Various mammals are available for immunization; such commonly used animals include rodents, lagomorphs and primates. Such animals include, for example, rodents, such as mice, rats and hamsters; lagomorphs, such as rabbits; and primates, including monkeys such as cynomolgus monkeys, macaques, baboons, and chimpanzees. In addition, transgenic animals carrying human antibody gene banks are also known, and human antibodies can be obtained using these animals (see WO 96/34096; Mendez et al, nat. Genet. (1997) 15: 146-56). As an alternative to the use of such transgenic animals, a desired human antibody having a binding activity to an antigen can be obtained, for example, by sensitizing human lymphocytes with a desired antigen or cells expressing a desired antigen in vitro and then fusing the sensitized lymphocytes with human myeloma cells (e.g., U266) (see Japanese patent application Kokoku publication No. (JP-B) H01-59878 (published unexamined approved Japanese patent application for objection)). Furthermore, a desired human antibody can be obtained by immunizing a transgenic animal carrying a complete human antibody gene bank with a desired antigen (see WO93/12227, WO 92/03918, WO 94/02602, WO 96/34096 and WO 96/33735).
Immunization of animals can be carried out as follows: the sensitizing antigen is appropriately diluted and suspended in Phosphate Buffered Saline (PBS), physiological saline, or the like, and if necessary, mixed with an adjuvant to emulsify. It is then injected intraperitoneally or subcutaneously into the animal. The sensitizing antigen mixed with Freund's incomplete adjuvant is then preferably administered several times every 4-21 days. Antibody production can be confirmed by measuring the titer of the antibody of interest in the serum of the animal using conventional methods.
Antigen binding molecule producing cells obtained from lymphocytes or animals immunized with the desired antigen can be fused with myeloma cells using conventional fusing agents (e.g., polyethylene glycol) to produce hybridomas (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) 59-103). If desired, the hybridoma cells can be cultured and grown, and the binding specificity of the antigen-binding molecules produced from these hybridomas can be measured using known analytical methods, such as immunoprecipitation, Radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). Thereafter, if necessary, the hybridomas producing the antigen-binding molecules of interest (whose specificity, affinity or activity has been determined) can be subcloned, e.g., by limiting dilution.
Next, the gene encoding the selected antigen binding molecule can be cloned from a hybridoma or a cell producing the antigen binding molecule (sensitized lymphocytes, etc.) using a probe that specifically binds to the antigen binding molecule (e.g., an oligonucleotide complementary to a sequence encoding the constant region of the antibody). The gene can also be cloned from mRNA using RT-PCR. Immunoglobulins are divided into 5 different classes, IgA, IgD, IgE, IgG and IgM. These classes are further divided into several subclasses (isotypes) (e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1, and IgA-2, etc.). The H chain and L chain used in the production of the antigen-binding molecule of the present invention are not particularly limited, and may be derived from antibodies belonging to any of these classes or subclasses; however, IgG is particularly preferred.
In this context, genetic engineering techniques can be used to alter the H chain encoding gene and the L chain encoding gene. For antibodies such as mouse, rat, rabbit, hamster, sheep, and camel antibodies, genetically altered antibodies, such as chimeric and humanized antibodies, which are artificially altered for the purpose of reducing heterologous immunogenicity to humans, and the like, may be suitably produced. Chimeric antibodies are antibodies that include the H chain and L chain variable regions of a non-human mammalian antibody (e.g., a mouse antibody) and the H chain and L chain constant regions of a human antibody. Chimeric antibodies can be obtained by ligating a DNA encoding a mouse antibody variable region with a DNA encoding a human antibody constant region, inserting the same into an expression vector, and introducing the vector into an antibody-producing host. Several oligonucleotides produced so that they have overlapping portions at the ends of the DNA sequences designed to link the Complementarity Determining Regions (CDRs) of an antibody from a non-human mammal (e.g., a mouse) can be used to synthesize humanized antibodies by PCR, which also constitute human antibodies. The resulting DNA may be ligated with a DNA encoding a constant region of a human antibody. The ligated DNA may be inserted into an expression vector and the vector may be introduced into a host to produce antibodies (see EP 239400 and WO 96/02576). When the CDR forms a favorable antigen-binding site, the FR of the human antibody linked through the CDR is selected. Amino acids in the framework regions of the variable regions of the antibody can be replaced, if necessary, so that the CDRs of the reshaped human antibody form a suitable antigen binding site (K.Sato et al, Cancer Res. (1993) 53: 10.01-10.06).
In addition to the above humanization, the antibody may be altered to improve its biological properties, such as binding to an antigen. In the present invention, such alterations can be achieved by methods such as site-directed mutagenesis (see, e.g., Kunkel (1910.0) Proc. Natl. Acad. Sci. USA 82: 488), PCR mutagenesis, and cassette mutagenesis. In general, mutant antibodies having improved biological properties exhibit amino acid sequence homology and/or similarity of 70% or greater, more preferably 80% or greater, even more preferably 90% or greater (e.g., 95% or greater, 97%, 98% or 99%) when compared to the amino acid sequence of the variable region of the original antibody. Sequence homology and/or similarity is defined herein as the ratio of amino acid residues that are homologous (identical residues) or similar (amino acid residues grouped into the same group by the overall nature of the amino acid side chains) to the original antibody residues after maximizing the value of sequence homology by sequence alignment and gap introduction (if necessary). In general, natural amino acid residues are classified into the following groups according to the nature of their side chains as follows:
(1) hydrophobicity: alanine, isoleucine, valine, methionine and leucine;
(2) Neutral hydrophilicity: asparagine, glutamine, cysteine, threonine, and serine;
(3) acidity: aspartic acid and glutamic acid;
(4) alkalinity: arginine, histidine and lysine;
(5) residues that affect the orientation of the chain: glycine and proline; and
(6) aromatic: tyrosine, tryptophan and phenylalanine.
In general, a total of 6 complementarity determining regions (CDRs; hypervariable regions) present in the H chain and L chain variable regions interact with each other to form the antigen-binding site of an antibody. It is also known that only the variable region is able to recognize and bind antigen, but with a lower affinity than the intact binding site. Thus, antibody genes encoding the H chain and L chain of the present invention may encode fragments each comprising an H chain or L chain antigen binding site, so long as the polypeptide encoded by the gene retains the activity of binding to the desired antigen.
As mentioned above, the heavy chain variable region is typically composed of 3 CDRs and 4 FRs. In a preferred embodiment of the invention, the amino acid residues that are "altered" may suitably be selected from amino acid residues in e.g. a CDR or FR. Overall, changes in amino acid residues in the CDRs can reduce antigen binding capacity. Therefore, in the present invention, suitable amino acid residues that are "changed" are preferably selected from amino acid residues in FR, but not limited thereto. Amino acids in the CDRs may be selected as long as the changes are confirmed not to reduce binding capacity. Alternatively, by using public databases or the like, a person skilled in the art can obtain suitable sequences that can be used as antibody variable region FRs of organisms (e.g., human or mouse).
In addition, the present invention provides a gene encoding the antigen binding molecule of the present invention. The gene encoding the antigen binding molecule of the present invention may be any gene, and may be DNA, RNA, nucleic acid analogs, and the like.
In addition, the invention also provides a host cell carrying the gene. Host cells are not particularly limited and include, for example, Escherichia coli (E.coli) and various animal cells. Host cells may be used, for example, as a production system for producing and expressing the antibodies of the invention. In vitro and in vivo production systems are useful in polypeptide production systems. Such in vitro production systems include, for example, production systems using eukaryotic or prokaryotic cells.
Eukaryotic cells that can be used as host cells include, for example, animal cells, plant cells, and fungal cells. The animal cells include: mammalian cells such as CHO (J.Exp.Med. (1995) 108: 94.0), COS, HEK293, 3T3, myeloma, BHK (baby hamster kidney), HeLa and Vero; amphibian cells such as Xenopus laevis oocytes (Valle et al, Nature (1981) 291: 338-340); and insect cells such as Sf9, Sf21 and Tn 5. CHO-DG44, CHO-DX11B, COS7 cells, HEK293 cells and BHK cells are preferably used to express the antibody of the present invention. Among animal cells, CHO cells are particularly preferable for large-scale expression. The vector can be introduced into the host cell by, for example, the calcium phosphate method, the DEAE-dextran method, the method using cationic liposome DOTAP (Boehringer-Mannheim), the electroporation method and the lipofection method.
As plant cells, for example, tobacco (Nicotiana tabacum) -derived cells and duckweed (Lemna minor) are known as protein production systems. Callus may be cultured from these cells to produce the antigen binding molecules of the invention. As fungal cells, known protein expression systems are those using yeast cells, such as cells of the genus Saccharomyces (Saccharomyces), such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; and filamentous fungi such as Aspergillus (e.g.Aspergillus niger). These cells can be used as hosts to produce the antigen binding molecules of the invention.
Bacterial cells can be used in prokaryotic production systems. As for the bacterial cell, in addition to the production system using the above-mentioned Escherichia coli, a production system using Bacillus subtilis is known. Such systems can be used to produce the antigen binding molecules of the invention.
< screening method >
The present invention provides methods of screening for antigen binding molecules that have human FcRn binding activity in the acidic and neutral pH ranges. The invention also provides a method of screening for antigen binding molecules that have human FcRn binding activity in the acidic and neutral pH ranges and antigen binding activity in the acidic pH range is lower than antigen binding activity in the neutral pH range. The invention also provides methods of screening for antigen binding molecules that are capable of promoting uptake of an antigen by a cell. The invention also provides methods of screening for antigen binding molecules that are modified to allow binding of more antigen per molecule. The invention also provides methods of screening for antigen binding molecules that are capable of promoting antigen elimination. The invention also provides methods of screening for antigen binding molecules with improved pharmacokinetics. The invention also provides methods of screening for antigen binding molecules that promote intracellular dissociation from their extracellularly bound antigen. The invention also provides methods of screening for antigen binding molecules that promote extracellular release of antigen-free forms after uptake into cells in antigen-bound forms. The invention also provides methods of screening for antigen binding molecules particularly useful as pharmaceutical compositions. The above method can be used for screening antigen-binding molecules which are particularly excellent in plasma retention and have an excellent ability to eliminate antigens from plasma.
In particular, the present invention provides a method of screening for an antigen binding molecule, the method comprising the steps of:
(a) selecting an antigen binding molecule having greater human FcRn binding activity in the neutral pH range than before altering at least one amino acid in the human FcRn binding domain of an antigen binding molecule having human FcRn binding activity in the acidic pH range; and
(b) at least one amino acid in the antigen binding domain of the antigen binding molecule is altered, and an antigen binding molecule having a stronger antigen binding activity in the neutral pH range than in the acidic pH range is selected.
Steps (a) and (b) may be performed in either order. Further, each step may be repeated two or more times. The number of times steps (a) and (b) are repeated is not particularly limited; however, the number of times is usually 10 times or less.
In the screening method of the present invention, the antigen binding activity of the antigen binding molecule in the neutral pH range is not particularly limited as long as it is an antigen binding activity in the range of pH 6.7 to 10.0. For example, embodiments described in WO 2009/125825 are included. Preferred antigen binding activities include antigen binding activities in the range of pH 7.0-8.0. More preferred antigen binding activity includes antigen binding activity at pH 7.4. Meanwhile, the antigen binding activity of the antigen binding molecule in the acidic pH range is not particularly limited as long as it is in the range of pH 4.0 to 6.5. Preferred antigen binding activities include antigen binding activities in the range of pH5.5 and 6.5. More preferred antigen binding activity includes antigen binding activity at pH 5.8 or pH 5.5.
The human FcRn binding activity of the antigen binding molecule in the neutral pH range is not particularly limited as long as it is a human FcRn binding activity in the range of pH 6.7-10.0. Preferred human FcRn binding activities include human FcRn binding activity in the pH range of 7.0-8.0. More preferred human FcRn binding activities include human FcRn binding activity at pH 7.4.
The human FcRn binding activity of the antigen binding molecule in the acidic pH range is not particularly limited as long as it is a human FcRn binding activity in the range of pH 4.0 to 6.5. Preferred human FcRn binding activities include human FcRn binding activity in the pH range 5.5-6.5. More preferred human FcRn binding activities include human FcRn binding activity in the pH range of 5.8-6.0.
In this context, an acidic pH range generally means a pH of 4.0 to 6.5. The acidic pH range is preferably a range expressed by any pH value within the range of pH 5.5 to pH 6.5, preferably selected from 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5, particularly preferred pH 5.8 to pH6.0, which is close to the pH of early endosomes in vivo. Meanwhile, the neutral pH range herein generally means pH 6.7 to pH 10.0. The neutral pH range is preferably a range expressed by any pH value within the range of pH 7.0 to pH 8.0, 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, particularly preferably pH 7.4, which is close to the in vivo plasma (blood) pH. If it is difficult to assess the binding affinity between the human FcRn binding domain and human FcRn due to its low affinity at pH 7.4, pH 7.0 may be used instead of pH 7.4. As for the temperature used for the assay conditions, the binding affinity between the human FcRn binding domain and human FcRn can be assessed at any temperature from 10 ℃ to 50 ℃. Temperatures between 15 ℃ and 40 ℃ are preferably used to determine the binding affinity between the human FcRn binding domain and human FcRn. More preferably also any temperature between 20 ℃ and 35 ℃, such as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 ℃, is used to determine the binding affinity between the human FcRn binding domain and human FcRn. The 25 ℃ temperature described in example 5 is an example of an embodiment of the present invention.
The antigen binding activity of the antigen binding molecule and the human FcRn binding activity can be determined by methods known to those skilled in the art. One skilled in the art can select suitable conditions other than pH. KD (dissociation constant), apparent KD (apparent dissociation constant), dissociation rate k can be usedd(dissociation rate), apparent kd(apparent dissociation: apparent dissociation rate), and the like, to evaluate the antigen binding activity of the antigen-binding molecule and the human FcRn binding activity. They can be determined by methods known to those skilled in the art, for example using biacore (ge healthcare), Scatchard curves, flow cytometers, and the like.
According to Journal of Immunology (2009) 182: 7663-7671 the human FcRn binding activity of intact human IgG1 was KD 1.7 micromolar in the acidic pH range (pH 6.0), whereas little activity was detected in the neutral pH range. Thus, in preferred embodiments, antigen binding molecules of the invention can be screened for human FcRn binding activity in both acidic and neutral pH ranges, including antigen binding molecules having human FcRn binding activity in the acidic pH range of KD 20 micromolar or greater, which is equal to or greater than the human FcRn binding activity of intact human IgG in the neutral pH range. In a more preferred embodiment, antigen binding molecules of the invention, including antigen binding molecules with human FcRn binding activity having a KD 2.0 micromolar or greater in the acidic pH range and a KD 40 micromolar or greater in the neutral pH range, can be screened. In an even more preferred embodiment, antigen binding molecules of the invention, including antigen binding molecules with a binding activity for human FcRn in the acidic pH range of KD 0.5 micromolar or stronger and a binding activity for human FcRn in the neutral pH range of KD 15 micromolar or stronger, can be screened. The above-mentioned KD value is determined by Journal of Immunology (2009) 182: 7663-7671 by immobilizing antigen binding molecules on a chip and loading human FcRn as an analyte.
The present invention provides a method of screening for an antigen binding molecule, the method comprising the steps of:
(a) selecting an antigen binding molecule having a human FcRn binding activity greater than KD3.2 micromolar over a neutral pH range obtained by altering at least one amino acid in the human FcRn binding domain of the antigen binding molecule,
(b) obtaining a gene encoding an antigen binding molecule wherein the human FcRn binding domain prepared in (a) is linked to an antigen binding domain; and
(c) generating an antigen binding molecule using the gene generated in (b).
In one embodiment, an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain can be screened for human FcRn binding activity in both acidic and neutral pH ranges according to the methods employed by those skilled in the art described above, wherein the antigen binding activity in the acidic pH range is less than the antigen binding activity of human FcRn in the neutral pH range by more than 3.2 micromolar KD. In a more preferred embodiment, the human FcRn binding activity at pH 7.0 and 25 ℃ is stronger than KD3.2 micromolar.
The present invention provides a method of screening for an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, which has human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is greater than KD 2.3 micromolar. The invention also provides a method of screening for an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, which has human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is 38 times that of intact human IgG.
The antigen-binding molecules of the present invention having human FcRn binding activity in the neutral pH range are not particularly limited as long as they have human FcRn binding activity at pH 6.7 to 10.0. However, preferred antigen binding molecules have a stronger human FcRn binding activity at pH 6.7-10.0 than that of intact human IgG.
The antigen-binding molecules of the present invention having human FcRn binding activity in the acidic pH range are not particularly limited as long as they have human FcRn binding activity at pH 4.0 to 6.5. However, preferred antigen binding molecules have human FcRn binding activity at pH 5.5-6.5 that is comparable to or stronger than that of intact human IgG.
Herein, the step of selecting an antigen binding molecule having a stronger antigen binding activity in the neutral pH range than in the acidic pH range is synonymous with the step of selecting an antigen binding molecule having a lower antigen binding activity in the acidic pH range than in the neutral pH range.
The ratio of the antigen-binding activity between the neutral and acidic pH ranges is not particularly limited as long as the antigen-binding activity in the neutral pH range is stronger than the antigen-binding activity in the acidic pH range. However, the antigen-binding activity at pH 6.7 to 10.0 is preferably 2 times or more, more preferably 10 times or more, still more preferably 40 times or more the antigen-binding activity at pH 4.0 to 6.5.
In the screening method of the present invention, a library, for example, a phage library, may be used.
In the method of the present invention, the antigen and the antigen-binding molecule may be bound together in any state, and thus the state is not particularly limited. For example, an antigen can be contacted with an immobilized antigen binding molecule to effect binding thereof. Alternatively, the antigen binding molecule may be contacted with an immobilized antigen to effect binding thereof. Alternatively, the antigen binding molecule and antigen may be contacted with each other in solution to effect binding thereof.
The antigen binding molecules to be screened by the screening methods of the invention may be prepared by any method. For example, a pre-existing antibody, a pre-existing antigen-binding domain library (phage library or the like), an antibody or antigen-binding domain library prepared from B cells of an immunized animal or a hybridoma prepared by an immunized animal, an antibody or antigen-binding domain library obtained by introducing random amino acid changes into the above antibody or antigen-binding domain library, an antibody or antigen-binding domain library introduced with histidine mutations or non-natural amino acid mutations (a library with a high content of histidine or non-natural amino acids, an antigen-binding domain library introduced with histidine or non-natural amino acid mutations at specific sites, or the like) or the like may be used.
An antigen-binding molecule that binds to an antigen multiple times can be obtained by the screening method of the present invention, which is therefore excellent in plasma retention. Therefore, the screening method of the present invention can be used as a screening method for obtaining an antigen-binding molecule excellent in plasma retention.
Furthermore, an antigen-binding molecule that can bind to an antigen two or more times when administered to an animal (e.g., a human, mouse, or monkey) can be obtained by the screening method of the present invention. Thus, the screening method of the present invention can be used as a screening method for obtaining an antigen-binding molecule that can bind to an antigen two or more times.
Furthermore, an antigen-binding molecule capable of binding to more antigens than the number of antigen-binding sites thereof when administered to an animal (e.g., human, mouse or monkey) can be obtained by the screening method of the present invention. Therefore, the screening method of the present invention can be used as a screening method for obtaining an antigen-binding molecule capable of binding to more antigens than its antigen-binding site. For example, when the antibody is a neutralizing antibody, the screening method of the present invention can be used as a screening method for obtaining an antigen-binding molecule that: it can neutralize more antigen than the number of antigen binding sites of the antigen binding molecule.
Furthermore, such antigen-binding molecules obtainable by the screening method of the present invention are capable of dissociating intracellularly from an extracellularly bound antigen when administered to an animal (e.g., human, mouse or monkey). Thus, the screening method of the present invention can be used as a screening method for obtaining an antigen-binding molecule capable of dissociating intracellularly from an extracellularly bound antigen.
Furthermore, an antigen-binding molecule which, when administered to an animal (e.g., a human, mouse or monkey), binds to an antigen and is taken into a cell and released outside the cell in a form free from the antigen can be obtained by the screening method of the present invention. Therefore, the screening method of the present invention can be used as a screening method for obtaining an antigen-binding molecule that binds to an antigen and is taken into a cell and released outside the cell in a form free from the antigen.
Furthermore, antigen-binding molecules that can rapidly eliminate antigens in plasma when administered to an animal (e.g., human, mouse, or monkey) can be obtained by the screening method of the present invention. Therefore, the screening method of the present invention can be used as a screening method for obtaining an antigen-binding molecule having an increased ability to eliminate a plasma antigen (high ability).
Furthermore, such antigen binding molecules are expected to be particularly advantageous as drugs because the dosage and frequency of administration to a patient, and thus the total dose, can be reduced. Thus, the screening method of the present invention can be used as a method for screening an antigen-binding molecule for use as a pharmaceutical composition.
< method for producing antigen-binding molecule >
The present invention provides methods for producing antigen binding molecules that have human FcRn binding activity at endosomal pH and plasma pH, and that have lower antigen binding activity at endosomal pH than at plasma pH. The invention also provides methods for producing antigen binding molecules that when administered exhibit superior pharmacokinetics and in promoting a decrease in plasma antigen concentration. The invention also provides methods for producing antigen binding molecules that are particularly beneficial when used as pharmaceutical compositions.
In particular, the present invention provides a method for producing an antigen binding molecule, the method comprising the steps of:
(a) selecting an antigen binding molecule having greater human FcRn binding activity in the neutral pH range than before altering at least one amino acid in the human FcRn binding domain of an antigen binding molecule having human FcRn binding activity in the acidic pH range;
(b) altering at least one amino acid in the antigen binding domain of the antigen binding molecule and selecting an antigen binding molecule having a stronger antigen binding activity in the neutral pH range than in the acidic pH range;
(c) Obtaining a gene encoding an antigen binding molecule wherein the human FcRn binding domain prepared in (a) and (b) is linked to an antigen binding domain; and
(d) generating an antigen binding molecule using the gene prepared in (c).
Steps (a) and (b) may be performed in either order. Further, each step may be repeated two or more times. The number of times steps (a) and (b) are repeated is not particularly limited; however, the number of times is usually 10 times or less.
The linker that operably links the human FcRn binding domain prepared in (a) and (b) to the antigen binding domain is not limited to any form. The human FcRn binding domain and the antigen binding domain may be linked by a covalent or non-covalent force. In particular, the linker may be a peptide linker or a chemical linker or binding pair, such as a combination of biotin and streptavidin. Modifications of polypeptides comprising a human FcRn binding domain and an antigen binding domain are known in the art. In another embodiment, the human FcRn binding domain of the present invention may be linked to the antigen binding domain by forming a fusion protein between the human FcRn binding domain and the antigen binding domain. To construct a fusion protein between a human FcRn binding domain and an antigen binding domain, genes encoding the human FcRn binding domain and the antigen binding domain are operably linked such that an in-frame fusion polypeptide is formed. A linker comprising a peptide consisting of several amino acids may be suitably inserted between the human FcRn binding domain and the antigen binding domain. Various flexible linkers, e.g. those of the sequence consisting of (GGGGS) nConstitutive linkers are known in the art.
The antigen-binding molecule used in the production method of the present invention can be prepared by any method. For example, a pre-existing antibody, a pre-existing library (phage library, etc.), an antibody and library prepared from a hybridoma obtained by immunizing an animal or from a B cell of an immunized animal, an antibody and library prepared by introducing random amino acid changes into the above antibody and library, an antibody and library prepared by introducing histidine or unnatural amino acid mutations into the above antibody and library (a library with a high content of histidine or unnatural amino acids, a library with histidine or unnatural amino acids introduced at a specific site, etc.), and the like can be used.
In the above production method, the human FcRn binding activity of the antigen-binding molecule in the neutral pH range is not particularly limited as long as it is a human FcRn binding activity in the pH range of 6.7 to 10.0. Preferred human FcRn binding activities include human FcRn binding activity in the pH range of 7.0-8.0. More preferred human FcRn binding activities include human FcRn binding activity at pH 7.4.
The human FcRn binding activity of the antigen binding molecule in the acidic pH range is not particularly limited as long as it is a human FcRn binding activity in the range of pH 4.0 to 6.5. Preferred human FcRn binding activities include human FcRn binding activity in the pH range 5.5-6.5. More preferred human FcRn binding activities include human FcRn binding activity at pH 6.0.
In this context, an acidic pH range generally means a pH of 4.0 to 6.5. The acidic pH range is preferably a range expressed by any pH value within the range of pH 5.5 to pH 6.5, preferably selected from the group consisting of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 and 6.5, particularly preferably pH 5.8 to pH6.0, which is close to the pH of early endosomes in vivo. Meanwhile, the neutral pH range herein generally means pH 6.7 to pH 10.0. The neutral pH range is preferably a range expressed by any pH value within the range of pH 7.0 to pH 8.0, 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, particularly preferably pH 7.4, which is close to the in vivo plasma (blood) pH. If it is difficult to assess the binding affinity between the human FcRn binding domain and human FcRn due to its low affinity at pH 7.4, pH 7.0 may be used instead of pH 7.4. As for the temperature used for the assay conditions, the binding affinity between the human FcRn binding domain and human FcRn can be assessed at any temperature from 10 ℃ to 50 ℃. Temperatures between 15 ℃ and 40 ℃ are preferably used to determine the binding affinity between the human FcRn binding domain and human FcRn. More preferably also any temperature between 20 ℃ and 35 ℃, such as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 ℃, is used to determine the binding affinity between the human FcRn binding domain and human FcRn. The 25 ℃ temperature described in example 5 is an example of an embodiment of the present invention.
The present invention provides a method for producing an antigen binding molecule, the method comprising the steps of:
(a) selecting an antigen binding molecule having a human FcRn binding activity of greater than 3.2 micromolar over a neutral pH range obtained by altering at least one amino acid in the human FcRn binding domain of the antigen binding molecule;
(b) obtaining a gene encoding an antigen binding molecule wherein the human FcRn binding domain prepared in (a) is linked to an antigen binding domain; and
(c) producing an antigen binding molecule using the gene prepared in (b).
In preferred embodiments, the antigen binding molecules of the invention can be produced to have human FcRn binding activity in both acidic and neutral pH ranges, including antigen binding molecules having human FcRn binding activity in the acidic pH range of KD 20 micromolar or greater, which is equal to or greater than the human FcRn binding activity of intact human IgG in the neutral pH range. In a more preferred embodiment, antigen binding molecules of the invention can also be produced that include antigen binding molecules that have a binding activity for human FcRn in the acidic pH range of KD 2.0 micromolar or stronger and a binding activity for human FcRn in the neutral pH range of KD 40 micromolar or stronger. In an even more preferred embodiment, it may be preferred to produce antigen binding molecules of the invention that include antigen binding molecules that have human FcRn binding activity with KD 0.5 micromolar or greater in the acidic pH range and KD 15 micromolar or greater in the neutral pH range. The above KD value is determined by Journal of immunology (2009) 182: 7663-. In one embodiment, an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain having human FcRn binding activity in both acidic and neutral pH ranges may be produced according to the methods employed by those skilled in the art described above, wherein the antigen binding activity of human FcRn in the acidic pH range is greater than KD 3.2 micromolar below that in the neutral pH range. In a more preferred embodiment, the antigen binding molecule thus produced has a binding activity for human FcRn at pH 7.0 and 25 ℃ that is greater than KD 3.2 micromolar.
The present invention provides a method for the production of an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, said antigen binding molecule having human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is stronger than KD 2.3 micromolar. The invention also provides a method for producing an antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, said antigen binding molecule having human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is 38 times greater than that of intact human IgG.
In the above production method, the antigen binding activity of the antigen binding molecule in the neutral pH range is not particularly limited as long as the antigen binding activity is an antigen binding activity at a pH between pH 6.7 and pH 10.0, including, for example, the embodiment described in WO 2009/125825. The preferred antigen binding activity is an antigen binding activity at a pH between pH 7.0 and pH 8.0, and the more preferred antigen binding activity is an antigen binding activity at pH 7.4. Alternatively, the antigen binding activity of the antigen binding molecule in the acidic pH range is not particularly limited as long as the antigen binding activity is an antigen binding activity at a pH between pH 4.0 and pH 6.5. Preferred antigen binding activity is antigen binding activity at a pH between pH 5.5-pH 6.5, more preferred antigen binding activity is antigen binding activity at pH 5.8 or pH 5.5.
The antigen binding activity of the antigen binding molecule and the human FcRn binding activity can be determined by methods known to those skilled in the art. Conditions other than pH may be appropriately determined by those skilled in the art.
In the production method of the present invention, the antigen-binding molecules having human FcRn binding activity in the neutral pH range are not particularly limited as long as they have human FcRn binding activity at pH 6.7 to 10.0. However, the human FcRn binding activity of the antigen binding molecule at pH 6.7-10.0 is preferably stronger than that of intact human IgG. More preferably, the antigen binding molecule has a human FcRn binding activity greater than 40 micromolar KD, even more preferably greater than 15 micromolar KD.
In the production method of the present invention, the antigen-binding molecules having human FcRn binding activity in the acidic pH range are not particularly limited as long as they have human FcRn binding activity at pH 4.0 to 6.5. However, at pH 5.5-6.5, the antigen binding molecule preferably has human FcRn binding activity greater than KD 20 micromolar. More preferably, the human FcRn binding activity is comparable to or stronger than that of intact human IgG1 (stronger than KD 1.7 micromolar), more preferably stronger than KD 0.5 micromolar.
The above-mentioned KD value can be determined by "The Journal of Immunology, (2009) 182: 7663-.
In the production method of the present invention, the step of selecting an antigen-binding molecule having an antigen-binding activity at pH 6.7 to pH 10.0 that is stronger than the antigen-binding activity at pH 4.0 to pH 6.5 is synonymous with the step of selecting an antigen-binding molecule having an antigen-binding activity at pH 4.0 to pH 6.5 that is lower than the antigen-binding activity at pH 6.7 to pH 10.0.
The ratio between the antigen-binding activity in the neutral pH range and the antigen-binding activity in the acidic pH range is not particularly limited as long as the antigen-binding activity in the neutral pH range is stronger than the antigen-binding activity in the acidic pH range. The antigen-binding activity at pH 6.7 to pH 10.0 is preferably 2-fold or more, more preferably 10-fold or more, still more preferably 40-fold or more, the antigen-binding activity at pH 4.0 to pH 6.5.
In the above production method, the antigen and the antigen-binding molecule may be bound to each other in any state, and the human FcRn and the antigen-binding molecule may be bound to each other in any state. The state is not particularly limited; for example, an antigen or human FcRn can be contacted with an immobilized antigen binding molecule to bind the antigen binding molecule. Alternatively, the antigen binding molecule may be contacted with an immobilized antigen or human FcRn to bind the antigen binding molecule. Alternatively, the antigen binding molecule may be contacted with the antigen or human FcRn in solution to bind the antigen binding molecule.
The antigen binding molecule produced by the above method may be any antigen binding molecule; preferred antigen binding molecules include, for example, those having an antigen binding domain and a human FcRn binding domain comprising in the human FcRn binding domain: at least one amino acid change, and a substitution of one or more amino acids with histidine or an insertion of at least one histidine.
Amino acid changes in the human FcRn binding domain are not particularly limited as long as they enhance human FcRn binding activity in the neutral pH range. Alterations include, for example, alterations in the amino acids at the following positions in the IgG Fc domain described above: 221-. More specifically, amino acid changes include changes at the amino acid positions shown in Table 1, Table 2, tables 6-1, and tables 6-2 (by EU numbering). Preferably, the binding activity of human FcRn in the neutral pH range can be increased by altering at least one amino acid selected from the amino acids at the following positions: 237. 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436(EU numbering). The number of amino acids to be changed is not particularly limited; the amino acids may be changed at only a single site or at two or more sites. Combinations of two or more amino acid changes include, for example, those shown in Table 3, tables 4-1 to 4-5, Table 6-1, and Table 6-2.
Meanwhile, the site in which the histidine mutation is introduced is not particularly limited, and thus it may be introduced at any position as long as the histidine mutation reduces the antigen binding activity in an acidic pH range to the antigen binding activity in a less than neutral pH range. Such histidine mutations may be introduced at a single site or at two or more sites.
Therefore, the production method of the present invention may further comprise the steps of changing the above amino acids and substituting or inserting histidine. In the production method of the present invention, an unnatural amino acid may be used in place of histidine. Thus, the present invention is also understood to be the replacement of the above histidine with an unnatural amino acid.
Furthermore, in another embodiment, the antigen binding molecule produced by the above production method includes, for example, an antigen binding molecule comprising an altered antibody constant region. Therefore, the production method of the present invention may further comprise a step of changing the amino acids of the constant region of the antibody.
The antigen-binding molecule produced by the production method of the present invention is administered to promote a decrease in plasma antigen concentration. Thus, the production method of the present invention can be used as a method for producing an antigen-binding molecule that promotes a decrease in plasma antigen concentration when administered.
Alternatively, the antigen-binding molecules produced by the production methods of the invention have improved pharmacokinetics. Therefore, the production method of the present invention can be used as a method for producing an antigen-binding molecule having improved pharmacokinetics.
Alternatively, the antigen-binding molecules produced by the production methods of the invention can increase the number of antigens to which a single antigen-binding molecule can bind when administered to an animal (e.g., human, mouse, and monkey). Thus, the production method of the present invention can be used as a method for producing an antigen-binding molecule in which the number of antigens to which a single antigen-binding molecule can bind is increased.
Furthermore, it is contemplated that the antigen-binding molecules produced by the production methods of the invention are capable of dissociating intracellularly from an extracellularly bound antigen when administered to an animal (e.g., a human, mouse, or monkey). Thus, the production method of the present invention can be used as a method for producing an antigen-binding molecule capable of dissociating intracellularly from an extracellularly bound antigen.
Furthermore, it is expected that the antigen-binding molecule produced by the production method of the present invention is capable of binding to an antigen and being taken into cells and being released outside the cells in a form free from the antigen when administered to an animal (e.g., human, mouse, or monkey). Therefore, the production method of the present invention can be used as a method for producing an antigen-binding molecule capable of binding to an antigen and being taken into a cell and released outside the cell in an antigen-free form.
In addition, since such antigen-binding molecules have a greater activity of reducing the plasma antigen concentration by administration than typical antigen-binding molecules, they are expected to be particularly excellent as a drug. Thus, the production method of the present invention can be used as a method for producing an antigen-binding molecule for use as a pharmaceutical composition.
The gene obtained by the production method of the present invention is usually carried by an appropriate vector (inserted into an appropriate vector) and then introduced into a host cell. The vectors are not particularly limited as long as they stably retain the inserted nucleic acid. For example, when Escherichia coli is used as the host, preferred cloning vectors include pBluescript vector (Stratagene); however, various commercially available carriers can be used. Expression vectors are particularly useful when vectors are used to produce the antigen binding molecules of the invention. The expression vector is not particularly limited so long as the vector expresses the antigen-binding molecule in vitro, in E.coli, in cultured cells, or in vivo. For example, the pBEST vector (Promega) is preferred for in vitro expression; pET vector (Invitrogen) is preferably used for E.coli; the pME18S-FL3 vector (GenBank accession number AB009864) is preferably used for culturing cells; the pME18S vector (Mol Cell Biol. (1988) 8: 466-472) is preferably used in vivo. The DNA of the present invention can be inserted into a vector by a conventional method, for example, by ligation using restriction enzyme sites (Current protocols in Molecular Biology, eds. Ausubel et al (1987), publish. John Wiley & Sons, sections 11.4-11.11).
The host cell is not particularly limited, and various host cells can be used according to the purpose. Examples of cells for expressing antigen binding molecules include bacterial cells (e.g., cells of the genera Streptococcus (Streptococcus), Staphylococcus (Staphylococcus), Escherichia coli, Streptomyces (Streptomyces) and Bacillus subtilis), eukaryotic cells (e.g., cells of the genera Yeast and Aspergillus), insect cells (e.g., Drosophila S2 and Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293 and Bowes melanoma cells), and plant cells. The vector can be introduced into the host cell by known methods, for example, calcium phosphate precipitation method, electroporation method (Current protocols in Molecular Biology, eds. Ausubel et al (1987), publish. John Wiley & Sons, sections 9.1-9.9), lipofection method, and microinjection method.
The host cell can be cultured by known methods. For example, when animal cells are used as the host, DMEM, MEM, RPMI1640, or IMDM can be used as the medium. They can be used with serum supplements such as FBS or Fetal Calf Serum (FCS). The cells may be cultured in serum-free cultures. During the cultivation, the preferred pH is about 6-8. The incubation is typically carried out at 30-40 ℃ for about 15-200 hours. When necessary, the medium is replaced, aerated or stirred.
Appropriate secretion signals can be incorporated into the polypeptide of interest to allow secretion of the antigen binding molecule expressed in the host cell into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment. These signals may be endogenous to the antigen binding molecule of interest, or may be heterologous signals.
In another aspect, for example, a production system using animals or plants can be used as a system for producing a polypeptide in vivo. Introducing the target polynucleotide into an animal or plant, producing a polypeptide in the animal or plant, and collecting the polypeptide. The "host" of the present invention includes such animals and plants.
Production systems using animals include systems using mammals or insects. Mammals, such as goats, pigs, sheep, mice and cattle, can be used (Vicki Glaser SPECTRUMBIOtechnologics Applications (1993)). The mammal may be a transgenic animal.
For example, polynucleotides encoding the antigen binding molecules of the invention are prepared as fusion genes with genes encoding polypeptides specifically produced in milk (e.g., goat β -casein). Next, goat embryos are injected with the polynucleotide fragments containing the fusion genes, and then implanted into female goats. The desired antigen binding molecule may be obtained from milk produced from a transgenic goat (which is born from a goat receiving embryos) or its offspring. Hormones may be administered to increase the amount of milk containing the antigen binding molecule produced by the transgenic goat, as appropriate (Ebert et al, Bio/Technology (1994) 12: 699-).
Insects (e.g., silkworm) can be used to produce the antigen binding molecules of the invention. When silkworms are used, the silkworms can be infected with baculovirus carrying a polynucleotide encoding the antigen-binding molecule of interest, and the antigen-binding molecule of interest is obtained from body fluid.
In addition, for example, when plants are used to produce the antigen binding molecules of the invention, tobacco can be used. When tobacco is used, the polynucleotide encoding the antigen binding molecule of interest is inserted into a plant expression vector, such as pMON 530, and the vector is then inserted into a bacterium, such as Agrobacterium tumefaciens (Agrobacterium tumefaciens). The bacteria are then allowed to infect tobacco, such as tobacco (Nicotiana tabacum), and the desired antigen binding molecule is collected from its leaves (Ma et al, Eur. J. Immunol. (1994) 24: 131-. Alternatively, duckweed (Lemnaminor) may be infected with similar bacteria. After cloning, the desired antigen binding molecule can be obtained from duckweed cells (Cox KM et al, nat. Biotechnol.2006, 12 months; 24 (12): 1591-.
The antigen binding molecules thus obtained can be isolated from the inside and outside of the host cell (e.g., culture medium and milk) and purified to a substantially pure and homogeneous antigen binding molecule. The method for separating and purifying the antigen-binding molecule is not particularly limited, and separation and purification methods commonly used for polypeptide purification may be employed. The antigen-binding molecule can be isolated and purified by appropriate selection and combination, for example, chromatography column, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization.
Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography and adsorption chromatography (stratgies for Protein purification and chromatography: A Laboratory Course Manual, eds. Daniel R. Marshak et al (1996) Cold Spring Harbor Laboratory Press). Such chromatographic methods can be carried out using liquid chromatography methods such as HPLC and FPLC. Columns used for affinity chromatography include protein a columns and protein G columns. Columns using protein A include, for example, Hyper D, POROS and Sepharose F.F (Pharmacia).
If desired, the antigen-binding molecule may be modified arbitrarily, and the peptide portion may be deleted by allowing an appropriate protein-modifying enzyme to act before or after purification of the antigen-binding molecule. Such protein modifying enzymes include, for example, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, and glucosidase.
< pharmaceutical composition >
The invention also relates to pharmaceutical compositions comprising the antigen binding molecules of the invention, antigen binding molecules isolated by the screening methods of the invention or antigen binding molecules produced by the production methods of the invention. The antigen-binding molecule of the present invention and the antigen-binding molecule produced by the production method of the present invention have a greater activity of reducing plasma antigen concentration by administration than typical antigen-binding molecules, and thus can be used as a pharmaceutical composition. The pharmaceutical compositions of the present invention may include a pharmaceutically acceptable carrier.
In the present invention, the pharmaceutical composition generally refers to an agent for treating or preventing, or detecting and diagnosing a disease.
The pharmaceutical compositions of the present invention may be formulated by methods known to those skilled in the art. For example, they may be administered parenterally in the form of injections of sterile solutions or suspensions, including water or other pharmaceutically acceptable liquids. For example, such compositions may be formulated as follows: in suitable admixture with a pharmaceutically acceptable carrier or vehicle, particularly sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binding agents and the like, in the required unit dosage form as is commonly recognized in the practice of pharmaceutical manufacture. In such formulations, the amount of active ingredient is adjusted to give the appropriate amount within the predetermined range.
Sterile compositions for injection may be formulated according to standard formulation practice using vehicles such as distilled water for injection. Aqueous solutions for injection include, for example, physiological saline and isotonic solutions containing glucose or other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. It can also be used in combination with suitable solubilizing agents such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.), nonionic surfactants (polysorbate 80(TM), HCO-50, etc.).
The oil comprises sesame oil and soybean oil. Benzyl benzoate and/or benzyl alcohol may be used in combination as a solubilizer. Buffers (e.g., phosphate buffers and sodium acetate buffers), moderators (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and/or antioxidants can also be mixed. The appropriate ampoule is filled with the prepared injection.
Preferably, the pharmaceutical compositions of the present invention are administered parenterally. For example, the composition may be in a dosage form for injection, nasal administration, pulmonary administration, or transdermal administration. For example, they can be administered systemically by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc., or locally.
The administration method may be appropriately selected in consideration of the age and symptoms of the patient. For each administration, the dosage of the pharmaceutical composition containing the antigen binding molecule may be, for example, 0.0001 to 1,000 mg/kg. Alternatively, the dosage may be, for example, from 0.001 to 100,000mg per patient. However, the present invention is not limited by the above numerical values. The dose and administration method may vary depending on the body weight, age, symptoms, etc. of the patient. In view of the above, one skilled in the art can set the appropriate dosage and method of administration.
The amino acids comprised in the amino acid sequence of the present invention may be post-translationally modified. For example, modification of the N-terminal glutamine to pyroglutamic acid by pyroglutamic acidification is well known to those skilled in the art. Clearly, 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 herein incorporated by reference.
Examples
Hereinafter, the present invention will be described specifically with reference to examples, but it should not be construed as being limited thereto.
EXAMPLE 1 investigation of accelerated antigen Elimination Effect of accelerated antibody
anti-IL-6 receptor antibodies
Preparation of anti-human IL-6 receptor antibody having FcRn binding activity under neutral conditions
H54/L28-IgG1 comprising H54(SEQ ID NO: 1) and L28(SEQ ID NO: 2) described in WO 2009/125825 is a humanized anti-IL-6 receptor antibody. Mutations were introduced into H54(SEQ ID NO: 1) to increase FcRn binding under neutral pH conditions (pH 7.4). Specifically, H54-IgG1-F14(SEQ ID NO: 3) was prepared from the heavy chain constant region of IgG1 by substituting Trp for Met at position 252 and Trp for Asn at position 434 (numbering by EU). Amino acid substitutions were introduced by methods known to those skilled in the art as described in reference example 1.
H54/L28-IgG1 comprising H54(SEQ ID NO: 1) and L28(SEQ ID NO: 2) and H54/L28-IgG1-F14 comprising H54-IgG1-F14(SEQ ID NO: 3) and L28(SEQ ID NO: 2) were expressed and purified by methods known to those skilled in the art described in reference example 2.
In vivo antibody production using human FcRn transgenic mouse strain 276 by steady state infusion model
Study of
In vivo experiments were performed using the H54/L28-IgG1 and H54/L28-IgG1-F14 prepared above, using the human FcRn transgenic mouse strain 276 via a steady state infusion model. An infusion PUMP (MINI-OSMOTIC PUMP MODEL 2004; alzet) containing a soluble human IL-6 receptor was implanted under the back skin of a human FcRn transgenic mouse strain 276(B6.mFcRn-/-. hFcRn Tg strain 276+/+ mouse (B6.mFcRn-/-hFCRN Tg276 B6.Cg-Fcgrt < tm1Dcr > Tg (FCGRT)276Dcr (Jackson #4919)), Jackson Laboratories; MethodsMol Biol. (2010) 602: 93-104) to prepare MODEL animals in which the plasma concentration of the soluble human IL-6 receptor was kept constant. Anti-human IL-6 receptor antibodies were administered to model animals to evaluate in vivo kinetics following administration of soluble human IL-6 receptor. Monoclonal anti-mouse CD4 antibody (R & D) was administered at 20mg/kg to inhibit neutralizing antibody production against soluble human IL-6 receptor prior to implantation of the infusion pump and 14 days after administration of the antibody to the tail vein. Then, an infusion pump containing 92.8 microgram/ml of soluble human IL-6 receptor was implanted under the skin of the back of the mice. Anti-human IL-6 receptor antibodies (H54/L28-IgG1 and H54/L28-IgG1-F14) were administered once at 1mg/kg to the tail vein 3 days after implantation of the infusion pump. Blood was collected 15 minutes, 7 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 14 days, 21 days, and 28 days after administration of the anti-human IL-6 receptor antibody. Immediately, the collected blood was centrifuged at 15,000rpm and 4 ℃ for 15 minutes to separate plasma. The separated plasma was stored in a refrigerator at-20 ℃ or lower before the measurement.
By electricityDetermination of plasma hsIL-6R concentration by chemiluminescence assay
The hsIL-6R concentration in the plasma of mice was measured by electrochemiluminescence. Mouse plasma samples diluted 50-fold or more were prepared by adjusting hsIL-6R calibration curve samples to concentrations of 2,000, 1,000, 500, 250, 125, 62.5, and 31.25 pg/ml. The sample was mixed with a solution of monoclonal anti-human IL-6R antibody (R & D), biotinylated anti-human IL-6R antibody (R & D) and WT-IgG1 labeled with Sulfo-Tag NHS Ester (Meso Scale Discovery) ruthenium, and then allowed to react overnight at 37 ℃. The final concentration of WT-IgG1, which is an anti-human IL-6 receptor antibody comprising H (WT) (SEQ ID NO: 4) and L (WT) (SEQ ID NO: 5), was 333 micrograms/ml, which exceeded the concentration of anti-human IL-6 receptor antibody contained in the sample, with the aim of allowing almost all hsIL-6R molecules in the sample to bind to WT-IgG 1. Subsequently, the sample was dispensed in a MA400PR streptavidin plate (Meso Scale Discovery), allowed to react at room temperature for 1 hour, and washed. Just after dispensing the read buffer T (ReadBuffer T) (x4) (Meso Scale Discovery), measurements were performed by a Sector PR 400 reader (Meso Scale Discovery). Using the analytical software SOFTMax PRO (Molecular Devices), hsIL-6R concentration was calculated from the response of the calibration curve. The time course of plasma hsIL-6R concentration after intravenous administration of H54/L28-IgG1 and H54/L28-IgG1-F14, measured according to this method, is shown in FIG. 1.
As shown in FIG. 1, administration of H54/L28-IgG1 resulted in a significant increase in plasma hsIL-6R concentration compared to the baseline hsIL-6R concentration in the absence of antibody. On the other hand, administration of H54/L28-IgG1-F14 resulted in a decrease in the increase in plasma hsIL-6R concentration compared to H54/L28-IgG 1. This increased reduction in comparison to H54/L28-IgG1 stems from increased human FcRn binding at neutral pH in H54/L28-IgG 1-F14. This demonstrates that increasing the binding affinity of the antibody to FcRn at neutral pH promotes antigen clearance, but that H54/L28-IgG1-F14 promotes antigen clearance to a lesser extent than H54/L28-IgG 1.
EXAMPLE 2 investigation of the acceleration of antigen Elimination by pH-dependent antigen-binding antibody (preparation of antibody)
Antibodies related to pH-dependent human IL-6 receptor binding
H54/L28-IgG1 comprising H54(SEQ ID NO: 1) and L28(SEQ ID NO: 2) described in WO 2009/125825 is a humanized anti-IL-6 receptor antibody. Fv4-IgG1 comprising VH3-IgG1(SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 7) is a humanized anti-IL-6 receptor antibody (which binds at pH 7.4 but dissociates at pH 5.8) produced by conferring the property of H54/L28-IgG1 to bind to soluble human IL-6 receptor in a pH-dependent manner. In vivo experiments using mice described in WO 2009/125825 demonstrated that elimination of soluble human IL-6 receptor was greatly accelerated in the group to which a mixture of Fv4-IgG1 and soluble human IL-6 receptor was administered as an antigen, compared to the group to which a mixture of H54/L28-IgG1 and soluble human IL-6 receptor was administered as an antigen.
Soluble human IL-6 receptor bound to normal antibodies, which bind to soluble human IL-6 receptor, is recycled to plasma via FcRn along with the antibody. Meanwhile, the antibody binding to the soluble human IL-6 receptor in a pH-dependent manner dissociates the soluble human IL-6 receptor bound to the antibody under acidic conditions of the endosome. The dissociated soluble human IL-6 receptor is degraded in lysosomes. This can greatly accelerate the elimination of soluble human IL-6 receptor. The antibody bound to the soluble human IL-6 receptor in a pH-dependent manner is then recycled to the plasma via FcRn. The recycled antibody can then bind to other soluble human IL-6 receptors. By repeating this cycle, a single antibody molecule can repeatedly bind to the soluble human IL-6 receptor multiple times (fig. 2).
Antibodies that bind to antigens in a pH-dependent manner accelerate the elimination of soluble antigens. Antibodies produce this effect by repeatedly binding to soluble antigen multiple times. Thus, such antibodies are very useful. Methods for increasing FcRn binding under neutral conditions (pH 7.4) were tested to further enhance proantigen elimination.
PH-dependent human IL-6 receptor binding antibodies with FcRn binding activity under neutral conditions
Preparation of
Mutations were introduced into Fv4-IgG1 comprising VH3-IgG1(SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 7) to increase FcRn binding under neutral conditions (pH 7.4). Specifically, VH3-IgG1-v1(SEQ ID NO: 8) was prepared from the heavy chain constant region of IgG1 by substituting Met at position 252 with Tyr, Ser at position 254 with Tyr and Thr at position 256 with Glu (numbering according to EU), while VH3-IgG1-v2(SEQ ID NO: 9) was constructed from the heavy chain constant region of IgG1 by substituting Asn at position 434 with Trp (numbering according to EU). Amino acid substitutions were introduced by methods known to those skilled in the art as described in reference example 1.
H54/L28-IgG1 comprising H54(SEQ ID NO: 1) and L28(SEQ ID NO: 2), Fv4-IgG1 comprising VH3-IgG1(SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 7), Fv4-IgG1-v1 comprising VH3-IgG1-v1(SEQ ID NO: 8) and VL3-CK (SEQ ID NO: 7), and Fv4-IgG1-v2 comprising VH3-IgG1-v2(SEQ ID NO: 9) and VL3-CK (SEQ ID NO: 7) were expressed and purified by methods known to those skilled in the art described with reference to example 2.
EXAMPLE 3 investigation of the accelerated action of pH-dependent antigen-binding antibody on antigen Elimination (in vivo test)
In vivo assay using human FcRn transgenic and normal mice
The in vivo kinetics of hsIL-6R (soluble human IL-6 receptor: prepared as described in reference example 3) and anti-human IL-6 receptor antibodies were evaluated after administration of hsIL-6R alone or a combination of hsIL-6R and anti-human IL-6 receptor antibodies to human FcRn transgenic mice (B6.mFcRn-/-. hFcRn Tg strain 276+/+ mice, Jackson laboratories; Methods Mol Biol. (2010) 602: 93-104) and normal mice (C57BL/6J mice; Charles River Japan). hsIL-6R solution (5 microgram/ml) or a solution containing a mixture of hsIL-6R and anti-human IL-6 receptor antibody (5 microgram/ml and 0.1mg/ml, respectively) was administered to the tail vein once at a dose of 10 ml/kg. In this case, anti-human IL-6 receptor antibody is present in excess of hsIL-6R, so it is assumed that almost every hsIL-6R binds to the antibody. Blood was collected at 15 minutes, 7 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 14 days, 21 days, and 28 days after administration. The collected blood was immediately centrifuged at 15,000rpm and 4 ℃ for 15 minutes to separate plasma. The separated plasma was stored at-20 ℃ or below-20 ℃ in a refrigerator prior to the assay. The anti-human IL-6 receptor antibodies used were: H54/L28-IgG1, Fv4-IgG1, and Fv4-IgG1-v2 as described above for human FcRn transgenic mice; the normal mice were H54/L28-IgG1, Fv4-IgG1, Fv4-IgG1-v1, and Fv4-IgG1-v 2.
Measurement of plasma concentration of anti-human IL-6 receptor antibody by ELISA
The concentration of anti-human IL-6 receptor antibody in mouse plasma was measured by ELISA. Anti-human IgG (gamma chain specific) F (ab') 2 antibody fragment (Sigma) was dispensed on Nunc-ImmunoplatemMaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4 ℃ to prepare anti-human IgG-immobilized plates. Calibration curve samples with plasma concentrations of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025 and 0.0125 micrograms/ml and mouse plasma samples diluted 100-fold or more were prepared. 200 microliters (microL) of 20ng/ml hsIL-6R were added to 100 microliters of the calibration curve sample and the plasma sample, and the sample was allowed to stand at room temperature for 1 hour. Subsequently, the sample was dispensed into an anti-human IgG-immobilized plate and allowed to stand at room temperature for 1 hour. Then, a biotinylated anti-human IL-6R antibody (R & D) was added thereto, and the reaction was carried out at room temperature for 1 hour. Subsequently, streptavidin-PolyHRP 80(Stereospecific detection technologies) was added, reacted at room temperature for 1 hour, and a color reaction was performed using TMP One Component HRMicrowell Substrate (BioFX Laboratories) as a Substrate. After the reaction was terminated with 1N sulfuric acid (Showa Chemica), the absorbance at 450nm was measured by a microplate reader. The concentration in the mouse plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTMax PRO (Molecular Devices). The time course of plasma concentrations after intravenous administration as measured by this method is shown in figure 3 for human FcRn transgenic mice and figure 5 for normal mice.
Measurement of hsIL-6R plasma concentration by electrochemiluminescence assay
The hsIL-6R concentration in the plasma of mice was measured by electrochemiluminescence. Mouse plasma samples diluted 50-fold or more were prepared by adjusting hsIL-6R calibration curve samples to concentrations of 2,000, 1,000, 500, 250, 125, 62.5, and 31.25 pg/ml. The sample was mixed with a solution of monoclonal anti-human IL-6R antibody (R & D), biotinylated anti-human IL-6R antibody (R & D) and WT-IgG1 labeled with Sulfo-Tag NHS Ester (Meso Scale Discovery) ruthenium, and then allowed to react overnight at 37 ℃. The final concentration of WT-IgG1, which is an anti-human IL-6 receptor antibody comprising H (WT) (SEQ ID NO: 4) and L (WT) (SEQ ID NO: 5), was 333 micrograms/ml, which exceeded the concentration of anti-human IL-6 receptor antibody contained in the sample, with the aim of allowing almost all hsIL-6R molecules in the sample to bind to WT-IgG 1. Subsequently, the sample was dispensed in a MA400PR streptavidin plate (Meso Scale Discovery), allowed to react at room temperature for 1 hour, and washed. Just after dispensing the reading buffer T (x4) (Meso Scale Discovery), measurements were performed by a Sector PR 400 reader (Meso Scale Discovery). The hsIL-6R concentration was calculated from the response of the calibration curve using the analytical software SOFTMax PRO (molecular devices). The time course of plasma hsIL-6R concentration after intravenous administration as measured by this method is shown in FIG. 4 for human FcRn transgenic mice and in FIG. 6 for normal mice.
Determination of free hsIL-6R concentration in plasma by electrochemiluminescence assay
To assess the extent of soluble human IL-6 receptor neutralization in plasma, the concentration of soluble human IL-6 receptor (free hsIL-6R concentration) in mouse plasma without (without neutralization by) anti-human IL-6 receptor antibody was determined by electrochemiluminescence assay. All IgG type antibodies (mouse IgG, anti-human IL-6 receptor antibody, and anti-human IL-6 receptor antibody-soluble human IL-6 receptor complex) in plasma were adsorbed onto protein A by adding 12 microliters of each of the hsIL-6R standard sample and the mouse plasma sample prepared at 10,000, 5,000, 2,500, 1,250, 625, 312.5, or 156.25pg/ml to an appropriate amount of rProtein A Sepharose Flow (GE Healthcare) resin dried in a 0.22 micron filter bowl (Millipore). The solution in the cup was then centrifuged using a high speed centrifuge to collect the flow through (passaged through) solution. The flow-through solution was free of protein A-bound anti-human IL-6 receptor antibody-soluble human IL-6 receptor complex. Thus, the concentration of free hsIL-6R in plasma can be determined by measuring the concentration of hsIL-6R in the flow-through solution. Then, the flow-through solution was mixed together with a monoclonal anti-human IL-6R antibody (R & D) labeled with Sulfo-Tag NHSEster (Meso Scale Discovery) ruthenium and a biotinylated anti-human IL-6R antibody (R & D). The resulting mixture was incubated at room temperature for 1 hour and then aliquoted into MA400PR streptavidin plates (Meso scale discovery). After an additional 1 hour incubation at room temperature, the plates were washed and read buffer T (x4) (Meso Scale Discovery) was aliquoted into them. Plates were immediately measured in a SECTOR PR 400 reader (Meso Scale Discovery). Using the analytical software SOFTMax PRO (Molecular Devices), hsIL-6R concentration was calculated from the reactions in the standard curve. The time course of free hsIL-6R concentration in plasma of normal mice as determined by the above method after intravenous administration is shown in FIG. 7.
PH-dependent binding to human IL-6 receptor
H54/L28-IgG1 and Fv4-IgG that bind to the human IL-6 receptor in a pH-dependent manner were tested in vivo and the results were compared between them. As shown in fig. 3 and 5, the antibody retention in plasma was comparable. Meanwhile, as shown in FIGS. 4 and 6, it was found that hsIL-6R administered simultaneously with Fv4-IgG1 that binds to human IL-6 receptor in a pH-dependent manner accelerates the elimination of hsIL-6R, compared to hsIL-6R administered simultaneously with H54/L28-IgG 1. The above trend was observed in both human FcRn transgenic and normal mice; thus, by conferring pH-dependent binding capacity to human IL-6 receptor, it was shown that plasma hsIL-6R concentrations were reduced by approximately 17 and 34-fold, respectively, 4 days after administration.
Effect of FcRn binding under neutral conditions (pH 7.4)
It was reported that under neutral conditions (pH 7.4), intact human IgG1 bound little (with very low affinity) to human FcRn. It was reported that human FcRn binding under neutral conditions (pH 7.4) was increased by substitution of Trp for Asn at position 434 (EU numbering) in intact human IgG1 (JImmunol. (2009)182 (12): 7663-71). Fv4-IgG1-v2 produced by introducing the above amino acid substitutions into Fv4-IgG1 was tested by in vivo testing using human FcRn transgenic mice. The results of the assay were compared to those of Fv4-IgG 1. As shown in FIG. 3, the antibody plasma retention between the two was comparable. Meanwhile, as shown in FIG. 4, it was found that hsIL-6R administered simultaneously with Fv4-IgG1-v2, which has improved binding to human FcRn under neutral conditions (pH 7.4), was eliminated more rapidly than hsIL-6R administered simultaneously with Fv4-IgG 1. Thus, by having the ability to bind human FcRn under neutral conditions (pH 7.4) it was shown that plasma concentrations of hsIL-6R were reduced by about 4-fold 4 days after administration.
Based on the homology between human and mouse FcRn, it was postulated that substitution of Asn at position 434 with Trp (numbering according to EU) increased binding to mouse FcRn under neutral conditions (pH 7.4). Meanwhile, it has been reported that binding to mouse FcRn under neutral conditions (pH 7.4) is increased by substituting Met at position 252 with Tyr, Ser at position 254 with Tyr, and Thr at position 256 with Glu (numbering according to EU) (J Immunol. (2002)169 (9): 5171-80). Fv4-IgG1-v1 and Fv4-IgG1-v2, which were produced by introducing the above amino acid substitutions into Fv4-IgG1, were tested in vivo using normal mice. The results of the assay were compared to those of Fv4-IgG 1. As shown in fig. 5, the plasma retention times of Fv4-IgG1-v1 and Fv4-IgG1-v2, which were improved to increase binding to mouse FcRn under neutral conditions (pH 7.4), were slightly shortened (approximately 1.5 and 1.9-fold reduction in the concentration of neutralizing antibody in plasma 1 day after administration), compared to Fv4-IgG 1.
As shown in FIG. 6, hsIL-6R administered simultaneously with either Fv4-IgG1-v1 or Fv4-IgG1-v2, which were modified to increase binding to mouse FcRn under neutral conditions (pH 7.4), demonstrated significantly faster elimination compared to hsIL-6R administered simultaneously with Fv4-IgG 1. 1 day after administration, Fv4-IgG1-v1 and Fv4-IgG1-v2 reduced plasma hsIL-6R concentrations by approximately 32-fold and 80-fold, respectively. Thus, it was revealed that plasma concentrations can be reduced by allowing it to have mouse FcRn binding ability under neutral conditions (pH 7.4). As described above, by allowing it to have mouse FcRn binding ability under neutral conditions (pH 7.4), plasma antibody concentrations were slightly reduced; however, a reduction in plasma hsIL-6R concentration was produced which greatly exceeded the reduction in antibody concentration. Furthermore, it was found that hsIL-6R administered simultaneously with either Fv4-IgG1-v1 or Fv4-IgG1-v2 was eliminated more rapidly even when compared to the group administered with hsIL-6R alone. As shown in FIG. 6, it was shown that hsIL-6R administered simultaneously with either Fv4-IgG1-v1 or Fv4-IgG1-v2 reduced plasma hsIL-6R concentrations by about 4-fold or 11-fold, respectively, 1 day after administration compared to hsIL-6R alone. Specifically, this means that the elimination of soluble IL-6 receptor can be accelerated by administering an antibody that binds to soluble IL-6 receptor in a pH-dependent manner and that is made to have a mouse FcRn binding ability under neutral conditions (pH 7.4). In particular, plasma antigen concentrations can be reduced in vivo by administering such antibodies to the body.
As shown in FIG. 7, free hsIL-6R was within detectable concentrations 7 days after H54/L28-IgG1 administration, whereas free hsIL-6R was not detectable 1 day after Fv4-IgG1 administration. On the other hand, 7 hours after administration of Fv4-IgG1-v1 or Fv4-IgG1-v2, free hsIL-6R could not be detected. Specifically, the lower concentration of free hsIL-6R in the presence of Fv4-IgG1 that binds to hsIL-6R in a pH-dependent manner, as compared to H54/L28-IgG1, indicates that strong hsIL-6R neutralization occurs by conferring pH-dependent binding ability to hsIL-6R. Furthermore, free hsIL-6R concentrations were much lower in the presence of either Fv4-IgG1-v1 or Fv4-IgG1-v2 (both modified from Fv4-IgG1 to increase FcRn binding capacity at pH 7.4). This demonstrates that much stronger hsIL-6R neutralization can be produced by increasing FcRn binding at pH 7.4.
When administered, common neutralizing antibodies such as H54/L28-IgG1 reduce clearance of bound antigen, resulting in prolonged retention of antigen plasma. It is not preferred that the administered antibody prolong plasma retention of the antigen whose effect is expected to be neutralized by the antibody. Antigen plasma retention can be shortened by imparting a pH dependence of antigen binding (antibodies bind under neutral conditions but dissociate under acidic conditions). In the present invention, the antigen retention time of plasma can be further shortened by additionally imparting human FcRn binding ability under neutral conditions (pH 7.4). Furthermore, it was demonstrated that antigen clearance can be improved by administering an antibody that binds to an antigen in a pH-dependent manner and is made FcRn binding ability under neutral conditions (pH 7.4) compared to clearance of the antigen alone. To date, there is no available method for increasing antigen clearance by administering antibodies relative to antigen-only clearance. Therefore, the method established as described in this example is very useful as a method for eliminating an antigen from plasma by administering an antibody. Furthermore, the present inventors have for the first time found the advantage of improving FcRn binding capacity under neutral conditions (pH 7.4). Furthermore, both v4-IgG1-v1 and Fv4-IgG1-v2, which have different amino acid substitutions and increase the FcRn binding ability under neutral conditions (pH 7.4), produce comparable effects. This suggests that each amino acid substitution that enhances human FcRn binding under neutral conditions (pH 7.4) may have an effect of accelerating antigen elimination regardless of the type of amino acid substitution. In particular, antibody molecules that eliminate antigen from plasma when administered can be produced using the following individual amino acid substitutions or combinations:
Amino acid substitution in which Pro at position 257 is substituted by Ile, and Gln at position 311 is substituted by Ile (numbering according to EU), J Biol chem.2007, 282 (3): 1709-17, both are reported; an amino acid substitution in which Asn at position 434 is substituted with Ala, Tyr or Trp, an amino acid substitution in which Met at position 252 is substituted with Tyr, an amino acid substitution in which Thr at position 307 is substituted with gin, an amino acid substitution in which Val at position 308 is substituted with Pro, an amino acid substitution in which Thr at position 250 is substituted with gin, an amino acid substitution in which Met at position 428 is substituted with Leu, an amino acid substitution in which Glu at position 380 is substituted with Ala, an amino acid substitution in which Ala at position 378 is substituted with Val, an amino acid substitution in which Tyr at position 436 is substituted with Ile (numbering by EU), J Immunol (2009)182 (12): 7663-71 all of these substitutions are reported; an amino acid substitution in which Met at position 252 is substituted with Tyr, Ser at position 254 is substituted with Thr, an amino acid substitution in which Thr at position 256 is substituted with Glu (numbering according to EU), J Biol chem.2006, 8/8 th, 281 (33): 23514-24, all of which are described; an amino acid substitution of His at position 433 with Lys, an amino acid substitution of Asn at position 434 with Phe, and an amino acid substitution of His at position 436 with Tyr (numbering by EU), Nat biotechnol.2005, month 10, 23 (10): all of these substitutions are reported in 1283-8; and the like.
EXAMPLE 4 evaluation of human FcRn binding Activity
J Immunol. (2009)182 (12): 7663-71 a Biacore based assay system for testing the interaction between antibodies and FcRn, a system for immobilising antibodies on a sensor chip and using human FcRn as an analyte, is reported. For this purpose, human FcRn was prepared as described in reference example 4. Fv4-IgG1, Fv4-IgG1-v1, and Fv4-IgG1-v2 were evaluated for human FcRn binding activity (dissociation constant (KD)) at pH 6.0 and pH 7.4 by using the above system. After being directly immobilized on an S-Series sensor Chip CM5(Series SSensor Chip CM5), the antibody was tested as a test substance. The antibody was immobilized on the sensor chip using the amino coupling kit according to the supplier's instruction manual to ensure a fixed amount of 500 RU. The running buffer used was 50mmol/l sodium phosphate/150 mmol/l NaCl (pH 6.0) containing 0.05% (v/v%) surfactant P20.
Using the prepared sensor chip, measurement was performed using 50mmol/l sodium phosphate/150 mmol/l NaCl (pH 6.0) containing 0.05% surfactant P20 or 50mmol/l sodium phosphate/150 mmol/l NaCl (pH 7.4) containing 0.05% surfactant P20 as a running buffer. The measurement was carried out exclusively at 25 ℃. Diluted human FcRn solution and running buffer as reference solution were injected at a flow rate of 5 μ l/min for 10 min to allow human FcRn to interact with the antibody on the chip. Next, the running buffer was injected at a flow rate of 5 μ l/min for 1 min to monitor the dissociation of FcRn. Then, the sensor chip was regenerated by injecting two 20mmol/l Tris-HCl/150mmol/l NaCl (pH 8.1) at a flow rate of 30. mu.l/min for 15 seconds.
The assay results were analyzed using Biacore T100 evaluation software (version 2.0.1). The dissociation constant (KD) was calculated from the measurements of 6 different FcRn concentrations by the steady-state affinity method. The results of the human FcRn binding activity (dissociation constant (KD)) of Fv4-IgG1, Fv4-IgG1-v1 and Fv4-IgG1-v2 at pH 6.0 and pH 7.4 are shown in Table 5 below.
[ Table 5]
At pH 7.4, human FcRn bound too weakly to Fv4-IgG1 to detect the KD value (NA). Meanwhile, binding of Fv4-IgG1-v1 and Fv4-IgG1-v2 to human FcRn was observed at pH 7.4, and the measured KD values were 36.55 and 11.03 micromoles, respectively. KD values for human FcRn at pH 6.0 were determined to be 1.99, 0.32 and 0.11 micromolar. As shown in FIG. 3, Fv4-IgG1-v2 accelerated the elimination of hsIL-6R in human FcRn transgenic mice when compared to Fv4-IgG 1. Thus, accelerated antigen elimination can be predicted by altering human IgG1 to increase human FcRn binding at pH 7.4 by at least 11.03 micromolar. Meanwhile, as in J Immunol. (2002)169 (9): 5171-80 human IgG1 bound about 10-fold more to mouse FcRn than to human FcRn. To this end, Fv4-IgG1-v1 and Fv4-IgG1-v2 were also predicted to bind to mouse FcRn about 10-fold more than human FcRn at pH 7.4. The acceleration of hsIL-6R elimination by either Fv4-IgG1-v1 or Fv4-IgG1-v2 in normal mice shown in FIG. 6 was more pronounced than that by Fv4-IgG1-v2 in human FcRn transgenic mice shown in FIG. 4. This indicates an increase in the acceleration of hsIL-6R elimination depending on the strength of FcRn binding at pH 7.4.
EXAMPLE 5 preparation of pH-dependent human IL-6 receptor-binding antibodies with increased human FcRn binding under neutral conditions
Various alterations that increase human FcRn binding under neutral conditions were introduced into Fv4-IgG1 to further increase the antigen elimination of pH-dependent human IL-6 receptor binding antibodies in human FcRn transgenic mice. Specifically, the amino acid changes shown in tables 6-1 and 6-2 were introduced into the heavy chain constant region of Fv4-IgG1 to generate various mutants (amino acid numbering of the mutation site is provided by EU numbering). Amino acid substitutions were introduced by methods known to those skilled in the art as described in reference example 1.
[ Table 6-1]
| Mutant name | KD(M) | Amino acid changes |
| IgG1 | ND | NONE |
| IgG1-v1 | 3.2E-06 | M252Y/S254T/T256E |
| IgG1-v2 | 8.1E-07 | N434W |
| IgG1-F3 | 2.5E-06 | N434Y |
| IgG1-F4 | 5.8E-06 | N434S |
| IgG1-F5 | 6.8E-06 | N434A |
| IgG1-F7 | 5.6E-06 | M252Y |
| IgG1-F8 | 4.2E-06 | M252W |
| IgG1-F9 | 1.4E-07 | M252Y/S254T/T256E/N434Y |
| IgG1-F10 | 6.9E-08 | M252Y/S254T/T256E/N434W |
| IgG1-F11 | 3.1E-07 | M252Y/N434Y |
| IgG1-F12 | 1.7E-07 | M252Y/N434W |
| IgG1-F13 | 3.2E-07 | M252W/N434Y |
| IgG1-F14 | 1.8E-07 | M252W/N434W |
| IgG1-F19 | 4.6E-07 | P257L/N434Y |
| IgG1-F20 | 4.6E-07 | V308F/N434Y |
| IgG1-F21 | 3.0E-08 | M252Y/V308P/N434Y |
| IgG1-F22 | 2.0E-06 | M428L/N434S |
| IgG1-F25 | 9.2E-09 | M252Y/S254T/T256E/V308P/N434W |
| IgG1-F26 | 1.0E-06 | I332V |
| IgG1-F27 | 7.4E-06 | G237M |
| IgG1-F29 | 1.4E-06 | I332V/N434Y |
| IgG1-F31 | 2.8E-06 | G237M/V308F |
| IgG1-F32 | 8.0E-07 | S254T/N434W |
| IgG1-F33 | 2.3E-06 | S254T/N434Y |
| IgG1-F34 | 2.8E-07 | T256E/N434W |
| IgG1-F35 | 8.4E-07 | T256E/N434Y |
| IgG1-F36 | 3.6E-07 | S254T/T256E/N434W |
| IgG1-F37 | 1.1E-06 | S254T/T256E/N434Y |
| IgG1-F38 | 1.0E-07 | M252Y/S254T/N434W |
| IgG1-F39 | 3.0E-07 | M252Y/S254T/N434Y |
| IgG1-F40 | 8.2E-08 | M252Y/T256E/N434W |
| IgG1-F41 | 1.5E-07 | M252Y/T256E/N434Y |
| IgG1-F42 | 1.0E-06 | M252Y/S254T/T256E/N434A |
| IgG1-F43 | 1.7E-06 | M252Y/N434A |
| IgG1-F44 | 1.1E-06 | M252W/N434A |
| IgG1-F47 | 2.4E-07 | M252Y/T256Q/N434W |
| IgG1-F48 | 3.2E-07 | M252Y/T256Q/N434Y |
| IgG1-F49 | 5.1E-07 | M252F/T256D/N434W |
| IgG1-F50 | 1.2E-06 | M252F/T256D/N434Y |
| IgG1-F51 | 8.1E-06 | N434F/Y436H |
| IgG1-F52 | 3.1E-06 | H433K/N434F/Y436H |
| IgG1-F53 | 1.0E-06 | I332V/N434W |
| IgG1-F54 | 8.4E-08 | V308P/N434W |
| IgG1-F56 | 9.4E-07 | I332V/M428L/N434Y |
| IgG1-F57 | 1.1E-05 | G385D/Q386P/N389S |
| IgG1-F58 | 7.7E-07 | G385D/Q386P/N389S/N434W |
| IgG1-F59 | 2.4E-06 | G385D/Q386P/N389S/N434Y |
| IgG1-F60 | 1.1E-05 | G385H |
| IgG1-F61 | 9.7E-07 | G385H/N434W |
| IgG1-F62 | 1.9E-06 | G385H/N434Y |
| IgG1-F63 | 2.5E-06 | N434F |
| IgG1-F64 | 5.3E-06 | N434H |
Table 6-2 is a continuation of Table 6-1.
[ tables 6-2]
| IgG1-F65 | 2.9E-07 | M252Y/S254T/T256E/N434F |
| IgG1-F66 | 4.3E-07 | M252Y/S254T/T256E/N434H |
| IgG1-F67 | 6.3E-07 | M252Y/N434F |
| IgG1-F68 | 9.3E-07 | M252Y/N434H |
| IgG1-F69 | 5.1E-07 | M428L/N434W |
| IgG1-F70 | 1.5E-06 | M428L/N434Y |
| IgG1-F71 | 8.3E-08 | M252Y/S254T/T256E/M428L/N434W |
| IgG1-F72 | 2.0E-07 | M252Y/S254T/T256E/M428L/N434Y |
| IgG1-F73 | 1.7E-07 | M252Y/M428L/N434W |
| IgG1-F74 | 4.6E-07 | M252Y/M428L/N434Y |
| IgG1-F75 | 1.4E-06 | M252Y/M428L/N434A |
| IgG1-F76 | 1.0E-06 | M252Y/S254T/T256E/M428L/N434A |
| IgG1-F77 | 9.9E-07 | T256E/M428L/N434Y |
| IgG1-F78 | 7.8E-07 | S254T/M428L/N434W |
| IgG1-F79 | 5.9E-06 | S254T/T256E/N434A |
| IgG1-F80 | 2.7E-06 | M252Y/T256Q/N434A |
| IgG1-F81 | 1.6E-06 | M252Y/T256E/N434A |
| IgG1-F82 | 1.1E-06 | T256Q/N434W |
| IgG1-F83 | 2.6E-06 | T256Q/N434Y |
| IgG1-F84 | 2.8E-07 | M252W/T256Q/N434W |
| IgG1-F85 | 5.5E-07 | M252W/T256Q/N434Y |
| IgG1-F86 | 1.5E-06 | S254T/T256Q/N434W |
| IgG1-F87 | 4.3E-06 | S254T/T256Q/N434Y |
| IgG1-F88 | 1.9E-07 | M252Y/S254T/T256Q/N434W |
| IgG1-F89 | 3.6E-07 | M252Y/S254T/T256Q/N434Y |
| IgG1-F90 | 1.9E-08 | M252Y/T256E/V308P/N434W |
| IgG1-F91 | 4.8E-08 | M252Y/V308P/M428L/N434Y |
| IgG1-F92 | 1.1E-08 | M252Y/S254T/T256E/V308P/M428L/N434W |
| IgG1-F93 | 7.4E-07 | M252W/M428L/N434W |
| IgG1-F94 | 3.7E-07 | P257L/M428L/N434Y |
| IgG1-F95 | 2.6E-07 | M252Y/S254T/T256E/M428L/N434F |
| IgG1-F99 | 6.2E-07 | M252Y/T256E/N434H |
The variants each comprising the produced heavy chain and L (WT) (SEQ ID NO: 5) were expressed and purified by methods known to those skilled in the art as described in reference example 2.
Evaluation of human FcRn binding
Binding between the antibody and human FcRn was kinetically analyzed using Biacore T100(GE Healthcare). For this purpose, human FcRn was prepared as described in reference example 4. An appropriate amount of L protein (ACTIGEN) was immobilized on a sensor chip CM4(GEHealthcare) by an amino-coupling method, and the target antibody was captured by the chip. Then, diluted FcRn solution and running buffer (as reference solution) were injected to let human FcRn interact with the antibody captured on the sensor chip. The running buffer used contained 50mmol/l sodium phosphate, 150mmol/l NaCl and 0.05% (w/v) Tween 20(pH 7.0). FcRn was diluted with each buffer. The chips were regenerated using 10mmol/l glycine-HCl (pH 1.5). The measurement was carried out exclusively at 25 ℃. Calculating the association rate constant ka (1/Ms) and the dissociation rate constant k from sensorgrams obtained in the assay d(1/s), both kinetic parameters, from which the kd (m) for human FcRn was determined for each antibody. The parameters were calculated using Biacore T100 evaluation software (GE Healthcare).
The results of the evaluation by Biacore on human FcRn binding under neutral conditions (pH 7.0) are shown in tables 6-1 and 6-2. KD for intact IgG1 could not be calculated because it only had very weak binding. Thus, KD is denoted as ND in Table 6-1.
EXAMPLE 6 in vivo assay of pH-dependent human IL-6 receptor-binding antibodies with increased human FcRn binding under neutral conditions
Using heavy chains with human FcRn binding capacity under neutral conditions prepared as described in example 4, pH-dependent human IL-6 receptor binding antibodies with human FcRn binding capacity under neutral conditions were generated. Antibodies were evaluated for their antigen-elimination in vivo. Specifically, the following antibodies were expressed and purified by methods known to those skilled in the art as described in reference example 2:
fv4-IgG1 comprising VH3-IgG1 and VL 3-CK;
fv4-IgG1-v2 comprising VH3-IgG1-v2 and VL 3-CK;
fv4-IgG1-F14 comprising VH3-IgG1-F14 and VL 3-CK;
fv4-IgG1-F20 comprising VH3-IgG1-F20 and VL 3-CK;
fv4-IgG1-F21 comprising VH3-IgG1-F21 and VL 3-CK;
Fv4-IgG1-F25 comprising VH3-IgG1-F25 and VL 3-CK;
fv4-IgG1-F29 comprising VH3-IgG1-F29 and VL 3-CK;
fv4-IgG1-F35 comprising VH3-IgG1-F35 and VL 3-CK;
fv4-IgG1-F48 comprising VH3-IgG1-F48 and VL 3-CK;
fv4-IgG1-F93 comprising VH3-IgG1-F93 and VL 3-CK; and
fv4-IgG1-F94 comprising VH3-IgG1-F94 and VL 3-CK.
The prepared pH-dependent human IL-6 receptor binding antibodies were tested in vivo by the same method described in example 3 using human FcRn transgenic mice (B6.mFcRn-/-. hFcRn Tg strain 276+/+ mice, Jackson Laboratories; Methods Mol Biol. (2010) 602: 93-104).
The time course of soluble human IL-6 receptor plasma concentrations following intravenous administration to human FcRn transgenic mice is shown in FIG. 8. The results of the assay show that plasma concentrations of soluble human IL-6 receptor remain low over time in the presence of any of the pH-dependent human IL-6 receptor binding antibodies with increased human FcRn binding under neutral conditions, compared to the presence of Fv4-IgG1 with little human FcRn binding capacity under neutral conditions. Among them, antibodies that produce significant effects include, for example, Fv4-IgG 1-F14. Plasma concentrations of soluble human IL-6 receptor administered concurrently with Fv4-IgG1-F14 showed an approximately 54-fold decrease 1 day after administration compared to plasma concentrations of soluble human IL-6 receptor administered concurrently with Fv4-IgG 1. Furthermore, the plasma concentration of soluble human IL-6 receptor administered concurrently with Fv4-IgG1-F21 showed an approximately 24-fold decrease at 7 hours post-administration compared to the plasma concentration of soluble human IL-6 receptor administered concurrently with Fv4-IgG 1. Furthermore, the plasma concentration of soluble human IL-6 receptor administered simultaneously with Fv4-IgG1-F25 was below the detection limit (1.56ng/ml) 7 hours after administration. Thus, Fv4-IgG1-F25 is expected to be able to significantly reduce the concentration of soluble human IL-6 receptor by 200-fold or more relative to the concentration of soluble human IL-6 receptor administered concurrently with Fv4-IgG 1. The results of the above studies indicate that an increase in human FcRn binding under neutral conditions of pH-dependent antigen-binding antibodies is very effective for enhancing antigen elimination. Meanwhile, the type of amino acid change (which is introduced to enhance antigen elimination) that increases human FcRn binding under neutral conditions is not particularly limited; such changes include those shown in Table 6-1 and Table 6-2. It is predicted that by any introduced change, in vivo antigen elimination will be enhanced.
Furthermore, the plasma concentration of soluble human IL-6 receptor administered simultaneously with one of the 4 types of pH-dependent human IL-6 receptor-binding antibodies (Fv4-IgG1-F14, Fv4-IgG1-F21, Fv4-IgG1-F25, and Fv4-IgG1-F48) remains low over time compared to the plasma concentration of soluble human IL-6 receptor administered alone. Such pH-dependent human IL-6 receptor-binding antibodies can be administered to an organism in which the plasma concentration of soluble human IL-6 receptor remains constant (steady state) to maintain the plasma concentration of soluble human IL-6 receptor below the steady state concentration in plasma. In particular, the concentration of antigen in plasma in vivo can be reduced by administering such antibodies to the body.
EXAMPLE 7 evaluation of the efficacy of Low dose (0.01mg/kg) Fv4-IgG1-F14
A low dose (0.01mg/kg) of Fv4-IgG1-F14 prepared as described in example 6 was tested according to the same in vivo assay method as described in example 6. The results (see FIG. 9) were compared with those described in example 6, which were obtained by administering Fv4-IgG1 and Fv4-IgG1-F14 at 1 mg/kg.
The results showed that, although the plasma antibody concentration in the group administered with 0.01mg/kg Fv4-IgG1-F14 was about 1/100 (FIG. 10) of the group administered with 1mg/kg, the time course of the plasma concentration of the soluble human IL-6 receptor was comparable to each other. Furthermore, it was confirmed that the plasma concentration of the soluble human IL-6 receptor was reduced by about 3-fold in the group to which 0.01mg/kg of Fv4-IgG1-F14 was administered, compared with the group to which 1mg/kg of Fv4-IgG1 was administered. Furthermore, the plasma concentrations of soluble human IL-6 receptor were lower over time in both groups given at different doses in the presence of Fv4-IgG1-F14 when compared to the group given only soluble human IL-6 receptor.
The results of the study indicate that v4-IgG1-F14, which is generated by modification of Fv4-IgG1 to increase human FcRn binding under neutral conditions, is effective in reducing the plasma concentration of soluble human IL-6 receptor, even when administered at doses of 1/100, which is Fv4-IgG1 doses. In particular, when pH-dependent antigen-binding antibodies are modified to increase their FcRn binding capacity under neutral conditions, it is predicted that the antigen can be effectively eliminated even at lower doses.
Example 8 Steady-State model-based in vivo assay Using Normal mice
Evaluation of binding to mouse FcRn under neutral conditions
The following were all prepared as described in example 5: VH3/L (WT) -IgG1 comprising VH3-IgG1(SEQ ID NO: 6) and L (WT) (SEQ ID NO: 5), VH3/L (WT) -IgG1-v2 comprising VH3-IgG1-v2(SEQ ID NO: 9) and L (WT) (SEQ ID NO: 5), and VH3/L (WT) -IgG1-F20 comprising VH3-IgG1-F20(SEQ ID NO: 10) and L (WT) (SEQ ID NO: 5) were evaluated for mouse FcRn binding under neutral conditions (pH 7.4) by the following methods.
Binding between the antibody and mouse FcRn was kinetically analyzed using Biacore T100(GE Healthcare). Tong (Chinese character of 'tong')An appropriate amount of L protein (ACTIGEN) was immobilized on a sensor chip CM4(GE Healthcare) by an amino coupling method, and the target antibody was captured by the chip. Then, diluted FcRn solution and running buffer (as reference solution) were injected to allow the mouse FcRn to interact with the antibody captured on the sensor chip. The running buffer used contained 50mmol/l sodium phosphate, 150mmol/l NaCl and 0.05% (w/v) Tween 20(pH 7.4). FcRn was diluted with each buffer. The chips were regenerated using 10mmol/l glycine-HCl (pH 1.5). The measurement was carried out exclusively at 25 ℃. Calculating the association rate constant ka (1/Ms) and the dissociation rate constant k from sensorgrams obtained in the assay d(1/s), both kinetic parameters, and from these values the kd (m) for each antibody to mouse FcRn was determined. The parameters were calculated using Biacore T100 evaluation software (GE Healthcare).
The results are shown in table 7 (affinity of mouse FcRn at pH 7.4). VH3/l (wt) -IgG1 (IgG 1 in table 7), whose constant region has intact IgG1, showed only very weak binding to mouse FcRn. Therefore, KD cannot be calculated and is represented by ND in table 7. The assay results show that altered antibodies with increased human FcRn binding under neutral conditions also show increased binding to mouse FcRn under neutral conditions.
[ Table 7]
| KD(M) | |
| IgG1 | ND |
| IgG1-v2 | 1.04E-06 |
| IgG1-F20 | 1.17E-07 |
In vivo assay using normal mice with constant plasma concentration of soluble human IL-6 receptor
In vivo experiments were performed using H54/L28-IgG1, Fv4-IgG1, Fv4-IgG1-v2, and Fv4-IgG1-F20 prepared as described in examples 1 and 5, by the following methods.
In vivo infusion assay using normal mice
An infusion pump (MINI-OSMOTIC PUMMODEL 2004; alzet) containing a soluble human IL-6 receptor was implanted under the back skin of normal mice (C57BL/6J mice; Charles River Japan) to prepare model animals in which the plasma concentration of the soluble human IL-6 receptor was kept constant. Anti-human IL-6 receptor antibodies were administered to model animals to evaluate in vivo kinetics following administration of soluble human IL-6 receptor. Monoclonal anti-mouse CD4 antibody (R & D) was administered once at 20mg/kg to the tail vein to inhibit the production of neutralizing antibodies against the soluble human IL-6 receptor. Then, an infusion pump containing 92.8 microgram/ml of soluble human IL-6 receptor was implanted under the skin of the back of the mice. Anti-human IL-6 receptor antibody was administered to the tail vein once at 1mg/kg 3 days after implantation of the infusion pump. Blood was collected 15 minutes, 7 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 14 days, 21 days, and 28 days after administration of the anti-human IL-6 receptor antibody. Immediately, the collected blood was centrifuged at 15,000rpm and 4 ℃ for 15 minutes to separate plasma. The separated plasma was stored at-20 ℃ or below-20 ℃ in a refrigerator prior to the assay.
Determination of plasma concentration of anti-human IL-6 receptor antibody by ELISA
The procedure used was the same as described in example 3.
Determination of plasma hsIL-6R concentration by electrochemiluminescence assay
The procedure used was the same as described in example 1.
As shown in FIG. 11, when H54/L28-IgG1, a neutralizing antibody against soluble human IL-6 receptor, was administered to normal mice (hsIL-6R group) in which the plasma concentration of soluble human IL-6 receptor was kept constant at about 40ng/ml, the plasma concentration of soluble human IL-6 receptor increased to 650ng/ml (15-fold before administration). On the other hand, in the group to which Fv4-IgG1 produced by imparting H54/L28-IgG1 pH-dependent antigen-binding ability was administered, the plasma concentration of soluble human IL-6 receptor was kept at about 70 ng/ml. This indicates that the increase in soluble human IL-6 receptor plasma concentration caused by the administration of H54/L28-IgG1 (a common neutralizing antibody) can be suppressed to about 1/10 by conferring pH-dependent binding capacity.
Furthermore, by administering either Fv-IgG1-v2 or Fv-IgG1-F20, both of which result from the introduction of an alteration to the pH-dependent human IL-6 receptor binding antibody to increase FcRn binding under neutral conditions, it was shown that plasma concentrations of soluble human IL-6 receptor remained 1/10 or less at steady state concentrations. When Fv-IgG1-v2 was administered, the plasma concentration of soluble human IL-6 receptor was about 2ng/ml at 14 days post-administration. Thus, Fv-IgG1-v2 reduced the concentration to 1/20 at the pre-dose level. Meanwhile, when Fv-IgG1-F20 was administered, the plasma concentration of soluble human IL-6 receptor was below the detection limit (1.56ng/ml) at 7 hours, 1 day, 2 days, and 4 days after the administration. This indicates that Fv-IgG1-F20 reduced the concentration to 1/25 or below 1/25 at the pre-dose level.
The above-described results of the study show that by administering an antibody having both pH-dependent antigen-binding ability and FcRn-binding ability under neutral conditions to a model animal in which the plasma antigen concentration is kept constant, the plasma antigen concentration can be significantly reduced by increasing the plasma antigen elimination rate.
Typical antibodies such as H54/L28-IgG1 can only neutralize the effects of the target antigen by binding to the target antigen, increasing plasma antigen concentrations even worse. In contrast, it was found that an antibody having pH-dependent antigen binding ability and FcRn binding ability under neutral conditions is capable of not only neutralizing a target antigen but also reducing the plasma concentration of the target antigen. The effect of removing antigen from plasma can be expected to be more beneficial than neutralization. In addition, antigen removal may also act on target antigens that are not sufficiently efficient by neutralization alone.
Example 9 determination of the threshold value for binding affinity to human FcRn at neutral pH and the relationship between antigen Elimination and binding affinity to human FcRn at neutral pH required for improved antigen Elimination
Antibody preparation for in vivo studies
Fc variants of Fv4-IgG1 comprising VH3-IgG1(SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 7) with increased FcRn binding at neutral pH were generated. Specifically, VH3-M73(SEQ ID NO: 15) and VH3-IgG1-v1(SEQ ID NO: 8) were prepared. Amino acid substitutions were introduced by methods known to those skilled in the art as described in reference example 1.
By referring to the methods known to those skilled in the art described in example 2, Fv4-IgG 869 comprising VH3-IgG1(SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 7), Fv 8672-M3 comprising VH3-M73(SEQ ID NO: 15) and VL3-CK (SEQ ID NO: 7), Fv 3-IgG 3-v 3 comprising VH3-IgG 3-v 3(SEQ ID NO: 8) and VL3-CK (SEQ ID NO: 7), and IgG 3-v 3 comprising VH3-IgG 3-v 3(SEQ ID NO: 9) and VL3-CK (SEQ ID NO: 7) were expressed and purified.
Evaluation of binding affinity of antibody to human FcRn under neutral pH conditions
The following were all prepared as described in example 2: VH3/L (WT) -IgG1 comprising VH3-IgG1(SEQ ID NO: 6) and L (WT) (SEQ ID NO: 5), VH3/L (WT) -M73 comprising VH3-M73(SEQ ID NO: 15) and L (WT) (SEQ ID NO: 5), VH3/L (WT) -IgG1-v1 comprising VH3-IgG1-v1(SEQ ID NO: 8) and L (WT) (SEQ ID NO: 5), and VH3/L (WT) -IgG1-v2 comprising VH3-IgG1-v2(SEQ ID NO: 9) and L (WT) (SEQ ID NO: 5), and these were evaluated for human FcRnFcRn binding at neutral pH (pH 7.0).
The binding activity of VH3/L (WT) -IgG1-v1 and VH3/L (WT) -IgG1-v2 to human FcRn was measured using the method described in example 5. These antibodies were evaluated by the following method, since the binding activity to human FcRn could not be measured by the method described in example 5 due to the low binding activity of VH3/l (wt) -IgG1 and VH3/l (wt) -M73 to human FcRn. Binding between the antibody and human FcRn was kinetically analyzed using Biacore T100(GE Healthcare). An appropriate amount of L protein (ACTIGEN) was immobilized on a sensor chip CM4(GE Healthcare) by an amine coupling method, and the target antibody was captured by the chip. Then, diluted FcRn solution and running buffer as reference solution were injected to let human FcRn interact with the antibody captured on the sensor chip. The running buffer used contained 50mmol/l sodium phosphate, 150mmol/l NaCl and 0.05% (w/v) Tween 20(pH 7.0). FcRn was diluted with each buffer. The chips were regenerated using 10mmol/l glycine-HCl (pH 1.5). The measurement was carried out at 25 ℃.
Kd (m) for each antibody was derived from sensorgram data using Biacore T100 evaluation software (GE Healthcare) that simultaneously fits all curves for the association and dissociation phases of the sensorgram and the ensemble of the working set. Sensorgrams were fitted to a 1: 1 binding model ("Langmuir binding" model provided by Biacore T100 evaluation software). For certain binding interactions, a equilibrium-based approach is employed, via ReqNonlinear regression analysis of the curve, balancing the logarithm of the binding reaction versus the analyte concentration yields the KD.
The results obtained by Biacore for human FcRn binding under neutral (pH 7.0) conditions are shown in table 8.
[ Table 8]
| KD(M) | |
| IgG1 | 8.8E-05 |
| M73 | 1.4E-05 |
| IgG1-v1 | 3.2E-06 |
| IgG1-v2 | 8.1E-07 |
Antibody-to-antigen elimination in a co-injection model using human FcRn transgenic mouse strain 276
In vivo study of effects
In vivo studies of antibodies were performed using the coinjection model as described in example 3. The anti-human IL-6 receptor antibodies used in this study were H54/L28-IgG1, Fv4-IgG1, Fv4-M73, Fv4-IgG1-v1, and Fv4-IgG1-v2, as described above. The mice used in this study were human FcRn transgenic mice (B6.mFcRn-/-. hFcRn Tg strain 276+/+ mice, Jackson laboratories; Methods Mol Biol. (2010) 602: 93-104).
As shown in FIG. 12, the pharmacokinetics of H54/L28-IgG1, Fv4-IgG1, Fv4-M73, Fv4-IgG1-v1, and Fv4-IgG1-v2 were comparable, and these antibodies maintained similar plasma concentrations during the study.
The time course of plasma hsIL-6R concentration is shown in FIG. 13. hsIL-6R administered with Fv4-IgG1-v2 showed increased clearance compared to hsIL-6R administered with Fv4-IgG1, while hsIL-6R administered with Fv4-M73 and Fv4-IgG1-v1 showed decreased clearance. Although all Fc variants M73, v1 and v2 showed increased binding affinity for human FcRn under neutral pH conditions (pH 7.0), only Fv4-IgG1-v2, but not Fv4-M73 and Fv4-IgG1-v1, was shown to show increased hsIL-6R clearance. This indicates that, to increase antigen clearance, the binding affinity of the antibody to human FcRn at pH7.0 must be at least stronger than IgG1-v1, which has KD 3.2 micromolar binding affinity to human FcRn or 28-fold greater affinity to intact human IgG1 (KD 88 micromolar binding affinity to human FcRn) at pH 7.0.
FIG. 14 depicts the relationship between binding affinity of Fc variants to human FcRn and plasma hsIL-6R concentration at pH7.0 on day 1 after co-injection of hsIL-6R and Fc variants. The Fc variants described in this example and example 6 (Fv4-IgG1, Fv4-M73, Fv4-IgG1-v1, Fv4-IgG1-v2, Fv4-IgG1-F14, Fv4-IgG1-F20, Fv4-IgG1-F21, Fv4-IgG1-F25, Fv4-IgG1-F29, Fv4-IgG1-F35, Fv4-IgG1-F48, Fv4-IgG1-F93, and Fv4-IgG 1-94) were plotted. By increasing the binding affinity of the antibody to human FcRn at ph7.0, the plasma concentration of hsIL-6R, which reflects antigen clearance, initially increases and then decreases rapidly. This indicates that in order to increase antigen clearance compared to fully human IgG1, the binding affinity of the antibody to human FcRn at pH7.0 must preferably be stronger than KD 2.3 micromolar (the value obtained from the curve fit of fig. 14). Binding affinity of antibodies between KD 88 micromolar and KD 2.3 micromolar to human FcRn reduces antigen clearance (higher hsIL-6R concentrations). In other words, the binding affinity of the antibody to human FcRn at pH7.0 must preferably be 38 times that of intact human IgG1 to improve antigen elimination, otherwise antigen clearance may be reduced.
FIG. 15 depicts the relationship between binding affinity of Fc variants to human FcRn and plasma antibody concentration at pH 7.0 on day 1 after co-injection of hsIL-6R and Fc variants. The Fc variants described in this example and example 6 (Fv4-IgG1, Fv4-M73, Fv4-IgG1-v1, Fv4-IgG1-v2, Fv4-IgG1-F14, Fv4-IgG1-F20, Fv4-IgG1-F21, Fv4-IgG1-F25, Fv4-IgG1-F29, Fv4-IgG1-F35, Fv4-IgG1-F48, Fv4-IgG1-F93, and Fv4-IgG 1-94) were plotted. By increasing the binding affinity of the antibody to human FcRn at pH 7.0, the plasma concentration of the antibody, which reflects the pharmacokinetics (clearance) of the antibody, is initially maintained, but then rapidly decreases. This indicates that to keep the pharmacokinetics of the antibody similar to that of intact human IgG1 (binding affinity for human FcRn of KD 88 micromolar), the affinity of the antibody for human FcRn at pH 7.0 must be weaker than KD 0.2 micromolar (values from the curve fit of fig. 15). Binding affinity of antibodies greater than KD 0.2 micromolar to human FcRn increases antibody clearance (i.e., antibody elimination from plasma is faster). In other words, the binding affinity of the antibody to human FcRn at pH 7.0 must be within 440-fold of that of intact human IgG1 to show similar antibody pharmacokinetics to intact human IgG1, which could otherwise result in rapid elimination of the antibody from the plasma.
For both fig. 14 and fig. 15, to improve antigen clearance (i.e. reduce antigen plasma concentration) compared to IgG1, while maintaining antibody pharmacokinetics similar to that of full human IgG1, the binding affinity of the antibody to human FcRn at pH 7.0 must be between 2.3 micromolar and 0.2 micromolar, or in other words, the binding affinity of the antibody to human FcRn at pH 7.0 must be in the range of between 38-fold and 440-fold that of full human IgG 1. Such antibodies with pharmacokinetics similar to IgG1, with long-term antigen elimination activity, may benefit antibody therapy (antibodyetherpeutic) requiring longer dosing intervals (e.g., chronic diseases) due to their long-lasting nature.
On the other hand, antigen clearance can be greatly improved in a short period by increasing the binding affinity of the antibody to human FcRn at pH 7.0 to be stronger than KD 0.2 micromolar, or in other words, by increasing the binding affinity of the antibody to human FcRn at pH 7.0 by more than 440-fold (compared to whole human IgG 1), although the elimination of the antibody from plasma is faster than that of whole human IgG 1. Such antibodies, which have the ability to induce a rapid and strong reduction in antigen concentration, may be beneficial for antibody therapy due to their fast-acting nature, e.g., acute diseases where disease-associated antigens must be removed from plasma.
The amount of antigen eliminated from plasma by each antibody is an important factor in evaluating the efficiency of antigen elimination by administration of an Fc variant of the antibody with increased binding affinity to human FcRn at pH 7.0. To evaluate the efficiency of antigen elimination for each antibody, the following calculations were performed at various time points of the in vivo studies described in this example and example 6.
A value: molar concentration of antigen at each time point
B value: antibody molarity at each time point
C value: molar concentration of antigen/molar concentration of antibody (antigen/molar ratio) at each time point
C＝A/B
The time course of the C value (antigen/antibody molar ratio) of each antibody is depicted in fig. 16. A smaller C value indicates a higher efficiency of eliminating antigen per antibody, and a higher C value indicates a lower efficiency of eliminating antigen per antibody. Lower C values compared to IgG1 indicate higher antigen elimination efficiency achieved by the Fc variant, while higher C values compared to IgG1 indicate that the Fc variant has a negative effect on antigen elimination efficiency. All Fc variants except Fv4-M73 and Fv4-IgG1-v1 showed improved antigen elimination efficiency compared to Fv4-IgG 1. Fv4-M73 and Fv4-IgG1-v1 showed a negative effect on antigen elimination efficiency, which is consistent with FIG. 14.
FIG. 17 depicts the relationship between binding affinity of Fc variants to human FcRn and C-value (antigen/antibody molar ratio) at pH 7.0 on day 1 after co-injection of hsIL-6R and Fc variants. The Fc variants described in this example and example 6 (Fv4-IgG1, Fv4-M73, Fv4-IgG1-v1, Fv4-IgG1-v2, Fv4-IgG1-F14, Fv4-IgG1-F20, Fv4-IgG1-F21, Fv4-IgG1-F25, Fv4-IgG1-F29, Fv4-IgG1-F35, Fv4-IgG1-F48, Fv4-IgG1-F93, and Fv4-IgG 1-94) were plotted. This indicates that in order to achieve higher antigen elimination efficiency compared to fully human IgG1, the affinity of the antibody to human FcRn at pH 7.0 must be stronger than KD 3.0 micromolar (the value obtained from the curve fit of fig. 17). In other words, the binding affinity of the antibody to human FcRn at pH 7.0 must be at least 29 times that of intact IgG1 to achieve higher antigen elimination efficiency compared to intact human IgG 1.
In summary, the group of antibody variants with binding affinities to FcRn at pH 7.0 between KD 3.0 micromolar and 0.2 micromolar, or in other words, within 29-fold to 440-fold of the binding affinity to FcRn at pH 7.0 of intact human IgG1, had similar antibody pharmacokinetics to IgG1, but increased ability to eliminate antibodies from plasma. Thus, such antibodies show improved antigen elimination efficiency compared to IgG 1. Pharmacokinetics similar to IgG1 will enable long-term antigen elimination from plasma (long-acting antigen elimination), so long dosing intervals may be preferred for antibody treatment of chronic diseases. The group of antibody variants with a binding affinity to FcRn at pH 7.0 that is stronger than KD 0.2 micromolar, or in other words, the group of antibody variants with a binding affinity to FcRn at pH 7.0 that is 440 times that of intact human IgG1, had rapid antibody clearance (short-term antibody elimination). However, because such antibodies are able to eliminate antigen even more rapidly (rapid antigen elimination), such antibodies also exhibit increased antigen elimination efficiency compared to IgG 1. As described in example 8, Fv4-IgG1-F20 induced massive elimination of antigen from plasma in a very short period of time in normal mice, but the antigen elimination was not long lasting. This feature may be preferred for acute diseases where the disease-associated antigen needs to be rapidly depleted from plasma in large amounts in a very short period of time.
EXAMPLE 10 in vivo study of Fv4-IgG1-F14 using human FcRn transgenic mouse strain 276 by steady state infusion model
In vivo studies using Fv4-IgG1-F14 of human FcRn transgenic mouse strain 276 via a steady state infusion model were performed as described in example 1. The study group consisted of: control (no antibody), Fv4-IgG1 at a dose of 1mg/kg, and Fv4-IgG1-F14 at doses of 1mg/kg, 0.2mg/kg, and 0.01 mg/kg.
FIG. 18 depicts a time profile of hsIL-6R plasma concentrations following administration of antibodies. Administration of 1mg/kg of Fv4-IgG1 resulted in a several-fold increase in plasma hsIL-6R concentration compared to the baseline hsIL-6R level without antibody. On the other hand, administration of 1mg/kg of Fv4-IgG1-F14 resulted in a significant decrease in plasma concentration compared to the Fv4-IgG1 group and the baseline group. On day 2, no plasma hsIL-6R concentration was detected (limit of quantitation for plasma hsIL-6R concentration in this measurement system was 1.56ng/mL), and this continued until day 14.
As described in example 1, H54/L28-IgG1-F14 showed a decrease in plasma hsIL-6R concentration, but to a lesser extent, compared to H54/L28-IgG 1. The degree of reduction was much higher for the Fv4 variable region, which has pH-dependent binding properties with hsIL-6R. This indicates that while increasing binding affinity to human FcRn at pH 7.0 effectively reduced plasma antigen concentration, the combination of pH-dependent antigen binding and increased binding affinity to human FcRn at neutral pH significantly increased antigen elimination.
Studies with lower doses of Fv4-IgG1-F14 showed that even at 0.01mg/kg (1/100 at 1 mg/kg) reduced antigen plasma concentrations below baseline, indicating a significant efficiency of the molecule in consuming antigen from plasma.
Example 11 comparison of human FcRn transgenic mouse lines 276 and 32 in a co-injection model
Previous in vivo studies have been performed using human FcRn transgenic mouse strain 276(Jackson Laboratories). To compare the differences between human FcRn transgenic mouse line 276 and the different transgenic line (line 32), we used human FcRn transgenic mouse line 32(b6.mfcrn-/-. hFcRn Tg line 32+/+ mice (b6.mfcrn-/-hFcRn Tg 32; b6.cg-Fcgrt < tm1Dcr > Tg (Fcgrt)32Dcr) (Jackson #4915)), Jackson laboratories; methods Mol Biol. (2010) 602: 93-104), co-injection studies of H54/L28-IgG1, Fv4-IgG1, and Fv4-IgG1-v2 were performed. The study procedure was the same as in example 3, except that human FcRn transgenic mouse line 32 was used instead of human FcRn transgenic mouse line 276.
Figure 19 depicts the time course of plasma hsIL-6R concentrations in both human FcRn transgenic mouse strain 276 and strain 32. H54/L28-IgG1, Fv4-IgG1, and Fv4-IgG1-v2 show similar temporal profiles of plasma hsIL-6R concentrations. In 2 mice, increasing binding affinity to human FcRn at pH 7.0 promotes antigen elimination from plasma (compare Fv4-IgG1 and Fv4-IgG1-v2) to the same extent.
Figure 20 depicts the time course of plasma antibody concentrations in both human FcRn transgenic mouse strain 276 and strain 32. H54/L28-IgG1, Fv4-IgG1, and Fv4-IgG1-v2 show similar temporal profiles of plasma antibody concentrations.
In summary, no significant difference was observed between line 276 and line 32, suggesting that Fc variants that increase binding affinity to human FcRn at pH 7.0 are effective in promoting elimination of antigen plasma concentrations in two different transgenic mouse lines expressing human FcRn.
Example 12 production of various antibody Fc variants with improved binding affinity to human FcRn at neutral pH
Production of Fc variants
Various mutations that increase binding affinity to human FcRn at neutral pH were introduced into Fv4-IgG1 to further improve antigen elimination characteristics. Specifically, the amino acid mutations shown in tables 9-1 to 9-14 were introduced into the heavy chain constant region of Fv4-IgG1 to generate Fc variants (amino acid numbering describing the mutation site in terms of EU numbering). Amino acid substitutions were introduced by methods known to those skilled in the art as described in reference example 1.
The prepared heavy chains and other variants (IgG1-F100 through IgG1-F599) of L (WT) (SEQ ID NO: 5) each were expressed and purified by methods known to those skilled in the art as described in reference example 2.
Evaluation of human FcRn binding
Kinetic analysis of binding between antibodies and human FcRn was performed as described in example 5 for IgG1-v1, IgG1-v2, and IgG1-F2 to IgG1-F599, or as described in example 9 for IgG1 and M73. The results obtained by Biacore for human FcRn binding under neutral conditions (pH 7.0) are shown in tables 9-1 to 9-14.
[ Table 9-1]
| Name of variants | KD(M) | Amino acid substitutions |
| IgG1 | 8.8E-05 | None |
| M73 | 1.4E-05 | (WO2009/125825) |
| IgG1-v1 | 3.2E-06 | M252Y/S254T/T256E |
| IgG1-v2 | 8.1E-07 | N434W |
| IgG1-F3 | 2.5E-06 | N434Y |
| IgG1-F4 | 5.8E-06 | N434S |
| IgG1-F5 | 6.8E-06 | N434A |
| IgG1-F7 | 5.6E-06 | M252Y |
| IgG1-F8 | 4.2E-06 | M252W |
| IgG1-F9 | 1.4E-07 | M252Y/S254T/T256E/N434Y |
| IgG1-F10 | 6.9E-08 | M252Y/S254T/T256E/N434W |
| IgG1-F11 | 3.1E-07 | M252Y/N434Y |
| IgG1-F12 | 1.7E-07 | M252Y/N434W |
| IgG1-F13 | 3.2E-07 | M252W/N434Y |
| IgG1-F14 | 1.8E-07 | M252W/N434W |
| IgG1-F19 | 4.6E-07 | P257L/N434Y |
| IgG1-F20 | 4.6E-07 | V308F/N434Y |
| IgG1-F21 | 3.0E-08 | M252Y/V308P/N434Y |
| IgG1-F22 | 2.0E-06 | M428L/N434S |
| IgG1-F25 | 9.2E-09 | M252Y/S254T/T256E/V308P/N434W |
| IgG1-F26 | 1.0E-06 | I332V |
| IgG1-F27 | 7.4E-06 | G237M |
| IgG1-F29 | 1.4E-06 | I332V/N434Y |
| IgG1-F31 | 2.8E-06 | G237M/V308F |
| IgG1-F32 | 8.0E-07 | S254T/N434W |
| IgG1-F33 | 2.3E-06 | S254T/N434Y |
| IgG1-F34 | 2.8E-07 | T256E/N434W |
| IgG1-F35 | 8.4E-07 | T256E/N434Y |
| IgG1-F36 | 3.6E-07 | S254T/T256E/N434W |
| IgG1-F37 | 1.1E-06 | S254T/T256E/N434Y |
| IgG1-F38 | 1.0E-07 | M252Y/S254T/N434W |
| IgG1-F39 | 3.0E-07 | M252Y/S254T/N434Y |
Table 9-2 is a continuation of Table 9-1.
[ tables 9-2]
| IgG1-F40 | 8.2E-08 | M252Y/T256E/N434W |
| IgG1-F41 | 1.5E-07 | M252Y/T256E/N434Y |
| IgG1-F42 | 1.0E-06 | M252Y/S254T/T256E/N434A |
| IgG1-F43 | 1.7E-06 | M252Y/N434A |
| IgG1-F44 | 1.1E-06 | M252W/N434A |
| IgG1-F47 | 2.4E-07 | M252Y/T256Q/N434W |
| IgG1-F48 | 3.2E-07 | M252Y/T256Q/N434Y |
| IgG1-F49 | 5.1E-07 | M252F/T256D/N434W |
| IgG1-F50 | 1.2E-06 | M252F/T256D/N434Y |
| IgG1-F51 | 8.1E-06 | N434F/Y436H |
| IgG1-F52 | 3.1E-06 | H433K/N434F/Y436H |
| IgG1-F53 | 1.0E-06 | I332V/N434W |
| IgG1-F54 | 8.4E-08 | V308P/N434W |
| IgG1-F56 | 9.4E-07 | I332V/M428L/N434Y |
| IgG1-F57 | 1.1E-05 | G385D/Q386P/N389S |
| IgG1-F58 | 7.7E-07 | G385D/Q386P/N389S/N434W |
| IgG1-F59 | 2.4E-06 | G385D/Q386P/N389S/N434Y |
| IgG1-F60 | 1.1E-05 | G385H |
| IgG1-F61 | 9.7E-07 | G385H/N434W |
| IgG1-F62 | 1.9E-06 | G385H/N434Y |
| IgG1-F63 | 2.5E-06 | N434F |
| IgG1-F64 | 5.3E-06 | N434H |
| IgG1-F65 | 2.9E-07 | M252Y/S254T/T256E/N434F |
| IgG1-F66 | 4.3E-07 | M252Y/S254T/T256E/N434H |
| IgG1-F67 | 6.3E-07 | M252Y/N434F |
| IgG1-F68 | 9.3E-07 | M252Y/N434H |
| IgG1-F69 | 5.1E-07 | M428L/N434W |
| IgG1-F70 | 1.5E-06 | M428L/N434Y |
| IgG1-F71 | 8.3E-08 | M252Y/S254T/T256E/M428L/N434W |
| IgG1-F72 | 2.0E-07 | M252Y/S254T/T256E/M428L/N434Y |
| IgG1-F73 | 1.7E-07 | M252Y/M428L/N434W |
| IgG1-F74 | 4.6E-07 | M252Y/M428L/N434Y |
| IgG1-F75 | 1.4E-06 | M252Y/M428L/N434A |
| IgG1-F76 | 1.0E-06 | M252Y/S254T/T256E/M428L/N434A |
| IgG1-F77 | 9.9E-07 | T256E/M428L/N434Y |
Table 9-3 is a continuation of Table 9-2.
[ tables 9 to 3]
| IgG1-F78 | 7.8E-07 | S254T/M428L/N434W |
| IgG1-F79 | 5.9E-06 | S254T/T256E/N434A |
| IgG1-F80 | 2.7E-06 | M252Y/T256Q/N434A |
| IgG1-F81 | 1.6E-06 | M252Y/T256E/N434A |
| IgG1-F82 | 1.1E-06 | T256Q/N434W |
| IgG1-F83 | 2.6E-06 | T256Q/N434Y |
| IgG1-F84 | 2.8E-07 | M252W/T256Q/N434W |
| IgG1-F85 | 5.5E-07 | M252W/T256Q/N434Y |
| IgG1-F86 | 1.5E-06 | S254T/T256Q/N434W |
| IgG1-F87 | 4.3E-06 | S254T/T256Q/N434Y |
| IgG1-F88 | 1.9E-07 | M252Y/S254T/T256Q/N434W |
| IgG1-F89 | 3.6E-07 | M252Y/S254T/T256Q/N434Y |
| IgG1-F90 | 1.9E-08 | M252Y/T256E/V308P/N434W |
| IgG1-F91 | 4.8E-08 | M252Y/V308P/M428L/N434Y |
| IgG1-F92 | 1.1E-08 | M252Y/S254T/T256E/V308P/M428L/N434W |
| IgG1-F93 | 7.4E-07 | M252W/M428L/N434W |
| IgG1-F94 | 3.7E-07 | P257L/M428L/N434Y |
| IgG1-F95 | 2.6E-07 | M252Y/S254T/T256E/M428L/N434F |
| IgG1-F99 | 6.2E-07 | M252Y/T256E/N434H |
| IgG1-F101 | 1.1E-07 | M252W/T256Q/P257L/N434Y |
| IgG1-F103 | 4.4E-08 | P238A/M252Y/V308P/N434Y |
| IgG1-F104 | 3.7E-08 | M252Y/D265A/V308P/N434Y |
| IgG1-F105 | 7.5E-08 | M252Y/T307A/V308P/N434Y |
| IgG1-F106 | 3.7E-08 | M252Y/V303A/V308P/N434Y |
| IgG1-F107 | 3.4E-08 | M252Y/V308P/D376A/N434Y |
| IgG1-F108 | 4.1E-08 | M252Y/V305A/V308P/N434Y |
| IgG1-F109 | 3.2E-08 | M252Y/V308P/Q311A/N434Y |
| IgG1-F111 | 3.2E-08 | M252Y/V308P/K317A/N434Y |
| IgG1-F112 | 6.4E-08 | M252Y/V308P/E380A/N434Y |
| IgG1-F113 | 3.2E-08 | M252Y/V308P/E382A/N434Y |
| IgG1-F114 | 3.8E-08 | M252Y/V308P/S424A/N434Y |
| IgG1-F115 | 6.6E-06 | T307A/N434A |
| IgG1-F116 | 8.7E-06 | E380A/N434A |
| IgG1-F118 | 1.4E-05 | M428L |
| IgG1-F119 | 5.4E-06 | T250Q/M428L |
Tables 9-4 are the continuation of tables 9-3.
[ tables 9 to 4]
| IgG1-F120 | 6.3E-08 | P257L/V308P/M428L/N434Y |
| IgG1-F121 | 1.5E-08 | M252Y/T256E/V308P/M428L/N434W |
| IgG1-F122 | 1.2E-07 | M252Y/T256E/M428L/N434W |
| IgG1-F123 | 3.0E-08 | M252Y/T256E/V308P/N434Y |
| IgG1-F124 | 2.9E-07 | M252Y/T256E/M428L/N434Y |
| IgG1-F125 | 2.4E-08 | M252Y/S254T/T256E/V308P/M428L/N434Y |
| IgG1-F128 | 1.7E-07 | P257L/M428L/N434W |
| IgG1-F129 | 2.2E-07 | P257A/M428L/N434Y |
| IgG1-F131 | 3.0E-06 | P257G/M428L/N434Y |
| IgG1-F132 | 2.1E-07 | P257I/M428L/N434Y |
| IgG1-F133 | 4.1E-07 | P257M/M428L/N434Y |
| IgG1-F134 | 2.7E-07 | P257N/M428L/N434Y |
| IgG1-F135 | 7.5E-07 | P257S/M428L/N434Y |
| IgG1-F136 | 3.8E-07 | P257T/M428L/N434Y |
| IgG1-F137 | 4.6E-07 | P257V/M428L/N434Y |
| IgG1-F139 | 1.5E-08 | M252W/V308P/N434W |
| IgG1-F140 | 3.6E-08 | S239K/M252Y/V308P/N434Y |
| IgG1-F141 | 3.5E-08 | M252Y/S298G/V308P/N434Y |
| IgG1-F142 | 3.7E-08 | M252Y/D270F/V308P/N434Y |
| IgG1-F143 | 2.0E-07 | M252Y/V308A/N434Y |
| IgG1-F145 | 5.3E-08 | M252Y/V308F/N434Y |
| IgG1-F147 | 2.4E-07 | M252Y/V308I/N434Y |
| IgG1-F149 | 1.9E-07 | M252Y/V308L/N434Y |
| IgG1-F150 | 2.0E-07 | M252Y/V308M/N434Y |
| IgG1-F152 | 2.7E-07 | M252Y/V308Q/N434Y |
| IgG1-F154 | 1.8E-07 | M252Y/V308T/N434Y |
| IgG1-F157 | 1.5E-07 | P257A/V308P/M428L/N434Y |
| IgG1-F158 | 5.9E-08 | P257T/V308P/M428L/N434Y |
| IgG1-F159 | 4.4E-08 | P257V/V308P/M428L/N434Y |
| IgG1-F160 | 8.5E-07 | M252W/M428I/N434Y |
| IgG1-F162 | 1.7E-07 | M252W/M428Y/N434Y |
| IgG1-F163 | 3.5E-07 | M252W/M428F/N434Y |
| IgG1-F164 | 3.7E-07 | P238A/M252W/N434Y |
| IgG1-F165 | 2.9E-07 | M252W/D265A/N434Y |
| IgG1-F166 | 1.5E-07 | M252W/T307Q/N434Y |
Tables 9-5 are continuation of tables 9-4.
[ tables 9 to 5]
| IgG1-F167 | 2.9E-07 | M252W/V303A/N434Y |
| IgG1-F168 | 3.2E-07 | M252W/D376A/N434Y |
| IgG1-F169 | 2.9E-07 | M252W/V305A/N434Y |
| IgG1-F170 | 1.7E-07 | M252W/Q311A/N434Y |
| IgG1-F171 | 1.9E-07 | M252W/D312A/N434Y |
| IgG1-F172 | 2.2E-07 | M252W/K317A/N434Y |
| IgG1-F173 | 7.7E-07 | M252W/E380A/N434Y |
| IgG1-F174 | 3.4E-07 | M252W/E382A/N434Y |
| IgG1-F175 | 2.7E-07 | M252W/S424A/N434Y |
| IgG1-F176 | 2.9E-07 | S239K/M252W/N434Y |
| IgG1-F177 | 2.8E-07 | M252W/S298G/N434Y |
| IgG1-F178 | 2.7E-07 | M252W/D270F/N434Y |
| IgG1-F179 | 3.1E-07 | M252W/N325G/N434Y |
| IgG1-F182 | 6.6E-08 | P257A/M428L/N434W |
| IgG1-F183 | 2.2E-07 | P257T/M428L/N434W |
| IgG1-F184 | 2.7E-07 | P257V/M428L/N434W |
| IgG1-F185 | 2.6E-07 | M252W/I332V/N434Y |
| IgG1-F188 | 3.0E-06 | P257I/Q311I |
| IgG1-F189 | 1.9E-07 | M252Y/T307A/N434Y |
| IgG1-F190 | 1.1E-07 | M252Y/T307Q/N434Y |
| IgG1-F191 | 1.6E-07 | P257L/T307A/M428L/N434Y |
| IgG1-F192 | 1.1E-07 | P257A/T307A/M428L/N434Y |
| IgG1-F193 | 8.5E-08 | P257T/T307A/M428L/N434Y |
| IgG1-F194 | 1.2E-07 | P257V/T307A/M428L/N434Y |
| IgG1-F195 | 5.6E-08 | P257L/T307Q/M428L/N434Y |
| IgG1-F196 | 3.5E-08 | P257A/T307Q/M428L/N434Y |
| IgG1-F197 | 3.3E-08 | P257T/T307Q/M428L/N434Y |
| IgG1-F198 | 4.8E-08 | P257V/T307Q/M428L/N434Y |
| IgG1-F201 | 2.1E-07 | M252Y/T307D/N434Y |
| IgG1-F203 | 2.4E-07 | M252Y/T307F/N434Y |
| IgG1-F204 | 2.1E-07 | M252Y/T307G/N434Y |
| IgG1-F205 | 2.0E-07 | M252Y/T307H/N434Y |
| IgG1-F206 | 2.3E-07 | M252Y/T307I/N434Y |
| IgG1-F207 | 9.4E-07 | M252Y/T307K/N434Y |
| IgG1-F208 | 3.9E-07 | M252Y/T307L/N434Y |
Tables 9-6 are continuation of tables 9-5.
[ tables 9 to 6]
| IgG1-F209 | 1.3E-07 | M252Y/T307M/N434Y |
| IgG1-F210 | 2.9E-07 | M252Y/T307N/N434Y |
| IgG1-F211 | 2.4E-07 | M252Y/T307P/N434Y |
| IgG1-F212 | 6.8E-07 | M252Y/T307R/N434Y |
| IgG1-F213 | 2.3E-07 | M252Y/T307S/N434Y |
| IgG1-F214 | 1.7E-07 | M252Y/T307V/N434Y |
| IgG1-F215 | 9.6E-08 | M252Y/T307W/N434Y |
| IgG1-F216 | 2.3E-07 | M252Y/T307Y/N434Y |
| IgG1-F217 | 2.3E-07 | M252Y/K334L/N434Y |
| IgG1-F218 | 2.6E-07 | M252Y/G385H/N434Y |
| IgG1-F219 | 2.5E-07 | M252Y/T289H/N434Y |
| IgG1-F220 | 2.5E-07 | M252Y/Q311H/N434Y |
| IgG1-F221 | 3.1E-07 | M252Y/D312H/N434Y |
| IgG1-F222 | 3.4E-07 | M252Y/N315H/N434Y |
| IgG1-F223 | 2.7E-07 | M252Y/K360H/N434Y |
| IgG1-F225 | 1.5E-06 | M252Y/L314R/N434Y |
| IgG1-F226 | 5.4E-07 | M252Y/L314K/N434Y |
| IgG1-F227 | 1.2E-07 | M252Y/N286E/N434Y |
| IgG1-F228 | 2.3E-07 | M252Y/L309E/N434Y |
| IgG1-F229 | 5.1E-07 | M252Y/R255E/N434Y |
| IgG1-F230 | 2.5E-07 | M252Y/P387E/N434Y |
| IgG1-F236 | 8.9E-07 | K248I/M428L/N434Y |
| IgG1-F237 | 2.3E-07 | M252Y/M428A/N434Y |
| IgG1-F238 | 7.4E-07 | M252Y/M428D/N434Y |
| IgG1-F240 | 7.2E-07 | M252Y/M428F/N434Y |
| IgG1-F241 | 1.5E-06 | M252Y/M428G/N434Y |
| IgG1-F242 | 8.5E-07 | M252Y/M428H/N434Y |
| IgG1-F243 | 1.8E-07 | M252Y/M428I/N434Y |
| IgG1-F244 | 1.3E-06 | M252Y/M428K/N434Y |
| IgG1-F245 | 4.7E-07 | M252Y/M428N/N434Y |
| IgG1-F246 | 1.1E-06 | M252Y/M428P/N434Y |
| IgG1-F247 | 4.4E-07 | M252Y/M428Q/N434Y |
| IgG1-F249 | 6.4E-07 | M252Y/M428S/N434Y |
| IgG1-F250 | 2.9E-07 | M252Y/M428T/N434Y |
| IgG1-F251 | 1.9E-07 | M252Y/M428V/N434Y |
Tables 9-7 are continuation of tables 9-6.
[ tables 9 to 7]
| IgG1-F252 | 1.0E-06 | M252Y/M428W/N434Y |
| IgG1-F253 | 7.1E-07 | M252Y/M428Y/N434Y |
| IgG1-F254 | 7.5E-08 | M252W/T307Q/M428Y/N434Y |
| IgG1-F255 | 1.1E-07 | M252W/Q311A/M428Y/N434Y |
| IgG1-F256 | 5.4E-08 | M252W/T307Q/Q311A/M428Y/N434Y |
| IgG1-F257 | 5.0E-07 | M252Y/T307A/M428Y/N434Y |
| IgG1-F258 | 3.2E-07 | M252Y/T307Q/M428Y/N434Y |
| IgG1-F259 | 2.8E-07 | M252Y/D270F/N434Y |
| IgG1-F260 | 1.3E-07 | M252Y/T307A/Q311A/N434Y |
| IgG1-F261 | 8.4E-08 | M252Y/T307Q/Q311A/N434Y |
| IgG1-F262 | 1.9E-07 | M252Y/T307A/Q311H/N434Y |
| IgG1-F263 | 1.1E-07 | M252Y/T307Q/Q311H/N434Y |
| IgG1-F264 | 2.8E-07 | M252Y/E382A/N434Y |
| IgG1-F265 | 6.8E-07 | M252Y/E382A/M428Y/N434Y |
| IgG1-F266 | 4.7E-07 | M252Y/T307A/E382A/M428Y/N434Y |
| IgG1-F267 | 3.2E-07 | M252Y/T307Q/E382A/M428Y/N434Y |
| IgG1-F268 | 6.3E-07 | P238A/M252Y/M428F/N434Y |
| IgG1-F269 | 5.2E-07 | M252Y/V305A/M428F/N434Y |
| IgG1-F270 | 6.6E-07 | M252Y/N325G/M428F/N434Y |
| IgG1-F271 | 6.9E-07 | M252Y/D376A/M428F/N434Y |
| IgG1-F272 | 6.8E-07 | M252Y/E380A/M428F/N434Y |
| IgG1-F273 | 6.5E-07 | M252Y/E382A/M428F/N434Y |
| IgG1-F274 | 7.6E-07 | M252Y/E380A/E382A/M428F/N434Y |
| IgG1-F275 | 4.2E-08 | S239K/M252Y/V308P/E382A/N434Y |
| IgG1-F276 | 4.1E-08 | M252Y/D270F/V308P/E382A/N434Y |
| IgG1-F277 | 1.3E-07 | S239K/M252Y/V308P/M428Y/N434Y |
| IgG1-F278 | 3.0E-08 | M252Y/T307Q/V308P/E382A/N434Y |
| IgG1-F279 | 6.1E-08 | M252Y/V308P/Q311H/E382A/N434Y |
| IgG1-F280 | 4.1E-08 | S239K/M252Y/D270F/V308P/N434Y |
| IgG1-F281 | 9.2E-08 | M252Y/V308P/E382A/M428F/N434Y |
| IgG1-F282 | 2.9E-08 | M252Y/V308P/E382A/M428L/N434Y |
| IgG1-F283 | 1.0E-07 | M252Y/V308P/E382A/M428Y/N434Y |
| IgG1-F284 | 1.0E-07 | M252Y/V308P/M428Y/N434Y |
| IgG1-F285 | 9.9E-08 | M252Y/V308P/M428F/N434Y |
| IgG1-F286 | 1.2E-07 | S239K/M252Y/V308P/E382A/M428Y/N434Y |
Tables 9-8 are continuation of tables 9-7.
[ tables 9 to 8]
| IgG1-F287 | 1.0E-07 | M252Y/V308P/E380A/E382A/M428F/N434Y |
| IgG1-F288 | 1.9E-07 | M252Y/T256E/E382A/N434Y |
| IgG1-F289 | 4.8E-07 | M252Y/T256E/M428Y/N434Y |
| IgG1-F290 | 4.6E-07 | M252Y/T256E/E382A/M428Y/N434Y |
| IgG1-F292 | 2.0E-08 | S239K/M252Y/V308P/E382A/M428I/N434Y |
| IgG1-F293 | 5.3E-08 | M252Y/V308P/E380A/E382A/M428I/N434Y |
| IgG1-F294 | 1.1E-07 | S239K/M252Y/V308P/M428F/N434Y |
| IgG1-F295 | 6.8E-07 | S239K/M252Y/E380A/E382A/M428F/N434Y |
| IgG1-F296 | 4.9E-07 | M252Y/Q311A/M428Y/N434Y |
| IgG1-F297 | 5.1E-07 | M252Y/D312A/M428Y/N434Y |
| IgG1-F298 | 4.8E-07 | M252Y/Q311A/D312A/M428Y/N434Y |
| IgG1-F299 | 9.4E-08 | S239K/M252Y/V308P/Q311A/M428Y/N434Y |
| IgG1-F300 | 8.3E-08 | S239K/M252Y/V308P/D312A/M428Y/N434Y |
| IgG1-F301 | 7.2E-08 | S239K/M252Y/V308P/Q311A/D312A/M428Y/N434Y |
| IgG1-F302 | 1.9E-07 | M252Y/T256E/T307P/N434Y |
| IgG1-F303 | 6.7E-07 | M252Y/T307P/M428Y/N434Y |
| IgG1-F304 | 1.6E-08 | M252W/V308P/M428Y/N434Y |
| IgG1-F305 | 2.7E-08 | M252Y/T256E/V308P/E382A/N434Y |
| IgG1-F306 | 3.6E-08 | M252W/V308P/E382A/N434Y |
| IgG1-F307 | 3.6E-08 | S239K/M252W/V308P/E382A/N434Y |
| IgG1-F308 | 1.8E-08 | S239K/M252W/V308P/E382A/M428Y/N434Y |
| IgG1-F310 | 9.4E-08 | S239K/M252W/V308P/E382A/M428I/N434Y |
| IgG1-F311 | 2.9E-08 | S239K/M252W/V308P/M428F/N434Y |
| IgG1-F312 | 4.5E-07 | S239K/M252W/E380A/E382A/M428F/N434Y |
| IgG1-F313 | 6.5E-07 | S239K/M252Y/T307P/M428Y/N434Y |
| IgG1-F314 | 3.2E-07 | M252Y/T256E/Q311A/D312A/M428Y/N434Y |
| IgG1-F315 | 6.8E-07 | S239K/M252Y/M428Y/N434Y |
| IgG1-F316 | 7.0E-07 | S239K/M252Y/D270F/M428Y/N434Y |
| IgG1-F317 | 1.1E-07 | S239K/M252Y/D270F/V308P/M428Y/N434Y |
| IgG1-F318 | 1.8E-08 | S239K/M252Y/V308P/M428I/N434Y |
| IgG1-F320 | 2.0E-08 | S239K/M252Y/V308P/N325G/E382A/M428I/N434Y |
| IgG1-F321 | 3.2E-08 | S239K/M252Y/D270F/V308P/N325G/N434Y |
| IgG1-F322 | 9.2E-08 | S239K/M252Y/D270F/T307P/V308P/N434Y |
| IgG1-F323 | 2.7E-08 | S239K/M252Y/T256E/D270F/V308P/N434Y |
| IgG1-F324 | 2.8E-08 | S239K/M252Y/D270F/T307Q/V308P/N434Y |
Tables 9-9 are continuation of tables 9-8.
[ tables 9 to 9]
| IgG1-F325 | 2.1E-08 | S239K/M252Y/D270F/T307Q/V308P/Q311A/N434Y |
| IgG1-F326 | 7.5E-08 | S239K/M252Y/D270F/T307Q/Q311A/N434Y |
| IgG1-F327 | 6.5E-08 | S239K/M252Y/T256E/D270F/T307Q/Q311A/N434Y |
| IgG1-F328 | 1.9E-08 | S239K/M252Y/D270F/V308P/M428I/N434Y |
| IgG1-F329 | 1.2E-08 | S239K/M252Y/D270F/N286E/V308P/N434Y |
| IgG1-F330 | 3.6E-08 | S239K/M252Y/D270F/V308P/L309E/N434Y |
| IgG1-F331 | 3.0E-08 | S239K/M252Y/D270F/V308P/P387E/N434Y |
| IgG1-F333 | 7.4E-08 | S239K/M252Y/D270F/T307Q/L309E/Q311A/N434Y |
| IgG1-F334 | 1.9E-08 | S239K/M252Y/D270F/V308P/N325G/M428I/N434Y |
| IgG1-F335 | 1.5E-08 | S239K/M252Y/T256E/D270F/V308P/M428I/N434Y |
| IgG1-F336 | 1.4E-08 | S239K/M252Y/D270F/T307Q/V308P/Q311A/M428I/N434Y |
| IgG1-F337 | 5.6E-08 | S239K/M252Y/D270F/T307Q/Q311A/M428I/N434Y |
| IgG1-F338 | 7.7E-09 | S239K/M252Y/D270F/N286E/V308P/M428I/N434Y |
| IgG1-F339 | 1.9E-08 | S239K/M252Y/D270F/V308P/L309E/M428I/N434Y |
| IgG1-F343 | 3.2E-08 | S239K/M252Y/D270F/V308P/M428L/N434Y |
| IgG1-F344 | 3.0E-08 | S239K/M252Y/V308P/M428L/N434Y |
| IgG1-F349 | 1.5E-07 | S239K/M252Y/V308P/L309P/M428L/N434Y |
| IgG1-F350 | 1.7E-07 | S239K/M252Y/V308P/L309R/M428L/N434Y |
| IgG1-F352 | 6.0E-07 | S239K/M252Y/L309P/M428L/N434Y |
| IgG1-F353 | 1.1E-06 | S239K/M252Y/L309R/M428L/N434Y |
| IgG1-F354 | 2.8E-08 | S239K/M252Y/T307Q/V308P/M428L/N434Y |
| IgG1-F356 | 3.4E-08 | S239K/M252Y/D270F/V308P/L309E/P387E/N434Y |
| IgG1-F357 | 1.6E-08 | S239K/M252Y/T256E/D270F/V308P/N325G/M428I/N434Y |
| IgG1-F358 | 1.0E-07 | S239K/M252Y/T307Q/N434Y |
| IgG1-F359 | 4.2E-07 | P257V/T307Q/M428I/N434Y |
| IgG1-F360 | 1.3E-06 | P257V/T307Q/M428V/N434Y |
| IgG1-F362 | 5.4E-08 | P257V/T307Q/N325G/M428L/N434Y |
| IgG1-F363 | 4.1E-08 | P257V/T307Q/Q311A/M428L/N434Y |
| IgG1-F364 | 3.5E-08 | P257V/T307Q/Q311A/N325G/M428L/N434Y |
| IgG1-F365 | 5.1E-08 | P257V/V305A/T307Q/M428L/N434Y |
| IgG1-F367 | 1.5E-08 | S239K/M252Y/E258H/D270F/T307Q/V308P/Q311A/N434Y |
| IgG1-F368 | 2.0E-08 | S239K/M252Y/D270F/V308P/N325G/E382A/M428I/N434Y |
| IgG1-F369 | 7.5E-08 | M252Y/P257V/T307Q/M428I/N434Y |
| IgG1-F372 | 1.3E-08 | S239K/M252W/V308P/M428Y/N434Y |
| IgG1-F373 | 1.1E-08 | S239K/M252W/V308P/Q311A/M428Y/N434Y |
Tables 9-10 are continuation of tables 9-9.
[ tables 9 to 10]
Tables 9-11 are continuation of tables 9-10.
[ tables 9 to 11]
| IgG1-F412 | 8.8E-08 | P257V/T307G/M428L/N434Y |
| IgG1-F413 | 1.2E-07 | P257V/T307P/M428L/N434Y |
| IgG1-F414 | 1.1E-07 | P257V/T307S/M428L/N434Y |
| IgG1-F415 | 5.6E-08 | P257V/N286E/T307A/M428L/N434Y |
| IgG1-F416 | 9.4E-08 | P257V/T307A/P387E/M428L/N434Y |
| IgG1-F418 | 6.2E-07 | S239K/M252Y/T307P/N325G/M428Y/N434Y |
| IgG1-F419 | 1.6E-07 | M252Y/T307A/Q311H/K360H/N434Y |
| IgG1-F420 | 1.5E-07 | M252Y/T307A/Q311H/P387E/N434Y |
| IgG1-F421 | 1.3E-07 | M252Y/T307A/Q311H/M428A/N434Y |
| IgG1-F422 | 1.8E-07 | M252Y/T307A/Q311H/E382A/N434Y |
| IgG1-F423 | 8.4E-08 | M252Y/T307W/Q311H/N434Y |
| IgG1-F424 | 9.4E-08 | S239K/P257A/V308P/M428L/N434Y |
| IgG1-F425 | 8.0E-08 | P257A/V308P/L309E/M428L/N434Y |
| IgG1-F426 | 8.4E-08 | P257V/T307Q/N434Y |
| IgG1-F427 | 1.1E-07 | M252Y/P257V/T307Q/M428V/N434Y |
| IgG1-F428 | 8.0E-08 | M252Y/P257V/T307Q/M428L/N434Y |
| IgG1-F429 | 3.7E-08 | M252Y/P257V/T307Q/N434Y |
| IgG1-F430 | 8.1E-08 | M252Y/P257V/T307Q/M428Y/N434Y |
| IgG1-F431 | 6.5E-08 | M252Y/P257V/T307Q/M428F/N434Y |
| IgG1-F432 | 9.2E-07 | P257V/T307Q/Q311A/N325G/M428V/N434Y |
| IgG1-F433 | 6.0E-08 | P257V/T307Q/Q311A/N325G/N434Y |
| IgG1-F434 | 2.0E-08 | P257V/T307Q/Q311A/N325G/M428Y/N434Y |
| IgG1-F435 | 2.5E-08 | P257V/T307Q/Q311A/N325G/M428F/N434Y |
| IgG1-F436 | 2.5E-07 | P257A/T307Q/M428V/N434Y |
| IgG1-F437 | 5.7E-08 | P257A/T307Q/N434Y |
| IgG1-F438 | 3.6E-08 | P257A/T307Q/M428Y/N434Y |
| IgG1-F439 | 4.0E-08 | P257A/T307Q/M428F/N434Y |
| IgG1-F440 | 1.5E-08 | P257V/N286E/T307Q/Q311A/N325G/M428L/N434Y |
| IgG1-F441 | 1.8E-07 | P257A/Q311A/M428L/N434Y |
| IgG1-F442 | 2.0E-07 | P257A/Q311H/M428L/N434Y |
| IgG1-F443 | 5.5E-08 | P257A/T307Q/Q311A/M428L/N434Y |
| IgG1-F444 | 1.4E-07 | P257A/T307A/Q311A/M428L/N434Y |
| IgG1-F445 | 6.2E-08 | P257A/T307Q/Q311H/M428L/N434Y |
| IgG1-F446 | 1.1E-07 | P257A/T307A/Q311H/M428L/N434Y |
| IgG1-F447 | 1.4E-08 | P257A/N286E/T307Q/M428L/N434Y |
Tables 9-12 are continuation of tables 9-11.
[ tables 9 to 12]
| IgG1-F448 | 5.3E-08 | P257A/N286E/T307A/M428L/N434Y |
| IgG1-F449 | 5.7E-07 | S239K/M252Y/D270F/T307P/N325G/M428Y/N434Y |
| IgG1-F450 | 5.2E-07 | S239K/M252Y/T307P/L309E/N325G/M428Y/N434Y |
| IgG1-F451 | 1.0E-07 | P257S/T307A/M428L/N434Y |
| IgG1-F452 | 1.4E-07 | P257M/T307A/M428L/N434Y |
| IgG1-F453 | 7.8E-08 | P257N/T307A/M428L/N434Y |
| IgG1-F454 | 9.6E-08 | P257I/T307A/M428L/N434Y |
| IgG1-F455 | 2.5E-08 | P257V/T307Q/M428Y/N434Y |
| IgG1-F456 | 3.4E-08 | P257V/T307Q/M428F/N434Y |
| IgG1-F457 | 4.0E-08 | S239K/P257V/V308P/M428L/N434Y |
| IgG1-F458 | 1.5E-08 | P257V/T307Q/V308P/N325G/M428L/N434Y |
| IgG1-F459 | 1.3E-08 | P257V/T307Q/V308P/Q311A/N325G/M428L/N434Y |
| IgG1-F460 | 4.7E-08 | P257V/T307A/V308P/N325G/M428L/N434Y |
| IgG1-F462 | 8.5E-08 | P257A/V308P/N325G/M428L/N434Y |
| IgG1-F463 | 1.3E-07 | P257A/T307A/V308P/M428L/N434Y |
| IgG1-F464 | 5.5E-08 | P257A/T307Q/V308P/M428L/N434Y |
| IgG1-F465 | 2.1E-08 | P257V/N286E/T307Q/N325G/M428L/N434Y |
| IgG1-F466 | 3.5E-07 | T256E/P257V/N434Y |
| IgG1-F467 | 5.7E-07 | T256E/P257T/N434Y |
| IgG1-F468 | 5.7E-08 | S239K/P257T/V308P/M428L/N434Y |
| IgG1-F469 | 5.6E-08 | P257T/V308P/N325G/M428L/N434Y |
| IgG1-F470 | 5.4E-08 | T256E/P257T/V308P/N325G/M428L/N434Y |
| IgG1-F471 | 6.6E-08 | P257T/V308P/N325G/E382A/M428L/N434Y |
| IgG1-F472 | 5.4E-08 | P257T/V308P/N325G/P387E/M428L/N434Y |
| IgG1-F473 | 4.5E-07 | P257T/V308P/L309P/N325G/M428L/N434Y |
| IgG1-F474 | 3.5E-07 | P257T/V308P/L309R/N325G/M428L/N434Y |
| IgG1-F475 | 4.3E-08 | T256E/P257V/T307Q/M428L/N434Y |
| IgG1-F476 | 5.5E-08 | P257V/T307Q/E382A/M428L/N434Y |
| IgG1-F477 | 4.3E-08 | P257V/T307Q/P387E/M428L/N434Y |
| IgG1-F480 | 3.9E-08 | P257L/V308P/N434Y |
| IgG1-F481 | 5.6E-08 | P257T/T307Q/N434Y |
| IgG1-F482 | 7.0E-08 | P257V/T307Q/N325G/N434Y |
| IgG1-F483 | 5.7E-08 | P257V/T307Q/Q311A/N434Y |
| IgG1-F484 | 6.2E-08 | P257V/V305A/T307Q/N434Y |
| IgG1-F485 | 9.7E-08 | P257V/N286E/T307A/N434Y |
Tables 9-13 are continuation of tables 9-12.
[ tables 9 to 13]
| IgG1-F486 | 3.4E-07 | P257V/T307Q/L309R/Q311H/M428L/N434Y |
| IgG1-F488 | 3.5E-08 | P257V/V308P/N325G/M428L/N434Y |
| IgG1-F490 | 7.5E-08 | S239K/P257V/V308P/Q311H/M428L/N434Y |
| IgG1-F492 | 9.8E-08 | P257V/V305A/T307A/N325G/M428L/N434Y |
| IgG1-F493 | 4.9E-07 | S239K/D270F/T307P/N325G/M428Y/N434Y |
| IgG1-F497 | 3.1E-06 | P257T/T307A/M428V/N434Y |
| IgG1-F498 | 1.3E-06 | P257A/M428V/N434Y |
| IgG1-F499 | 5.2E-07 | P257A/T307A/M428V/N434Y |
| IgG1-F500 | 4.3E-08 | P257S/T307Q/M428L/N434Y |
| IgG1-F506 | 1.9E-07 | P257V/N297A/T307Q/M428L/N434Y |
| IgG1-F507 | 5.1E-08 | P257V/N286A/T307Q/M428L/N434Y |
| IgG1-F508 | 1.1E-07 | P257V/T307Q/N315A/M428L/N434Y |
| IgG1-F509 | 5.8E-08 | P257V/T307Q/N384A/M428L/N434Y |
| 1gG1-F510 | 5.3E-08 | P257V/T307Q/N389A/M428L/N434Y |
| IgG1-F511 | 4.2E-07 | P257V/N434Y |
| IgG1-F512 | 5.8E-07 | P257T/N434Y |
| IgG1-F517 | 3.1E-07 | P257V/N286E/N434Y |
| IgG1-F518 | 4.2E-07 | P257T/N286E/N434Y |
| IgG1-F519 | 2.6E-08 | P257V/N286E/T307Q/N434Y |
| IgG1-F521 | 1.1E-08 | P257V/N286E/T307Q/M428Y/N434Y |
| IgG1-F523 | 2.6E-08 | P257V/V305A/T307Q/M428Y/N434Y |
| IgG1-F526 | 1.9E-08 | P257T/T307Q/M428Y/N434Y |
| IgG1-F527 | 9.4E-09 | P257V/T307Q/V308P/N325G/M428Y/N434Y |
| IgG1-F529 | 2.5E-08 | P257T/T307Q/M428F/N434Y |
| IgG1-F533 | 1.2E-08 | P257A/N286E/T307Q/M428F/N434Y |
| IgG1-F534 | 1.2E-08 | P257A/N286E/T307Q/M428Y/N434Y |
| IgG1-F535 | 3.9E-08 | T250A/P257V/T307Q/M428L/N434Y |
| IgG1-F538 | 9.9E-08 | T250F/P257V/T307Q/M428L/N434Y |
| IgG1-F541 | 6.0E-08 | T250I/P257V/T307Q/M428L/N434Y |
| IgG1-F544 | 3.1E-08 | T250M/P257V/T307Q/M428L/N434Y |
| IgG1-F549 | 5.4E-08 | T250S/P257V/T307Q/M428L/N434Y |
| IgG1-F550 | 5.9E-08 | T250V/P257V/T307Q/M428L/N434Y |
| IgG1-F551 | 1.2E-07 | T250W/P257V/T307Q/M428L/N434Y |
| IgG1-F552 | 1.1E-07 | T250Y/P257V/T307Q/M428L/N434Y |
| IgG1-F553 | 1.7E-07 | M252Y/Q311A/N434Y |
Tables 9-14 are continuation of tables 9-13.
[ tables 9 to 14]
| IgG1-F554 | 2.8E-08 | S239K/M252Y/S254T/V308P/N434Y |
| IgG1-F556 | 1.5E-06 | M252Y/T307Q/Q311A |
| IgG1-F559 | 8.0E-08 | M252Y/S254T/N286E/N434Y |
| IgG1-F560 | 2.8E-08 | M252Y/S254T/V308P/N434Y |
| IgG1-F561 | 1.4E-07 | M252Y/S254T/T307A/N434Y |
| IgG1-F562 | 8.3E-08 | M252Y/S254T/T307Q/N434Y |
| IgG1-F563 | 1.3E-07 | M252Y/S254T/Q311A/N434Y |
| IgG1-F564 | 1.9E-07 | M252Y/S254T/Q311H/N434Y |
| IgG1-F565 | 9.2E-08 | M252Y/S254T/T307A/Q311A/N434Y |
| IgG1-F566 | 6.1E-08 | M252Y/S254T/T307Q/Q311A/N434Y |
| IgG1-F567 | 2.2E-07 | M252Y/S254T/M428I/N434Y |
| IgG1-F568 | 1.1E-07 | M252Y/T256E/T307A/Q311H/N434Y |
| IgG1-F569 | 2.0E-07 | M252Y/T256Q/T307A/Q311H/N434Y |
| IgG1-F570 | 1.3E-07 | M252Y/S254T/T307A/Q311H/N434Y |
| IgG1-F571 | 8.1E-08 | M252Y/N286E/T307A/Q311H/N434Y |
| IgG1-F572 | 1.0E-07 | M252Y/T307A/Q311H/M428I/N434Y |
| IgG1-F576 | 1.6E-06 | M252Y/T256E/T307Q/Q311H |
| IgG1-F577 | 1.3E-06 | M252Y/N286E/T307A/Q311A |
| IgG1-F578 | 5.7E-07 | M252Y/N286E/T307Q/Q311A |
| IgG1-F580 | 8.6E-07 | M252Y/N286E/T307Q/Q311H |
| IgG1-F581 | 7.2E-08 | M252Y/T256E/N286E/N434Y |
| IgG1-F582 | 7.5E-07 | S239K/M252Y/V308P |
| IgG1-F583 | 7.8E-07 | S239K/M252Y/V308P/E382A |
| IgG1-F584 | 6.3E-07 | S239K/M252Y/T256E/V308P |
| IgG1-F585 | 2.9E-07 | S239K/M252Y/N286E/V308P |
| IgG1-F586 | 1.4E-07 | S239K/M252Y/N286E/V308P/M428I |
| IgG1-F587 | 1.9E-07 | M252Y/N286E/M428L/N434Y |
| IgG1-F592 | 2.0E-07 | M252Y/S254T/E382A/N434Y |
| IgG1-F593 | 3.1E-08 | S239K/M252Y/S254T/V308P/M428I/N434Y |
| IgG1-F595 | 1.8E-07 | S239K/M252Y/M428I/N434Y |
| IgG1-F596 | 4.0E-07 | M252Y/D312A/E382A/M428Y/N434Y |
| IgG1-F597 | 2.2E-07 | M252Y/E382A/P387E/N434Y |
| IgG1-F598 | 1.4E-07 | M252Y/D312A/P387E/N434Y |
| IgG1-F599 | 5.2E-07 | M252Y/P387E/M428Y/N434Y |
Example 13 in vivo study of various Fc variant antibodies using human FcRn transgenic mouse line 32 by steady-state infusion model
The Fc variants produced in example 12 were tested in a steady state infusion model using human FcRn transgenic mouse strain 32 for their ability to eliminate antigen from plasma. A steady state infusion model in vivo study was performed as described in example 1, except that human FcRn transgenic mouse strain 32 was used instead of strain 276 and monoclonal anti-mouse CD4 antibody was injected 2 times (14 days before and after infusion pump implantation) or 3 times (10 and 20 days before and after infusion pump implantation).
The following antibody Fc variants selected from the Fc variants described in tables 9-1 to 9-14 were expressed and purified by methods known to those skilled in the art described in reference example 2:
fv4-IgG1 comprising VH3-IgG1 and VL 3-CK;
fv4-IgG1-F11 comprising VH3-IgG1-F11 and VL 3-CK;
fv4-IgG1-F14 comprising VH3-IgG1-F14 and VL 3-CK;
fv4-IgG1-F39 comprising VH3-IgG1-F39 and VL 3-CK;
fv4-IgG1-F48 comprising VH3-IgG1-F48 and VL 3-CK;
fv4-IgG1-F140 comprising VH3-IgG1-F140 and VL 3-CK;
fv4-IgG1-F157 comprising VH3-IgG1-F157 and VL 3-CK;
fv4-IgG1-F194 comprising VH3-IgG1-F194 and VL 3-CK;
fv4-IgG1-F196 comprising VH3-IgG1-F196 and VL 3-CK;
fv4-IgG1-F198 comprising VH3-IgG1-F198 and VL 3-CK;
fv4-IgG1-F262 comprising VH3-IgG1-F262 and VL 3-CK;
fv4-IgG1-F264 comprising VH3-IgG1-F264 and VL 3-CK;
fv4-IgG1-F393 comprising VH3-IgG1-F393 and VL 3-CK;
fv4-IgG1-F424 comprising VH3-IgG1-F434 and VL 3-CK; and
fv4-IgG1-F447 comprising VH3-IgG1-F447 and VL 3-CK.
These antibodies were administered to human FcRn transgenic mouse line 32 at a dose of 1 mg/kg.
FIG. 21 depicts the time course of plasma hsIL-6R concentration in mice. All Fc variants with increased binding affinity to human FcRn at pH 7.0 showed reduced plasma hsIL-6R concentrations compared to Fv4-IgG1, thus facilitating antigen elimination from plasma. Although the extent and persistence of the reduction in antigen concentration varied among the Fc variants, the plasma hsIL-6R concentrations were consistently reduced for all variants compared to IgG1, suggesting that increasing binding affinity to human FcRn at pH 7.0 generally facilitates antigen elimination from plasma. Figure 22 depicts the time course of plasma antibody concentrations in mice. Antibody pharmacokinetics vary among Fc variants.
As described in example 9, the amount of antigen eliminated from plasma by each antibody was an important factor in evaluating the efficiency of antigen elimination by administration of the Fc variant of the antibody with increased binding affinity to human FcRn at pH 7.0. Thus, the time course of the C value (antigen/antibody molar ratio) of each antibody is depicted in fig. 23. Figure 24 depicts the relationship between binding affinity of Fc variants to human FcRn and C-value (antigen/antibody molar ratio) at pH 7.0 on day 1 after antibody administration. This indicates that all antibody Fc variants tested in this study have lower C values compared to Fv4-IgG 1. Since all Fc variants tested in this study had a stronger binding affinity to human FcRn than KD 3.0 micromolar at pH 7.0, they achieved higher antigen elimination efficiency compared to whole human IgG 1. This is in agreement with the results obtained in example 9 (fig. 17).
Figure 25 depicts antibodies with Fc variants of F11, F39, F48 and F264 that show similar pharmacokinetics to IgG1, among the Fc variants tested in this study. Since this study was performed using human FcRn transgenic mice, these Fc variants were expected to have long half-lives in humans similar to IgG1 as well. FIG. 26 depicts the time course of plasma hsIL-6R concentrations in mice injected with antibodies (F11, F39, F48, and F264) with pharmacokinetics similar to that of intact human IgG 1. These variants reduced plasma hsIL-6R concentrations by about 10-fold compared to IgG 1. In addition, these antibodies reduced hsIL-6R concentrations below baseline hsIL-6R concentrations (antibody-free concentrations). Thus, these antibodies are able to eliminate antigens in plasma for a long period of time, so long dosing intervals would be preferred for antibody treatment of chronic diseases.
FIGS. 27 and 28 depict the time course of plasma antibody concentrations and plasma hsIL-6R concentrations for IgG1 and Fc variants F157, F196, and F262, respectively. Surprisingly, although the antibody pharmacokinetics of F157 and F262 showed significantly faster clearance from plasma compared to intact human IgG1, F157 and F262 showed very substantial and sustained elimination of hsIL-6R from plasma. Specifically, from day 1 to day 28 (except day 14), the plasma hsIL-6R concentration of F157 was below the limit of detection (1.56ng/mL), while the plasma hsIL-6R concentration of F262 from day 14 to day 28 was below the limit of detection (1.56 ng/mL). On the other hand, for F196 with slower antibody clearance, the antigen concentration began to increase at day 14 and returned to baseline at day 28, compared to F157. Of the Fc variants tested in this study, F157 and F262 were the only Fc variants capable of reducing plasma hsIL-6R concentrations by less than 1.56ng/mL on day 28.
This long-lasting effect of F157 and F262 was not expected from the pharmacokinetics of the antibody, since the antibody was eliminated from plasma very rapidly compared to intact human IgG 1. Specifically, no plasma antibody concentration of F157 was detected on day 21. However, on days 21 and 28, plasma hsIL-6R concentrations continued to decrease to a level below the detection limit of 1.56 ng/mL. This unexpected effect is believed to be due to the presence of the antibody in FcRn-bound form on the surface of vascular endothelial cells. Although these antibodies show low concentrations in plasma, they are still present in the vascular compartment in FcRn-bound form (which cannot be measured as plasma antibody concentration). These FcRn-bound antibodies can still bind to antigens in plasma and following FcRn-mediated uptake of the antigen/antibody complex, the antigen is released in the endosomes and degraded by lysosomes while the antibody is recycled back to the cell surface in FcRn-bound form. These FcRn-binding antibodies therefore promote antigen elimination. This explains why these antibodies retain the antigen elimination ability even after the antibody concentration in plasma becomes low.
EXAMPLE 14 comparative computer simulation study on conventional antibody and antigen-eliminating antibody
Example 13 demonstrates that antibodies that bind pH-dependently to antigen and have increased binding affinity to human FcRn at neutral pH are able to eliminate antigen from plasma. Thus, such antigen-depleting antibodies may be used for antibodies targeting such antigens where simple binding and neutralization is insufficient to treat the disease, requiring depletion of the antigen from plasma.
Antigen-depleting antibodies may also be used to target antibodies that simply bind and neutralize enough antigen. Antibody binding and neutralization of the antigen requires at least the same molar amount of antibody as the antigen in plasma (if the antibody has unlimited affinity to the antigen, the antigen can be neutralized by the same molar amount of antibody as the antigen). Unlike conventional antibodies (antibodies without pH-dependent antigen binding and Fc engineering), antigen-depleting antibodies can reduce the antigen concentration in plasma. This means that the concentration of antibody required to neutralize the antigen can be reduced. If an antigen-eliminating antibody reduces plasma antigen concentration by 10-fold compared to conventional antibodies, the concentration of antibody required to neutralize the antigen can also be reduced by 10-fold. Thus, in therapeutic situations, an antigen-depleting antibody may reduce the antibody dose or extend the dosing interval compared to conventional antibodies.
Fc variants such as F11, F39, F48 and F264 were able to reduce plasma antigen concentrations by about 10-fold compared to IgG 1. To evaluate the effect of such antigen-eliminating antibodies over conventional antibodies, we performed in silico evaluation of the antibody dose required to maintain antigen neutralization in the treatment of both conventional and antigen-eliminating antibodies. We determined the dose required to maintain neutralization (i.e. the required dose of Q3M) every 3 month dosing interval.
Construction of pharmacokinetic model
We constructed a Pharmacokinetic (PK) model using PK analysis software SAAM II (The SAAM Institute, Inc.). PK models were constructed as described in the following documents: pharmacokinet pharmacodyn.2001 for 12 months; 28(6): 507-32 and Br J ClinPharmacol.2007 for 3 months; 63(5): 548-61. The PK model concept is shown in figure 29. The amount of each compartment is described by the following differential equation.
[Math.1]
Xsc: amount of antibody in subcutaneous tissue
Xmab: amount of serum free antibodies
Xcom: amount of immune complex (═ complex) of antibody and antigen
Xag: amount of serum free antigen
ka: absorption rate constant
In this model, for all antibodies, assuming a bioavailability (F) of 1, the antigen biosynthesis rate (R) is determined by the following equation.
[Math.2]
R＝CLag×Cpre
Cpre: steady state antigen concentration of serum.
The pharmacokinetic and antigen binding kinetic parameters used for this in silico study are described in table 10.
[ Table 10]
| CLmab | L/day/kg | 0.0025 |
| CLag | L/day/kg | 0.0243 |
| CLcom | L/day/kg | 0.0045 |
| Vmab＝Vag | L/kg | 0.0843 |
| Vcom | L/kg | 0.0519 |
| ka | 1/day | 0.4800 |
| koff | 1/day | 53.0496 |
| kon | 1/nM/day | 53.0496 |
| L/ug/day | 0.353664 |
Simulated calculation of antigen-eliminating antibodies and affinityEffect of cooking
The steady state concentration (Cpre) before antibody administration was set to 2,400 ng/mL. Using the constructed PK model, we estimated a minimum dose of antibody that maintained a free antigen concentration below 35ng/mL at 84 days after a single subcutaneous administration. The molecular weight of the antigen was set to 190kDa and the therapeutic antibodies were all set to 150 kDa.
As antibodies, conventional antibodies and antigen-eliminating antibodies with different binding affinities (different affinity maturation of the parent antibody from KD 1 nM) were used in this in silico study. The action of the antigomir antibody is cleared more rapidly at the antigen-antibody complex than at the conventional antibody. The clearance parameters for antigen-antibody complexes (CLcom) are described in table 11.
[ Table 11]
| Conventional Ab | Ab with antigen elimination | ||
| CLcom | L/day/kg | 0.0045 | 0.0729 |
The effect of affinity maturation from a parent antibody with a KD of 1nM was also considered (the affinity varied over a 100-fold range). KD's of 1nM, 300pM, 100pM, 30pM and 10pM were used for this in silico study. The effect of affinity maturation is reduced in koff. The koff value varies over a 100-fold range (koff 53.05, 17.68, 5.30, 1.77, 0.53[ l/day ].
To maintain free antigen concentrations below 35ng/mL at 84 days after a single subcutaneous administration, antibody doses per individual were obtained for conventional and antigen-depleting antibodies with binding affinities (KD) of 1nM, 300pM, 100pM, 30pM and 10 pM. The results are shown in Table 12.
[ Table 12]
| Dosage (mg/subject) | 1nM | 333pM | 100pM | 33pM | 10pM |
| Conventional Ab | 2868 | 1256 | 692 | 532 | 475 |
| Ab with antigen elimination | 180 | 81 | 46 | 36 | 33 |
The parent conventional antibody with a binding affinity of 1nM required 2,868mg to achieve the Q3M dosing. Although the antibody dose can be reduced by improving the binding affinity to the antigen, the reduction in dose reaches an upper limit. This upper limit stems from the fact that antibody binding and neutralization of the antigen requires at least the same molar amount of antibody as the antigen in plasma. Even with a binding affinity of 10pM, conventional antibodies require 475mg to achieve a Q3M dosing dose, which is a dose that cannot be injected subcutaneously by a single injection, because of limitations in formulation antibody concentrations and subcutaneous injectable volumes.
On the other hand, antibody doses can be significantly reduced by engineering conventional antibodies to antigen-eliminating antibodies by engineering pH-dependent antigen binding (or by directly generating antibodies with pH-dependent binding) and engineering FC regions with increased binding affinity to FcRn at neutral pH. An antigen-depleting antibody with a binding affinity of 1nM requires only 180mg to achieve the Q3M dose. Such dosage levels cannot be achieved even with conventional antibodies with unlimited affinity. By improving the binding affinity of the antigen-eliminating antibody to 10pM, the dose can be reduced to 33mg, which is a dose that can be easily injected subcutaneously.
Thus, this in silico study shows that antigen-depleting antibodies have significant advantages over conventional antibodies. The dosage of the antibody can be reduced to a level that cannot be achieved by even conventional antibodies with unlimited affinity. As for the dosing interval, when the antigen-eliminating antibody is injected at the same dose as the conventional antibody, the antigen-eliminating antibody may have a longer lasting effect, thus making the dosing interval significantly longer. Both dose reduction and extended dosing intervals by antigen-eliminating antibodies can provide significant advantages over conventional antibodies.
It should be noted that the antigen-depleting antibody does not necessarily require pH-dependent binding to the antigen as described in example 1. pH-dependent binding to antigen can significantly improve the antigen-eliminating activity of the antibody. Furthermore, the pH-dependent binding properties can be replaced by other factors whose concentrations differ in plasma and endosomes. Such factors can also be used to generate antibodies that bind antigen in plasma but dissociate antigen in endosomes.
EXAMPLE 15 study of the Effect of increasing the Elimination of human IL-6 by pH-dependent anti-human IL-6 antibody
production of pH-dependent human IL-6 binding antibodies
CLB8-IgG1 described in WO 2009/125825, which comprises CLB8H-IgG1(SEQ ID NO: 16) and CLB8L-CK (SEQ ID NO: 17), is a chimeric anti-IL-6 antibody. H16/L13-IgG1, comprising H16-IgG1(SEQ ID NO: 18) and L13-CK (SEQ ID NO: 19), is a chimeric anti-IL-6 antibody (which binds at pH 7.4 but dissociates at pH 5.8) that arises from the property of conferring on CLB8-IgG1 binding to human IL-6 in a pH-dependent manner.
Evaluation of pH-dependent binding Activity of chimeric anti-IL-6 antibody with human IL-6
CLB8-IgG1 and H16/L13-IgG1 were evaluated for human IL-6 binding activity (dissociation constant (KD)) at pH 5.5 and pH 7.4 using Biacore T100(GE Healthcare). The assay was performed using 10mmol/l ACES/150mmol/l NaCl (pH 7.4 and pH 6.0) containing 0.05% surfactant P20 as running buffer. After the antibody was bound to recombinant protein A/G (thermoscientific) immobilized on the sensor chip by an amino coupling method, human IL-6(TORAY) was injected at an appropriate concentration as an analyte. The measurement was carried out at 37 ℃. The measurement results were analyzed using Biacore T100 evaluation software (GE Healthcare), and the association rate constant ka (1/Ms) and dissociation rate constant k were calculated from the measurement resultsd(1/s). Then from ka and kdKd (m) was calculated (table 13). In addition, the pH-dependent binding of each antibody was re-evaluated to calculate the KD ratio between pH 7.4 and pH 6.0.
[ Table 13]
Preparation of pH-dependent anti-human IL-6 antibodies having FcRn binding Activity under neutral conditions
Mutations were introduced into H16/L13-IgG1 comprising H16-IgG1(SEQ ID NO: 18) and L13-CK (SEQ ID NO: 19) to increase FcRn binding under neutral conditions (pH 7.4). Specifically, H16-IgG1-v2(SEQ ID NO: 20) was prepared from the heavy chain constant region of IgG1 by substituting Trp for Asn at position 434 (numbering by EU), while H16-F14(SEQ ID NO: 21) was constructed from the heavy chain constant region of IgG1 by substituting Tyr for Met at position 252 and Trp for Asn at position 434 (numbering by EU). Amino acid substitutions were introduced by methods known to those skilled in the art as described in reference example 1.
CLB8-IgG1 comprising CLB8H-IgG1(SEQ ID NO: 16) and CLB8L-CK (SEQ ID NO: 17), H16/L13-IgG1 comprising H16-IgG1(SEQ ID NO: 18) and L13-CK (SEQ ID NO: 19), H16/L13-IgG1-v2 comprising H16-IgG1-v2(SEQ ID NO: 20) and L13-CK (SEQ ID NO: 19), and H16/L13-F14 comprising H16-F14(SEQ ID NO: 21) and L13-CK (SEQ ID NO: 19) were expressed and purified by referring to methods known to those skilled in the art described in example 2.
Evaluation of mouse FcRn binding Activity of Fc variants at neutral pH
The following were all prepared as described in example 5: VH3/l (wt) -IgG1 comprising VH3-IgG1 and l (wt), VH3/l (wt) -IgG1-v2 comprising VH3-IgG1-v2 and l (wt), and VH3/l (wt) -IgG1-F14 comprising VH3-IgG1-F14 and l (wt), and these were evaluated for mouse FcRn binding under neutral conditions (pH 7.4) by the method described in example 8.
The results are shown in Table 14. IgG1 showed very weak binding activity, whereas IgG1-v2 and IgG1-F14 showed stronger binding affinity to mouse FcRn at pH 7.4.
[ Table 14]
| KD | |
| IgG1 | ND |
| IgG1-v2 | 1.0E-06 |
| IgG1-F14 | 1.3E-07 |
In vivo assay using normal mice
After administering hIL-6 alone or hIL-6 and anti-human IL-6 antibodies to normal mice (C57BL/6J mice; Charles River Japan), the in vivo kinetics of human IL-6 (hIL-6; TORAY) and anti-human IL-6 antibodies were evaluated. hIL-6 solution (5. mu.g/ml) or a mixture solution containing hIL-6 and anti-human IL-6 antibody (CLB8-IgG1 group; 5. mu.g/ml hIL-6 and 0.025mg/ml CLB8-IgG1, H16/L13-IgG1, H16/L13-IgG1-v2 and H16/L13-IgG1-F14 groups, respectively; 5. mu.g/ml hIL-6 and 0.14mg/ml H16/L13-IgG1, H16/L13-IgG1-v2 and H16/L13-IgG1-F14) was administered once to the tail vein at a dose of 10 ml/kg. The antibody dose is set so that more than 99.8% of the human IL-6 in the dosing solution binds to the antibody. Blood was collected at 5 minutes, 30 minutes, 2 hours, 4 hours, 7 hours, 1 day after administration of hIL-6 alone and at 5 minutes, 7 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 14 days, 21 days, and 30 days after administration of the hIL-6 and anti-human IL-6 antibody solution mixture. The collected blood was immediately centrifuged at 15,000rpm and 4 ℃ for 15 minutes to separate plasma. The separated plasma was stored in a refrigerator at-20 ℃ or lower before the measurement.
Measurement of human IL-6 plasma concentration by ELISA
The concentration of human IL-6 in mouse plasma was measured by using a human IL-6Quantikine HS ELISA kit (R & D). Calibration curve samples with plasma concentrations of 20, 10, 5, 2.5, 1.25, 0.625 and 0.3125ng/ml and mouse plasma samples diluted 100-fold or more were prepared. To bind all human IL-6 in the sample to CLB8-IgG1, 150 microliters of 5 micrograms/ml CLB8-IgG1 were added to 150 microliters of the calibration curve sample and the plasma sample, and the sample was allowed to stand at room temperature for 1 hour. Subsequently, the samples were dispensed into plates provided by an ELISA kit (R & D) and allowed to stand at room temperature for 1 hour. Then, IL-6 conjugate supplied from ELISA kit (R & D) was added and reacted at room temperature for 1 hour, and substrate solution supplied from ELISA kit (R & D) was added and reacted at room temperature for 1 hour. Subsequently, a color reaction was performed using an amplification Solution (Amplifier Solution) supplied from an ELISA kit (R & D) as a substrate, and the reaction was carried out at room temperature for half an hour. After the reaction was terminated with a termination solution provided by an ELISA kit (R & D), absorbance at 490nm was measured by a microplate reader. The concentration in the mouse plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTMax PRO (Molecular Devices). For normal mice, the time course of plasma hIL-6 concentration after intravenous administration as measured by this method is shown in FIG. 30.
Measurement of plasma concentration of anti-human IL-6 antibody by ELISA
The concentration of anti-human IL-6 antibody in the plasma of mice was measured by ELISA. Anti-human IgG (gamma chain specific) F (ab') 2 antibody fragment (Sigma) was dispensed on Nunc-ImmunoPlateMaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4 ℃ to prepare anti-human IgG-immobilized plates. Calibration curve samples with plasma concentrations of 1.6, 0.8, 0.4, 0.2, 0.1, 0.05 and 0.025 microgram/ml and mouse plasma samples diluted 100-fold or more were prepared. In order to make the sample of all anti human IL-6 antibody and IL-6 binding, 200 microliter 1 micrograms/ml IL-6 added to 100 microliter calibration curve sample and plasma samples, then the sample at room temperature for 1 hours. Subsequently, the sample was dispensed into an anti-human IgG-immobilized plate and allowed to stand at room temperature for 1 hour. Then, goat anti-human IgG (gamma chain-specific) biotin (biott) conjugate (Southern Biotech Association) was added and reacted at room temperature for 1 hour. Subsequently, streptavidin-PolyHRP 80 (Stereospeic detection Technologies) was added, reacted at room temperature for 1 hour, and a color reaction was performed using TMB OneComponent HR Microwell Substrate (BioFX Laboratories) as a Substrate. After the reaction was terminated with 1N sulfuric acid (Showa Chemical), the absorbance at 450nm was measured by a microplate reader. The concentration in the mouse plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTmax PRO (molecular devices). For normal mice, the time course of plasma antibody concentration after intravenous administration measured by this method is shown in fig. 31.
Effect of pH-dependent binding to human IL-6
CLB8-IgG1 and H16/L13-IgG1 bound to human IL-6 in a pH-dependent manner were tested in vivo and the results were compared between them. As shown in fig. 31, the pharmacokinetics of the antibody showed linear clearance. Meanwhile, as shown in FIG. 30, it was found that hIL-6 administered simultaneously with H16/L13-IgG1 that binds human IL-6 in a pH-dependent manner accelerated elimination of hIL-6 compared to hIL-6 administered simultaneously with CLB8-IgG 1. Thus, it was demonstrated that by conferring pH-dependent human IL-6 binding capacity, plasma hIL-6 concentrations could be reduced by about 76-fold 4 days after administration.
Effect of FcRn binding under neutral conditions (pH 7.4)
In addition to H16/L13-IgG1, H16/L13-IgG1-v2 and H16/L13-F14 produced by introducing the above amino acid substitutions into H16/L13-IgG1 were tested in vivo using normal mice. The results of the assay were compared with those of H16/L13-IgG 1. As shown in FIG. 31, the plasma antibody concentration of H16/L13-IgG1-v2, which increased binding to mouse FcRn under neutral conditions (pH 7.4), was 1/2.9-fold higher than that of H16/L13-IgG1 1 day after administration. Alternatively, at 7 hours post-administration, binding to mouse FcRn under neutral conditions (pH 7.4) further increased plasma antibody concentration of H16/L13-F14 by 1/21 times that of H16/L13-IgG 1.
As shown in figure 30, hIL-6 administered concurrently with H16/L13-IgG1-v2 or H16/L13-F14, which had increased binding to mouse FcRn under neutral conditions (pH 7.4), demonstrated significantly faster elimination compared to hIL-6 administered concurrently with H16/L13-IgG 1. H16/L13-IgG1-v2 reduced plasma concentrations of hIL-6 by about 10-fold compared to H16/L13-IgG1 on day 1. H16/L13-F14 reduced plasma concentrations of hIL-6 by about 38-fold compared to H16/L13-IgG1 at 7 hours. Thus, it was revealed that plasma human IL-6 concentrations can be reduced by conferring mouse FcRn binding ability under neutral conditions (pH 7.4). As described above, by conferring mouse FcRn binding ability under neutral conditions (pH 7.4), plasma antibody concentrations were reduced; however, the effect of reducing plasma hIL-6 concentration was produced, which greatly exceeded the reduction in antibody concentration. Specifically, this means that the elimination of human IL-6 can be accelerated by administering an antibody that binds to human IL-6 in a pH-dependent manner and that is made to have mouse FcRn binding ability under neutral conditions (pH 7.4).
The results of the above studies indicate that plasma antigen concentrations of not only human soluble IL-6 receptor but also antigens such as human IL-6 can also be significantly reduced by administering antibodies having pH-dependent antigen binding capacity and FcRn binding capacity under neutral conditions.
EXAMPLE 16 study on the Effect of promoting the abrogation and acceleration of human IgA of receptor Fc fusion protein binding to human IgA in a pH-dependent manner
Production of receptor Fc fusion proteins that bind to human IgA in a pH-dependent manner
A0-IgG1, which comprises a dimer of A0H-IgG1(SEQ ID NO: 22), is a human CD89-Fc fusion protein. As described in j.mol.biol. (2003) 324: 645-657, human CD89, also known as human Fc α receptor I, binds to human IgA in a pH-dependent manner (i.e., strongly binds to human IgA at neutral pH but weakly binds to human IgA at acidic pH).
Evaluation of pH-dependent binding Activity of CD89-Fc fusion protein with human IgA
A0-IgG1 was evaluated against human IgA binding activity (dissociation constant (KD)) at pH 6.0 and pH 7.4 using Biacore T100(GE Healthcare). The assay was performed using 10mmol/l ACES/150mmol/l NaCl (pH 7.4 and pH 6.0) containing 0.05% surfactant P20 as running buffer. After the CD89-Fc fusion protein was bound to the recombinant protein A/G (thermo scientific) immobilized on the sensor chip by the amino coupling method, hIgA (human IgA: prepared as described in reference example 5) was injected at an appropriate concentration as an analyte. The measurement was carried out at 37 ℃. The measurement results were analyzed using Biacore T100 evaluation software (GEHealthcare), and the sensorgram obtained is shown in fig. 32. This clearly indicates that the CD89-Fc fusion protein has pH-dependent human IgA binding activity, which binds strongly to human IgA at neutral pH, but weakly at acidic pH.
Preparation of pH-dependent receptor Fc fusion protein having FcRn binding activity under neutral conditions
Prepare for
Mutations were introduced into A0-IgG1, which contains a dimer of A0H-IgG1(SEQ ID NO: 22), to increase FcRn binding under neutral conditions (pH 7.4). Specifically, A0-IgG1-v2 was prepared from the heavy chain constant region of IgG1 by substituting Trp for Asn at position 426 in A0-IgG 1. Amino acid substitutions were introduced by methods known to those skilled in the art as described in reference example 1.
A0-IgG1 comprising a dimer of A0H-IgG1(SEQ ID NO: 22) and A0-IgG1-v2 comprising a dimer of A0H-IgG1-v2(SEQ ID NO: 23) were expressed and purified by methods known to those skilled in the art and described in reference example 2.
In vivo assay using normal mice
In normal mice (C57BL/6J mice; Charles River Japan), the in vivo kinetics of human IgA (hIgA) and CD89-Fc fusion proteins were evaluated after administration of hIgA alone or hIgA and CD89-Fc fusion protein (A0H-IgG1 or A0H-IgG1-v 2). The tail vein was dosed once with hIgA solution (80. mu.g/ml) or a solution containing a mixture of hIgA and CD89-Fc fusion protein (80. mu.g/ml and 1.5mg/ml, respectively, where most of the hIgA binds to CD89-Fc fusion protein) at a dose of 10 ml/kg. Blood was collected 15 minutes, 7 hours, 1 day, 2 days, 4 days, and 7 days after administration. The collected blood was immediately centrifuged at 15,000rpm and 4 ℃ for 15 minutes to separate plasma. The separated plasma was stored in a refrigerator at-20 ℃ or lower before the measurement.
Measurement of human IgA plasma concentration by ELISA
The concentration of human IgA in the plasma of mice was measured by ELISA using hsIL-6R, since recombinant human IgA has a variable region for hsIL-6R. Goat anti-human IgA antibody (Bethyl laboratories) was dispensed onto Nunc-ImmunoPlate maxiSorp (Nalge Nunc International) and allowed to stand at 4 ℃ overnight to prepare anti-human IgA-immobilized plates. Calibration curve samples with plasma concentrations of 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, or 0.00625 micrograms/ml and mouse plasma samples diluted 100-fold or more were prepared. To bind all human IgA in the sample to hsIL-6R, 200 microliters of 10 micrograms/ml hsIL-6R were added to 100 microliters of the calibration curve sample and the plasma sample, and the samples were allowed to stand at room temperature for 1 hour. Subsequently, the sample was dispensed into an anti-human IgA-immobilized plate and allowed to stand at room temperature for 1 hour. Then, a biotinylated anti-human IL-6R antibody (R & D) was added thereto, and the reaction was carried out at room temperature for 1 hour. Subsequently, streptavidin-PolyHRP 80 (Stereospeic detection Technologies) was added and reacted at room temperature for 1 hour, and a color reaction was performed using TMB OneComponent HRP Microwell Substrate (BioFX Laboratories) as a Substrate. After the reaction was terminated with 1N sulfuric acid (Showa Chemical), the absorbance at 450nm was measured by a microplate reader. The concentration in the mouse plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTmax PRO (molecular devices). For normal mice, the time course of plasma hIgA concentration after intravenous administration as measured by this method is shown in FIG. 33.
CD89-Fc fusion protein plasma concentrations measured by ELISA
The concentration of CD89-Fc fusion protein in mouse plasma was measured by ELISA. Anti-human IgG (gamma chain specific) F (ab') 2 antibody fragment (Sigma) was dispensed onto Nunc-ImmunoPlateMaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4 ℃ to prepare anti-human IgG-immobilized plates. Calibration curve samples with plasma concentrations of 25.6, 12.8, 6.4, 3.2, 1.6, 0.8 and 0.4 micrograms/ml and mouse plasma samples diluted 100-fold or more were prepared. To bind all CD89-Fc fusion proteins in the sample to human IgA, 200 microliters of 5 micrograms/ml human IgA was added to 100 microliters of the calibration curve sample and plasma sample, and the sample was allowed to stand at room temperature for 1 hour. Subsequently, the sample was dispensed into an anti-human IgG-immobilized plate and allowed to stand at room temperature for 1 hour. Then, goat anti-human IgG (Fc-specific) -alkaline phosphatase conjugate (SIGMA) was added and reacted at room temperature for 1 hour. Subsequently, a color development reaction was carried out using a blue Phos Microwell phosphatase substrate System (blue Microwell phosphatase Substrates System) (Kirkegaard & Perry Laboratories) as a substrate, and absorbance at 650nm was measured by a microplate reader. The concentration in the mouse plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTMax PRO (Molecular Devices). For normal mice, the time course of the plasma concentration of CD89-Fc fusion protein after intravenous administration, measured in this manner, is shown in FIG. 34.
Effect of FcRn binding under neutral conditions (pH7.4)
A0-IgG1-v2 produced by introducing the above amino acid substitutions into A0-IgG1 was tested in vivo using normal mice, except for A0-IgG 1. The results of the assay were compared to those of A0-IgG 1. As shown in figure 34, plasma concentrations of a0-IgG1-v2 that increased binding to mouse FcRn under neutral conditions (pH7.4) were 1/1.8 times greater than a0-IgG1 2 days post-administration.
As shown in figure 33, the hIgA administered concurrently with a0-IgG1-v2, which had increased binding to mouse FcRn under neutral conditions (pH7.4), demonstrated significantly faster elimination compared to the hIgA administered concurrently with a0-IgG 1. On day 2, A0-IgG1-v2 reduced plasma hIgA concentrations by about 5.7-fold compared to A0-IgG 1. As described above, plasma antibody concentrations were reduced by conferring mouse FcRn binding ability under neutral conditions (pH 7.4); however, the effect of reducing plasma hIgA concentration is produced, which greatly exceeds the reduction in antibody concentration. Specifically, this means that elimination of human IgA can be accelerated by administering a receptor Fc fusion protein that binds human IgA in a pH-dependent manner and has a mouse FcRn binding ability under neutral conditions (pH 7.4).
The results of the above studies indicate that plasma antigen concentrations, such as that of human IgA, can also be significantly reduced by administering a receptor Fc fusion protein having both pH-dependent antigen binding ability and FcRn binding ability under neutral conditions. Thus, receptor Fc fusion proteins can also be engineered to have the ability to eliminate antigen (or ligand) plasma concentrations from plasma.
EXAMPLE 17 study of Plexin A1 Effect of promoting pH-dependent anti-human Plexin A1 antibody on abrogation and potentiation (preparation of antibody)
Antibodies related to pH-dependent human Plexin A1 binding
PX268-IgG1 comprising PX268H-IgG1(SEQ ID NO: 24) and PX268L-CK (SEQ ID NO: 25) is a chimeric anti-plexin A1 antibody. PX141-IgG1 comprising PX141H-IgG1(SEQ ID NO: 26) and PX141L-CK (SEQ ID NO: 27) is a chimeric anti-Plexin A1 antibody that binds to soluble Plexin A1 in a pH-dependent manner (i.e., binds strongly to soluble Plexin A1 at neutral pH, but binds weakly to soluble Plexin A1 at acidic pH).
Evaluation of pH-dependent binding Activity of anti-human Plexin A1 antibody with human Plexin A1
PX268-IgG1 and PX141-IgG1 were evaluated against human Plexin A1 binding activity (dissociation constant (KD)) at pH 6.0 and pH 7.4 using Biacore T100(GE Healthcare). The assay was performed using 10mmol/l ACES/150mmol/l NaCl (pH 7.4 and pH 6.0) containing 0.05% surfactant P20 as running buffer. After the antibody was bound to recombinant protein A/G (thermoscientific) immobilized on the sensor chip by the amino coupling method, hsPlexin A1 (soluble human Plexin A1: prepared as described in reference example 5) was injected at an appropriate concentration as an analyte. The measurement was carried out at 37 ℃. The measurement results were analyzed using Biacore T100 evaluation software (GE Healthcare), and the association rate constant, ka (1/Ms) and dissociation rate constant k were calculated from the measurement results d(1/s). Then according to ka and kdKd (m) was calculated (table 15). In addition, pH-dependent binding was evaluated to calculate the KD ratio between pH 7.4 and pH 6.0 for each antibody.
[ Table 15]
Method for producing pH-dependent anti-human Plexin A1 antibody having FcRn binding activity under neutral conditions
Preparation of
Mutations were introduced into PX141-IgG1 comprising PX141H-IgG1(SEQ ID NO: 26) and PX141L-CK (SEQ ID NO: 27) to increase FcRn binding under neutral conditions (pH 7.4). Specifically, PX141H-IgG1-v2(SEQ ID NO: 28) was prepared from the heavy chain constant region of IgG1 by substituting Trp for Asn at position 434 (numbering by EU). Amino acid substitutions were introduced by methods known to those skilled in the art as described in reference example 1.
PX268-IgG1 comprising PX268H-IgG1(SEQ ID NO: 24) and PX268L-CK (SEQ ID NO: 25), PX141-IgG1 comprising PX141H-IgG1(SEQ ID NO: 26) and PX141L-CK (SEQ ID NO: 27), and PX141-IgG1-v2 comprising PX141H-IgG1-v2(SEQ ID NO: 28) and PX141L-CK (SEQ ID NO: 27) were expressed and purified by referring to methods known to those skilled in the art as described in example 2.
In vivo assay using normal mice
In normal mice (C57BL/6J mice; Charles River Japan), the in vivo kinetics of soluble human Plexin A1(hsPlexin A1) and anti-human Plexin A1 antibodies were evaluated after administration of only hsPlexin A1 or hsPlexin A1 and anti-human Plexin A1 antibodies. An hsPlexin A1 solution (100 microgram/ml) or a solution containing a mixture of hsPlexin A1 and anti-human plexin A1 antibodies (PX268-IgG1 group; 100 microgram/ml hsPlexin A1 and 1.2mg/ml PX268-IgG1, PX141-IgG1, and PX141-IgG1-v2 groups, respectively; 100 microgram/ml hsPlexin A1 and 1.0mg/ml PX141-IgG1 and PX141-IgG1-v2) was administered once to the tail vein at a dose of 10 ml/kg.
In the dosing solution, the dose of antibody was set such that more than 99.9% of soluble human plexin a1 bound to the antibody. Blood was collected 15 minutes, 7 hours, 1 day, 2 days, 4 days, 7 days after administration of the hsPlexin A1 and anti-human plexin A1 antibody solution mixture. The collected blood was immediately centrifuged at 15,000rpm and 4 ℃ for 15 minutes to separate plasma. The separated plasma was stored in a refrigerator at-20 ℃ or lower before the measurement.
Measurement of human Plexin A1 plasma concentration by ELISA following administration of only hsPlexin A1
The concentration of human plexin a1 in mouse plasma was measured by ELISA using biotinylated anti-FLAG M2 antibody (Sigma), since recombinant human plexin a1 has a FLAG tag sequence end at the C-terminus. A rabbit anti-human Plexin A1 polyclonal antibody prepared by immunizing rabbits with Plexin A1 was dispensed onto Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International), and allowed to stand overnight at 4 ℃ to prepare an anti-human Plexin A1-immobilized plate. Calibration curve samples with plasma concentrations of 25.6, 12.8, 6.4, 3.2, 1.6, and 0.8 micrograms/ml and mouse plasma samples diluted 100-fold or more were prepared. Subsequently, the sample was dispensed onto an anti-human plexin A1-immobilized plate and allowed to stand at room temperature for 1 hour. Then, biotinylated anti-FLAG M2 antibody (Sigma) was added and the reaction was carried out at room temperature for 1 hour. Subsequently, streptavidin-PolyHRP 80 (Stereospeicic detection technologies) was added, reacted at room temperature for 1 hour, and developed using TMB One Component HRMicrowell Substrate (BioFX Laboratories) as a Substrate. After the reaction was terminated with 1N sulfuric acid (Showa Chemical), the absorbance at 450nm was measured by a microplate reader. The concentration in the mouse plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTMax PRO (Molecular Devices). The time course of plasma hsPlexin a1 concentration after intravenous administration as measured by this method is shown in figure 35.
Measurement of plasma concentration of human Plexin A1 in PX268-IgG1 group by ELISA
The concentration of human plexin a1 in mouse plasma was measured by ELISA using biotinylated anti-FLAG M2 antibody (Sigma), since recombinant human plexin a1 has a FLAG tag sequence end at the C-terminus. A rabbit anti-human Plexin A1 polyclonal antibody prepared by immunizing rabbits with Plexin A1 was dispensed onto Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International), and allowed to stand overnight at 4 ℃ to prepare an anti-human Plexin A1-immobilized plate. Calibration curve samples with plasma concentrations of 25.6, 12.8, 6.4, 3.2, 1.6 and 0.8 micrograms/ml and mouse plasma samples diluted 50-fold or more were prepared. To bind all human plexin A1 in the sample to PX268-IgG1, 150 microliters of 40 micrograms/ml PX268-IgG1 were added to 150 microliters of the calibration curve sample and the plasma sample, and the sample was allowed to stand overnight at 37 ℃. Subsequently, the sample was dispensed onto an anti-human Plexin A1-immobilized plate and allowed to stand at room temperature (or 4 ℃) for 1 hour. Then, biotinylated anti-FLAG M2 antibody (Sigma) was added and the reaction was carried out at room temperature (or 4 ℃ C.) for 1 hour. Subsequently, streptavidin-PolyHRP 80 (Stereospeicic Detection Technologies) was added, reacted at room temperature (or 4 ℃) for 1 hour, and a color reaction was performed using TMB One Component HRP Microwell Substrate (BioFXlaboratories) as a Substrate. After the reaction was terminated with 1N sulfuric acid (Showa Chemical), the absorbance at 450nm was measured by a microplate reader. The concentration in the mouse plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTMax PRO (Molecular Devices). The time course of plasma hsPlexin a1 concentration after intravenous administration as measured by this method is shown in figure 35.
Measurement of human Plexin A1 blood in the PX141-IgG1 and PX141-IgG1-v2 groups by ELISA
Pulp concentration
The concentration of human plexin a1 in mouse plasma was measured by ELISA using biotinylated anti-FLAG M2 antibody (Sigma), since recombinant human plexin a1 has a FLAG tag sequence end at the C-terminus. PX268-IgG1 was distributed onto Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4 ℃ to prepare anti-human plexin A1-immobilized plates. Calibration curve samples with plasma concentrations of 25.6, 12.8, 6.4, 3.2, 1.6, and 0.8 micrograms/ml and mouse plasma samples diluted 50-fold or more were prepared. To bind all human plexin A1 in the sample to PX141-IgG1 or PX141-IgG1-v2, 150 microliters of 40 micrograms/ml PX141-IgG1 or PX141-IgG1-v2 were added to 150 microliters of the calibration curve sample and plasma sample, and the samples were allowed to stand overnight at 37 ℃. Subsequently, the sample was dispensed onto an anti-human Plexin A1-immobilized plate and allowed to stand at room temperature (or 4 ℃) for 1 hour. Then, biotinylated anti-FLAG M2 antibody (Sigma) was added and the reaction was carried out at room temperature (or 4 ℃ C.) for 1 hour. Subsequently, streptavidin-PolyHRP 80 (Stereospeic detection Technologies) was added, reacted at room temperature (or 4 ℃) for 1 hour, and a color reaction was performed using TMBOne Component HRP Microwell Substrate (BioFX Laboratories) as a Substrate. After the reaction was terminated with 1N sulfuric acid (Showa Chemical), the absorbance at 450nm was measured by a microplate reader. The concentration in the mouse plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTMax PRO (Molecular Devices). Plasma hsPlexin a1 concentrations 7 hours after intravenous administration measured by this method are shown in figure 35.
Effect of pH-dependent binding to soluble human Plexin A1
PX268-IgG1 and PX141-IgG1 that bound human IL-6 in a pH dependent manner were tested in vivo and the results compared between them. Meanwhile, as shown in fig. 35, it was found that hsPlexin a1 administered simultaneously with PX141-IgG1 that binds soluble human plexin a1 in a pH-dependent manner reduced the total plasma concentration of hsPlexin a1 compared to hsPlexin a1 administered simultaneously with PX268-IgG 1.
Effect of FcRn binding under neutral conditions (pH 7.4)
In addition to PX141-IgG1, PX141-IgG1-v2 generated by introducing the above amino acid substitutions into PX141-IgG1 was tested in vivo using normal mice. The results of the assay were compared to the results for PX141-IgG 1.
As shown in figure 35, hsPlexin a1 administered concurrently with PX141-IgG1-v2, which increased binding to mouse FcRn under neutral conditions (pH 7.4), showed that total plasma concentrations of hsPlexin a1 were reduced to undetectable levels (detection limit of 0.8 micrograms/mL). Thus, it was revealed that the concentration of soluble human plexin a1 could be reduced by conferring binding ability to mouse FcRn under neutral conditions (pH 7.4). Specifically, this means that the elimination of soluble human plexin a1 can be accelerated by administering an antibody that binds to human plexin a1 in a pH-dependent manner and that is made to have mouse FcRn binding capacity under neutral conditions (pH 7.4).
The results of the above studies indicate that plasma antigen concentrations of not only human soluble IL-6 receptor but also antigens (e.g., human IL-6, human IgA, and human soluble Plexin A1) can also be significantly reduced by administering antibodies that have pH-dependent antigen binding and FcRn binding abilities under neutral conditions.
[ reference example 1] construction of expression vector for IgG antibody introduced with amino acid substitution
Mutants were generated using the QuikChange Site-Directed Mutagenesis Kit (QuikChange Site-Directed Mutagenesis Kit) (Stratagene) or the In-Fusion HD Cloning Kit (Clontech) according to the method described In the provided specification, and the resulting plasmid fragments were inserted into mammalian cell expression vectors to generate the desired H-chain expression vector and L-chain expression vector. The nucleotide sequence of the resulting expression vector is determined using conventional methods known to those skilled in the art.
[ reference example 2] expression and purification of IgG antibody
The antibody was expressed by the following method. Antibodies were expressed by FreestyleHEK293(Invitrogen) or HEK293H cell line (Invitrogen) as described by the manufacturer. The human embryonic kidney cancer-derived HEK293H cell line (Invitrogen) was suspended in dmem (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). For 5-6x10 5Adherent cells at a cell density of one cell/ml (diameter 10 cm; CORNING), the cells were plated in a petri dish at 10 ml/petri dish in CO2Incubator (37 ℃, 5% CO)2) Culturing for a whole day and night. Then, the medium was removed by aspiration, and 6.9ml of CHO-S-SFM-II medium (Invitrogen) was added. The prepared plasmid is introduced into cells by lipofection. The resulting culture supernatant was collected, centrifuged (about 2,000Xg, 5 minutes, room temperature) to remove the cells, and sterilized by filtration through a 0.22 micron filter MILLEX (registered trademark) -GV (Millipore) to give a supernatant. With rProtein A SepharoseTMFast Flow (Amersham Biosciences), antibodies were purified from the resulting culture supernatant by methods known to those skilled in the art. To determine the concentration of the purified antibody, the absorbance at 280nm was measured using a spectrophotometer. The method of using the Protein by Protein Science (1995) 4: 2411-2423, calculating the antibody concentration from the measured value.
[ reference example 3] preparation of soluble human IL-6 receptor (hsIL-6R)
Recombinant human IL-6 receptor as antigen was prepared as follows. Cell lines constitutively expressing a soluble human IL-6 receptor (hereinafter also referred to as hsIL-6R) having an amino acid sequence from the N-terminus from 1 to 357 (as reported in J.Immunol.152: 4958-4968 (1994)) were established by methods known to those skilled in the art. Cells were cultured to express hsIL-6R. hsIL-6R was purified from culture supernatants by the following two steps: blue Sepharose 6FF column chromatography and gel filtration chromatography. The fraction eluted as the main peak in the final stage was used as the final purified product.
[ reference example 4] preparation of human FcRn
FcRn is a complex of FcRn and β 2-microglobulin. Oligo DNA primers were prepared based on published human FcRn gene sequences (J Exp Med.1994, 12.1.; 180 (6): 2377-81). A DNA fragment encoding the entire gene was prepared by PCR using Human cDNA (Human planta Marathon-Ready cDNA, Clontech) as a template and prepared primers. Using the resulting DNA fragment as a template, a DNA fragment encoding the extracellular domain containing the signal region (Met1-Leu290) was amplified by PCR and inserted into a mammalian cell expression vector. Similarly, an oligomeric DNA primer was prepared based on the published human β 2-microglobulin gene sequence (Proc. Natl. Acad. Sci. U.S.A.99 (26): 16899-169903 (2002)). A DNA fragment encoding the entire gene was prepared by PCR using Human cDNA (Human planta Marathon-Ready cDNA, Clontech) as a template and prepared primers. Using the resulting DNA fragment as a template, a DNA fragment encoding the entire protein containing the signal region (Met1-Met119) was amplified by PCR and inserted into a mammalian cell expression vector.
Soluble human FcRn was expressed by the following method. Plasmids constructed for expressing human FcRn (SEQ ID NO: 30) and β 2-microglobulin (SEQ ID NO: 31) were introduced into cells of the human embryonic kidney cancer-derived cell line HEK293H (Invitrogen) by the lipofection method using PEI (Polyscience). The resulting culture supernatant was collected, and FcRn was purified using IgG Sepharose 6 Fastflow (Amersham biosciences), followed by further purification using HiTrap Q HP (GEHealthcare) (J Immunol.2002, 11/1; 169 (9): 5171-80).
[ reference example 5] preparation of human IgA (hIgA)
hIgA comprising H (WT) -IgA1(SEQ ID NO: 29) and L (WT) (SEQ ID NO: 5) was expressed and purified by methods known to those skilled in the art using rProtein L-Sepharose (ACTIgen) followed by gel filtration chromatography.
[ reference example 6] preparation of soluble human Plexin A1(hsPlexin A1)
Recombinant soluble human plexin a1 (hereinafter also referred to as hsPlexinA a1) was prepared as an antigen as follows. hsPlexin a1 was constructed with reference to the NCBI reference sequence (NP _ 115618). In particular, hsPlexin A1 consisting of amino acid sequence 27-1243 from the NCBI reference FLAG tag (DYKDDDDK) described above was ligated to its C-terminus. hsPlexin a1 was transiently expressed using FreeStyle293(Invitrogen) and purified from culture supernatants by the following two steps: anti-FLAG column chromatography and gel filtration chromatography. The fraction eluted as the main peak in the final stage was used as the final purified product.
Claims (57)
1. An antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, which has human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is greater than KD 3.2 micromolar.
2. An antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, having human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is 28-fold greater than the human FcRn binding activity of a complete human IgG.
3. An antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, which has human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is greater than KD 2.3 micromolar.
4. An antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, having human FcRn binding activity in the neutral pH range, wherein the human FcRn binding activity in the neutral pH range is 38 times greater than the human FcRn binding activity of a complete human IgG.
5. The antigen binding molecule of any one of claims 1-4, wherein the neutral pH range is pH 7.0-8.0.
6. An antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, wherein the total plasma antigen concentration following administration of the antigen binding molecule to a non-human animal is lower than the total plasma antigen concentration following administration of a reference antigen binding molecule to a non-human animal, the reference antigen binding molecule comprising the same antigen binding domain and a fully human IgG Fc domain as the human FcRn binding domain.
7. An antigen binding molecule, wherein the plasma antigen concentration following administration of the antigen binding molecule to a non-human animal is lower than the total plasma antigen concentration obtained from a non-human animal to which the antigen binding molecule has not been administered.
8. An antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, wherein the antigen/antigen binding molecule molar ratio (C) of the antigen binding molecule is calculated as follows:
C=A/B，
an antigen/antigen binding molecule molar ratio (C') lower than a reference antigen binding molecule comprising the same antigen binding domain and a fully human IgG Fc domain as human FcRn binding domain calculated as follows;
C'=A'/B'，
wherein;
a is the total antigen concentration in plasma following administration of the antigen binding molecule to a non-human animal,
b is the plasma concentration of the antigen binding molecule after administration of the antigen binding molecule to a non-human animal,
a' is the total antigen concentration in plasma after administration of the reference antigen binding molecule to a non-human animal,
b' is the plasma concentration of the antigen-binding molecule after administration of the reference antigen-binding molecule to a non-human animal.
9. An antigen binding molecule according to any of claims 6 to 8, wherein the non-human animal is a human FcRn transgenic mouse.
10. The antigen binding molecule of any one of claims 6-9, wherein said plasma antigen concentration is long term total plasma antigen concentration.
11. The antigen binding molecule of any one of claims 6-9, wherein said plasma antigen concentration is short term plasma total antigen concentration.
12. An antigen binding molecule comprising an antigen binding domain and a human FcRn binding domain, having human FcRn binding activity in acidic and neutral pH ranges and having lower antigen binding activity in the acidic pH range than in the neutral pH range, wherein human FcRn binding activity in the neutral pH range is stronger than the human FcRn binding activity of intact human IgG.
13. The antigen binding molecule of any one of claims 1-11, wherein the antigen binding domain has an antigen binding activity in the acidic pH range that is lower than the antigen binding activity in the neutral pH range.
14. An antigen binding molecule according to claim 12 or 13, wherein the ratio of antigen binding activity in the acidic pH range to the neutral pH range is at least 2 in KD (in the acidic pH range)/KD (in the neutral pH range) values.
15. The antigen binding molecule of any one of claims 12-14, comprising an amino acid mutation of the antigen binding domain, said mutation comprising a substitution of at least one amino acid of the antigen binding domain with histidine or an insertion of at least one histidine.
16. The antigen binding molecule of any one of claims 12-14, wherein the antigen binding domain is obtained from a library of antigen binding domains.
17. An antigen binding molecule according to any of claims 1 to 16, comprising as a human FcRn binding domain an Fc domain resulting from the substitution of at least one amino acid in the Fc domain of a parent IgG with a different amino acid.
18. The antigen binding molecule of any one of claims 1 to 17, wherein the human FcRn binding domain is a human FcRn binding domain comprising an amino acid sequence that replaces at least one of the following amino acids in the Fc domain selected from a parent IgG with a different amino acid: 237. 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering).
19. An antigen binding molecule according to any of claims 1 to 18, comprising a human FcRn binding domain comprising an amino acid substitution in the Fc domain of a parent IgG comprising at least one amino acid substitution selected from the following by EU numbering:
An amino acid substitution in which Gly at position 237 is substituted into Met;
amino acid substitution of Pro at position 238 to Ala;
an amino acid substitution wherein Ser at position 239 is substituted with Lys;
an amino acid substitution wherein Lys at position 248 is replaced with Ile;
an amino acid substitution at Thr at position 250 with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr;
an amino acid substitution of Met at position 252 with Phe, Trp or Tyr;
an amino acid substitution of Ser at position 254 to Thr;
amino acid substitution of Arg at position 255 with Glu;
an amino acid substitution at position 256 of Thr substituted with Asp, Glu or Gln;
an amino acid substitution at position 257 of Pro substituted with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val;
an amino acid substitution wherein Glu at position 258 is substituted with His;
an amino acid substitution at position 265 wherein Asp is substituted with Ala;
270 Asp substituted with an amino acid Phe;
an amino acid substitution wherein Asn at position 286 is replaced with Ala or Glu;
an amino acid substitution of Thr at position 289 with His;
an amino acid substitution wherein Asn at position 297 is substituted with Ala;
298 amino acid substitutions in which Ser is replaced with Gly;
an amino acid substitution wherein Val at position 303 is substituted with Ala;
An amino acid substitution wherein Val at position 305 is substituted with Ala;
a substitution of Thr at position 307 with an amino acid Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr;
an amino acid substitution of Val at position 308 with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr;
an amino acid substitution of Leu or Val at position 309 with Ala, Asp, Glu, Pro or Arg;
an amino acid substitution in which Gln at position 311 is replaced with Ala, His, or Ile;
an amino acid substitution wherein Asp at position 312 is substituted with Ala or His;
an amino acid substitution wherein Leu at position 314 is substituted with Lys or Arg;
an amino acid substitution wherein Asn at position 315 is substituted with Ala or His;
an amino acid substitution wherein Lys at position 317 is substituted with Ala;
an amino acid substitution wherein Asn at position 325 is replaced with Gly;
an amino acid substitution wherein Ile at position 332 is substituted with Val;
an amino acid substitution wherein Lys at position 334 is substituted with Leu;
an amino acid substitution wherein Lys at position 360 is replaced by His;
an amino acid substitution at position 376 of Asp to Ala;
an amino acid substitution in which Glu at position 380 is substituted with Ala;
an amino acid substitution wherein Glu at position 382 is substituted with Ala;
An amino acid substitution of Asn or Ser at position 384 with Ala;
an amino acid substitution wherein Gly at position 385 is substituted by Asp or His;
an amino acid substitution of Gln at position 386 with Pro;
amino acid substitution of Pro at position 387 with Glu;
an amino acid substitution wherein Asn at position 389 is substituted with Ala or Ser;
an amino acid substitution of Ser at position 424 with Ala;
an amino acid substitution of Met at position 428 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr;
an amino acid substitution of His at position 433 with Lys;
an amino acid substitution in which Asn at position 434 is substituted with Ala, Phe, His, Ser, Trp or Tyr;
and an amino acid substitution of Tyr or Phe at position 436 with His.
20. An antigen binding molecule according to any of claims 1 to 18, wherein the human FcRn binding domain comprises at least one amino acid selected from the following in the Fc domain of a parent IgG:
met at amino acid position 237;
ala at amino acid position 238;
lys at amino acid position 239;
an 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, 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;
phe at amino acid position 270;
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;
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 or His at amino acid position 315;
ala at amino acid position 317;
gly at amino acid position 325;
val at amino acid position 332;
a 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 at amino acid position 436 (EU numbering).
21. The antigen binding molecule of any one of claims 18-20, wherein the parent IgG is selected from IgG obtained from a non-human animal.
22. The antigen binding molecule of any of claims 18-20, wherein the parent IgG is a human IgG.
23. An antigen binding molecule as claimed in any one of claims 1 to 22 which has antagonistic activity.
24. The antigen binding molecule of any one of claims 1-23, which binds to a membrane antigen or a soluble antigen.
25. The antigen binding molecule of any one of claims 1-24, wherein the antigen binding domain comprises an artificial ligand that binds to a receptor.
26. The antigen binding molecule of any one of claims 1-24, wherein the antigen binding domain comprises an artificial receptor that binds to a ligand.
27. The antigen binding molecule of any one of claims 1-24, which is an antibody.
28. The antigen binding molecule of claim 27, wherein said antibody is selected from the group consisting of a chimeric antibody, a humanized antibody, or a human antibody.
29. A pharmaceutical composition comprising the antigen binding molecule of any one of claims 1-28.
30. A method for promoting antigen binding molecule mediated uptake of an antigen by a cell by increasing its human FcRn binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range.
31. A method for promoting antigen binding molecule mediated uptake of a cell to an antigen by increasing its human FcRn binding activity in the neutral pH range and decreasing its antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range.
32. A method for increasing the number of antigens to which a single antigen binding molecule can bind by increasing its human FcRn binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range.
33. A method for increasing the number of antigens to which a single antigen binding molecule can bind by increasing its human FcRn binding activity in the neutral pH range and decreasing its antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range.
34. A method for increasing the ability of an antigen binding molecule to eliminate antigen from plasma by increasing its human FcRn binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range.
35. A method for increasing the ability of an antigen binding molecule to eliminate antigen from plasma by increasing its human FcRn binding activity in the neutral pH range and decreasing its antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range.
36. A method for improving the pharmacokinetics of an antigen binding molecule by increasing its human FcRn binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain, and has human FcRn binding activity in the acidic pH range.
37. A method for improving the pharmacokinetics of an antigen binding molecule by increasing its human FcRn binding activity in the neutral pH range and decreasing its antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range.
38. A method for promoting intracellular dissociation of an antigen binding molecule from an antigen to which the antigen binding molecule binds extracellularly, by increasing its human FcRn binding activity in the neutral pH range and decreasing its antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range.
39. A method for promoting the extracellular release of an antigen binding molecule in antigen-bound form taken up into a cell in antigen-free form by increasing its human FcRn binding activity in the neutral pH range and decreasing its antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range.
40. A method for reducing the total plasma antigen concentration or the free plasma antigen concentration in plasma by increasing its human FcRn binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range.
41. A method for reducing the concentration of total plasma antigens or free plasma antigens in plasma by increasing their human FcRn binding activity in the neutral pH range and reducing their antigen binding activity in the acidic pH range to less than the antigen binding activity in the neutral pH range, wherein the antigen binding molecule comprises an antigen binding domain and a human FcRn binding domain and has human FcRn binding activity in the acidic pH range.
42. The method of any one of claims 30-41, wherein the acidic pH range is pH 5.5-6.5 and the neutral pH range is pH 7.0-8.0.
43. The method of any one of claims 30 to 41, wherein the increase in binding activity of human FcRn in the neutral pH range is an increase caused by the substitution of at least one amino acid in a parent IgG Fc domain of the human FcRn binding domain with a different amino acid.
44. The method of any one of claims 30 to 41, wherein the increase in binding activity of human FcRn in the neutral pH range is an increase caused by substitution of at least one of the following amino acids in a parent IgG Fc domain selected from the group consisting of the human FcRn binding domain with a different amino acid: 237. 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering).
45. The method of any one of claims 31, 33, 35, 37-39, and 41, wherein the antigen binding activity of the antigen binding molecule is reduced in the acidic pH range to below the antigen binding activity in the neutral pH range by substituting at least one amino acid of the antigen binding molecule with histidine or inserting at least one histidine.
46. The method of any one of claims 31, 33, 35, 37-39, and 41, wherein the antigen binding domain is obtained from a library of antigen binding domains.
47. The method of any one of claims 31, 33, 35, 37-39, and 41, wherein the decrease in antigen binding activity is expressed in terms of an increase in KD (in acidic pH range)/KD (in neutral pH range) value relative to the ratio of antigen binding activity in the acidic pH range and the neutral pH range prior to histidine substitution or insertion.
48. A method for producing an antigen binding molecule, the method comprising the steps of:
(a) selecting an antigen binding molecule having a human FcRn binding activity of greater than 3.2 micromolar over a neutral pH range obtained by altering at least one amino acid in the human FcRn binding domain of the antigen binding molecule;
(b) obtaining a gene encoding an antigen binding molecule, wherein the human FcRn binding domain prepared in (a) is linked to an antigen binding domain; and
(c) producing an antigen binding molecule using the gene prepared in (b).
49. A method for producing an antigen binding molecule, the method comprising the steps of:
(a) selecting an antigen binding molecule having greater human FcRn binding activity in the neutral pH range than before altering at least one amino acid in the human FcRn binding domain of an antigen binding molecule having human FcRn binding activity in the acidic pH range;
(b) Altering at least one amino acid in the antigen binding domain of the antigen binding molecule and selecting an antigen binding molecule having a stronger antigen binding activity in the neutral pH range than in the acidic pH range;
(c) obtaining a gene encoding an antigen binding molecule, wherein the human FcRn binding domain prepared in (a) and (b) is linked to an antigen binding domain; and
(d) generating an antigen binding molecule using the gene prepared in (c).
50. A method for producing an antigen binding molecule, the method comprising the steps of:
(a) selecting an antigen binding molecule having greater human FcRn binding activity in the neutral pH range than before altering at least one amino acid in the human FcRn binding domain of an antigen binding molecule having human FcRn binding activity in the acidic pH range;
(b) selecting an antigen binding molecule having a greater antigen binding activity in the neutral pH range than in the acidic pH range;
(c) obtaining a gene encoding an antigen binding molecule, wherein the human FcRn binding domain prepared in (a) and (b) is linked to an antigen binding domain; and
(d) generating an antigen binding molecule using the gene prepared in (c).
51. An antigen-binding molecule produced by the production method of any one of claims 48 to 50.
52. A method for screening for antigen binding molecules, the method comprising the steps of:
(a) selecting an antigen binding molecule having a human FcRn binding activity of greater than 3.2 micromolar over a neutral pH range obtained by altering at least one amino acid in the human FcRn binding domain of the antigen binding molecule;
(b) obtaining a gene encoding an antigen binding molecule, wherein the human FcRn binding domain prepared in (a) is linked to an antigen binding domain; and
(c) producing an antigen binding molecule using the gene prepared in (b).
53. A method for screening for antigen binding molecules, the method comprising the steps of:
(a) selecting an antigen binding molecule having greater human FcRn binding activity in the neutral pH range than before altering at least one amino acid in the human FcRn binding domain of an antigen binding molecule having human FcRn binding activity in the acidic pH range;
(b) altering at least one amino acid in the antigen binding domain of the antigen binding molecule and selecting an antigen binding molecule having a stronger antigen binding activity in the neutral pH range than in the acidic pH range;
(c) Obtaining a gene encoding an antigen binding molecule, wherein the human FcRn binding domain prepared in (a) and (b) is linked to an antigen binding domain; and
(d) generating an antigen binding molecule using the gene prepared in (c).
54. A method for screening for antigen binding molecules, the method comprising the steps of:
(a) selecting an antigen binding molecule having greater human FcRn binding activity in the neutral pH range than before altering at least one amino acid in the human FcRn binding domain of an antigen binding molecule having human FcRn binding activity in the acidic pH range;
(b) selecting an antigen binding molecule having a greater antigen binding activity in the neutral pH range than in the acidic pH range;
(c) obtaining a gene encoding an antigen binding molecule, wherein the human FcRn binding domain prepared in (a) and (b) is linked to an antigen binding domain; and
(d) generating an antigen binding molecule using the gene prepared in (c).
55. The method of any one of claims 30-54, wherein the antigen binding domain comprises an artificial ligand that binds to a receptor.
56. The method of any one of claims 30-54, wherein the antigen binding domain comprises an artificial receptor that binds to a ligand.
57. The method of any one of claims 30-54, wherein the antigen binding molecule is an antibody.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010-079667 | 2010-03-30 | ||
| JP2010-250830 | 2010-11-09 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1178173A true HK1178173A (en) | 2013-09-06 |
| HK1178173B HK1178173B (en) | 2021-09-03 |
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