HK1196623B - Therapeutic antigen-binding molecule with a fcrn-binding domain that promotes antigen clearance - Google Patents

Description

Therapeutic antigen-binding molecules with FcRn-binding domains that promote antigen clearance

Technical Field

The present invention relates to: a modified FcRn-binding domain with improved affinity for the neonatal Fc receptor (FcRn) at neutral pH; an antigen-binding molecule comprising the FcRn-binding domain, which has low immunogenicity, high stability, and forms only a few aggregates; a modified antigen-binding molecule with improved FcRn-binding activity at neutral or acidic pH without increased binding activity to pre-existing anti-drug antibodies at neutral pH; use of the antigen-binding molecule for improving antigen-binding molecule-mediated cellular uptake of an antigen; use of the antigen-binding molecule for reducing plasma specific antigen concentration; use of the modified FcRn-binding domain for increasing the total number of antigens that a single antigen-binding molecule can bind before its degradation; use of the modified FcRn-binding domain for improving the pharmacokinetics of an antigen-binding molecule; a method for reducing binding activity to pre-existing anti-drug antibodies; and a method for producing the antigen-binding molecule.

Background Art

Due to its high stability in blood plasma and few side effects, more and more antibodies are used as medicines. Conventional antibodies targeting soluble antigens bind the antigen in patient's blood plasma after injection, and then stably persist in the form of antibody-antigen complex until degradation. Although typical antibodies generally have a long half-life (1-3 weeks), antigen has a relatively short half-life of less than one day. Therefore, the antigen in the complex with the antibody has a significantly longer half-life than only antigen. Therefore, after conventional antibodies are injected, antigen concentration often increases. This has been reported for antibodies targeting various soluble antigens, such as IL-6 (J Immunotoxicol. 2005, 3, 131-9. (NPL 1)); beta-amyloid (MAbs. 2010 September-October; 2(5): 576-88 (NPL 2)); MCP-1 (ARTHRITIS & RHEUMATISM 2006, 54, 2387-92 (NPL 3)); hepcidin (AAPS J. 2010, 12(4): 646-57. (NPL 4)) and sIL-6 receptor (Blood. 2008 November 15; 112(10): 3959-64. (NPL 5)). The reports describe an increase in total plasma antigen concentration from baseline of approximately 10-1000 times (depending on the antigen) upon administration of the antibody.

Since this increase in total plasma antigen concentration is not required, strategies for eliminating antigens through therapeutic antibodies have been developed. One of these strategies is to use pH-dependent antigen-binding antibodies with improved binding affinity for the neonatal Fc receptor (FcRn) for IgG to quickly process antigens (see, for example, PCT application number PCT/JP2011/001888 (PTL1)). FcRn is a protein present in the membranes of many cells. Antibodies with improved binding activity to FcRn at neutral pH can bind to FcRn on the cell surface, whereby the antibody and receptor are internalized into cells in vesicles. Since the pH inside the vesicles gradually decreases, the antigen dissociates from the pH-dependent antigen-binding antibody due to its low affinity at acidic pH. The dissociated antigen is then degraded, and the FcRn and bound antibody are recycled back to the cell surface before degradation. Therefore, pH-dependent antigen-binding antibodies with improved binding activity to FcRn at neutral pH can be used to eliminate antigens from plasma and reduce their plasma concentrations.

Previous studies have also shown that Fc engineering to improve binding affinity for FcRn at acidic pH can also improve endosomal recycling efficiency and antibody pharmacokinetics. For example, the M252Y/S254T/T256E (YTE) variant (J Biol Chem, 2006, 281: 23514-23524. (NPL 6)), the M428L/N434S (LS) variant (Nat Biotechnol, 2010 28: 157-159. (NPL 7)), the T250Q/M428L (J Immunol. 2006, 176 (1): 346-56. (NPL 8)), and the N434H variant (Clinical Pharmacology & Therapeutics (2011) 89 (2): 283-290. (NPL 9)) showed an improvement in half-life relative to native IgG1.

However, such substitutions also carry the risk of altering antibody properties that are important for the development of therapeutic antibodies, such as antibody stability, immunogenicity, aggregation behavior, and binding affinity to pre-existing antibodies (e.g., rheumatoid factor). It is therefore a primary object of the present invention to provide modified FcRn binding domains that not only improve antibody clearance but also meet the criteria for developing therapeutic antigen-binding molecules. These developability criteria are, in particular, high stability, low immunogenicity, low percentage of aggregates, and low binding affinity to pre-existing anti-drug antibodies (ADA).

The following are prior art documents related to the present invention. All documents cited in this specification are incorporated herein by reference.

Reference List

Patent Literature

[PTL1] PCT/JP2011/00188 (WO/2011/122011), Antigen-Binding Molecules That Promote Antigen Clearance

Non-patent literature

[NPL1]Martin PL, Cornacoff J, Prabhakar U, Lohr T, Treacy G, Sutherland JE, Hersey S, Martin E; Reviews Preclinical Safety and Immune-Modulating Effects of Therapeutic Monoclonal Antibodies to Interleukin-6 and Tumor Necrosis Factor-alpha in Cynomolgus Macaques; J Immunotoxicol. 2005, 3, 131-9.

[NPL2] Davda JP, Hansen RJ.; Properties of a general PK/PD model of antibody-ligand interactions for therapeutic antibodies that bind to soluble endogenous targets; MAbs. 2010 Sep-Oct; 2(5): 576-88.

[NPL3] Haringman JJ, Gerlag DM, Smeets TJ, Baeten D, van den Bosch F, Bresnihan B, Breedveld FC, Dinant HJ, Legay F, Gram H, Loetscher P, Schmouder R, Woodworth T, Tak PP.; A randomized controlled trial with an anti-CCL2 (anti-monocyte chemotactic protein 1) monoclonal antibody in patients with rheumatoid arthritis; ARTHRITIS and RHEUMATISM 2006, 54, 2387-92.

[NPL4] Xiao JJ, Krzyzanski W, Wang YM, Li H, Rose MJ, Ma M, Wu Y, Hinkle B, Perez-Ruixo JJ.; Pharmacokinetics of anti-hepcidin monoclonal antibody Ab12B9m and hepcidin in cynomolgus monkeys; AAPSJ. 2010, 12(4), 646-57).

[NPL5] Nishimoto N, Terao K, Mima T, Nakahara H, Takagi N, Kakehi T.; Mechanisms and pathologic significances in increase in serum interleukin-6 (IL-6) and soluble IL-6 receptor after administration of an anti-IL-6 receptor antibody, tocilizumab, in patients with rheumatoid arthritis and Castleman disease; Blood. 2008 Nov 15;112(10):3959-64.

[NPL6]J Biol Chem,2006,281:23514-23524

[NPL7]Nat Biotechnol,201028:157-159

[NPL8]J Immunol.2006,176(1):346-56

[NPL9]Clinical Pharmacology&Therapeutics(2011)89(2):283-290

SUMMARY OF THE INVENTION

Technical issues

The present invention was conceived in light of the above circumstances. One object of the present invention is to provide a modified FcRn-binding domain with improved affinity for FcRn at neutral pH; an antigen-binding molecule comprising the FcRn-binding domain, wherein the antigen-binding molecule has low immunogenicity, high stability, and forms only a few aggregates; a modified antigen-binding molecule with improved FcRn-binding activity at neutral or acidic pH without increased binding activity to pre-existing anti-drug antibodies at neutral pH; use of the antigen-binding molecule for improving antigen-binding molecule-mediated cellular uptake of an antigen; use of the antigen-binding molecule for reducing plasma specific antigen concentration; use of the modified FcRn-binding domain for increasing the total number of antigens that a single antigen-binding molecule can bind before it is degraded; use of the modified FcRn-binding domain for improving the pharmacokinetics of an antigen-binding molecule; and a method for producing the antigen-binding molecule.

Solutions to the Problem

The present inventors have conducted intensive research into modified FcRn-binding domains with improved affinity for FcRn at neutral pH, and antigen-binding molecules comprising such FcRn-binding domains that have low immunogenicity, high stability, and form only a few aggregates. As a result, the present inventors discovered that substitutions at specific positions within the FcRn-binding domain improve affinity for FcRn at neutral pH without significantly increasing immunogenicity, significantly decreasing stability, and/or significantly increasing the proportion of high molecular weight species.

Furthermore, the present inventors have conducted intensive research into modified FcRn-binding domains that have improved affinity for FcRn at neutral or acidic pH without significantly increasing binding activity for pre-existing anti-drug antibodies, and antigen-binding molecules comprising such FcRn-binding domains. As a result, the present inventors discovered that substitutions at specific positions in the FcRn-binding domain reduce affinity for pre-existing anti-drug antibodies at neutral pH without significantly reducing FcRn-binding activity.

Specifically, the present invention relates to:

[1] An antigen-binding molecule comprising a modified FcRn-binding domain, wherein the modified FcRn-binding domain comprises an amino acid substitution at one or more positions selected from the group consisting of: EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436, wherein the numbers indicate the positions of the substitution according to EU numbering.

[2][1] The antigen-binding molecule, wherein the FcRn-binding domain has

a) amino acid substitutions of the amino acids at positions EU252 and EU434; and

b) an amino acid substitution at one or more positions selected from the group consisting of: EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU387, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436.

[3] The antigen-binding molecule of [1] or [2], wherein the modified FcRn-binding domain comprises:

Aspartic acid at position EU238,

Valine at position EU250,

Tyrosine at position EU252,

Threonine at position EU254,

Leucine at position EU255,

Glutamic acid at position EU256,

Aspartic acid or isoleucine at position EU258,

Glutamic acid at position EU286,

Glutamine at position EU307,

Proline at position EU308,

Glutamic acid at position EU309,

Alanine or histidine at position EU311,

Aspartic acid at position EU315,

Isoleucine at position EU428,

Alanine, lysine, proline, arginine or serine at position EU433,

Tyrosine or tryptophan at position EU434, and/or

Isoleucine, leucine, valine, threonine or phenylalanine at position EU436.

[4] The antigen-binding molecule of [2], wherein the FcRn-binding domain comprises an amino acid substitution at one or more positions selected from the group consisting of:

a) EU252, EU434 and EU436;

b) EU252, EU307, EU311 and EU434;

c) EU252, EU315 and EU434;

d) EU252, EU308 and EU434;

e) EU238, EU252 and EU434;

f) EU252, EU434, EU307, EU311 and EU436; and

g) EU252, EU387 and EU434.

[5][4] The antigen-binding molecule of [5][4], wherein the FcRn-binding domain comprises:

a) tyrosine at EU252, aspartic acid at EU315, and tyrosine at EU434; or

b) tyrosine at EU position 252, tyrosine at EU position 434, and isoleucine at EU position 436; or

c) tyrosine at EU position 252, tyrosine at EU position 434, and leucine at EU position 436; or

d) tyrosine at EU position 252, tyrosine at EU position 434, and valine at EU position 436; or

e) Tyrosine at EU position 252, Threonine at EU position 254, Tyrosine at EU position 434, and Isoleucine at EU position 436.

[6] The antigen-binding molecule of [2], wherein the FcRn-binding domain comprises amino acid substitutions at three or more positions, wherein the three or more positions are selected from one of the following combinations:

a)EU252/EU434/EU307/EU311/EU286;

b)EU252/EU434/EU307/EU311/EU286/EU254;

c)EU252/EU434/EU307/EU311/EU436;

d)EU252/EU434/EU307/EU311/EU436/EU254;

e)EU252/EU434/EU307/EU311/EU436/EU250;

f)EU252/EU434/EU308/EU250;

g) EU252/EU434/EU308/EU250/EU436; and

h)EU252/EU434/EU308/EU250/EU307/EU311.

[7][6] The antigen-binding molecule of [7][6], wherein the FcRn-binding domain comprises:

a) tyrosine at EU position 252, glutamic acid at EU position 286, glutamine at EU position 307, alanine at EU position 311, and tyrosine at EU position 434; or

b) tyrosine at EU position 252, threonine at EU position 254, glutamic acid at EU position 286, glutamine at EU position 307, alanine at EU position 311, and tyrosine at EU position 434; or

c) tyrosine at EU position 252, glutamine at EU position 307, alanine at EU position 311, tyrosine at EU position 434, and isoleucine at EU position 436; or

d) tyrosine at EU252, threonine at EU254, glutamic acid at EU286, glutamine at EU307, alanine at EU311, tyrosine at EU434, and isoleucine at EU436; or

e) valine at EU position 250, tyrosine at EU position 252, threonine at EU position 254, proline at EU position 308, tyrosine at EU position 434, and valine at EU position 436; or

f) valine at EU position 250, tyrosine at EU position 252, glutamine at EU position 307, alanine at EU position 311, tyrosine at EU position 434, and valine at EU position 436; or

g) tyrosine at EU252, glutamine at EU307, alanine at EU311, tyrosine at EU434, and valine at EU436; or

h) valine at EU position 250, tyrosine at EU position 252, proline at EU position 308, and tyrosine at EU position 434; or

i) Valine at EU250, Tyrosine at EU252, Glutamine at EU307, Proline at EU308, Alanine at EU311 and Tyrosine at EU434.

[8] The antigen-binding molecule of [2], wherein the FcRn-binding domain comprises amino acid substitutions at three or more positions, wherein the three or more positions are selected from one of the following combinations:

a) EU252 and EU434 and EU307 and EU311 and EU436 and EU286;

b) EU252 and EU434 and EU307 and EU311 and EU436 and EU250 and EU308;

c) EU252 and EU434 and EU307 and EU311 and EU436 and EU250 and EU286 and EU308;

d) EU252 and EU434 and EU307 and EU311 and EU436 and EU250 and EU286 and EU308 and EU428.

[9][8] The antigen-binding molecule of [9][8], wherein the FcRn-binding domain comprises:

a) tyrosine at EU position 252, glutamic acid at EU position 286, glutamine at EU position 307, alanine at EU position 311, tyrosine at EU position 434, and valine at EU position 436; or

b) valine at EU position 250, tyrosine at EU position 252, glutamine at EU position 307, proline at EU position 308, alanine at EU position 311, tyrosine at EU position 434, and valine at EU position 436; or

c) valine at EU position 250, tyrosine at EU position 252, glutamic acid at EU position 286, glutamine at EU position 307, proline at EU position 308, alanine at EU position 311, tyrosine at EU position 434 and valine at EU position 436; or

d) Valine at EU250, tyrosine at EU252, glutamic acid at EU286, glutamine at EU307, proline at EU308, alanine at EU311, tyrosine at EU434 and valine at EU436.

[10] The antigen-binding molecule of [2], wherein the FcRn-binding domain comprises amino acid substitutions at three or more positions, wherein the three or more positions are selected from one of the following combinations:

a) EU434, EU307 and EU311;

b) EU434 and EU307 and EU309 and EU311; or

c) EU434 and EU250 and EU252 and EU436.

[11][10] The antigen-binding molecule, wherein the FcRn-binding domain comprises:

a) glutamine at EU position 307, histidine at EU position 311, and tyrosine at EU position 434; or

b) glutamine at EU position 307, glutamic acid at EU position 309, alanine at EU position 311, and tyrosine at EU position 434; or

c) glutamine at EU position 307, glutamic acid at EU position 309, histidine at EU position 311, and tyrosine at EU position 434; or

d) Valine at EU position 250, tyrosine at EU position 252, tyrosine at EU position 434, and valine at EU position 436.

[12] The antigen-binding molecule of any one of [1] to [11], wherein the ratio of high molecular weight species is less than 2%.

[13] The antigen-binding molecule of any one of [1] to [12], wherein the antigen-binding molecule comprises an antigen-binding domain having the following:

a) The binding activity to the antigen is lower at pH 5.5-6.5 than at pH 7-8 or

b) "Calcium concentration-dependent binding" activity to antigens.

[14] The antigen-binding molecule of any one of [1]-[5], wherein the binding molecule has an FcRn binding activity of 50-150 nM at pH 7, a Tm greater than 63.0°C, and an Epibase score less than 250.

[15] The antigen-binding molecule of any one of [1]-[3] and [6]-[7], wherein the binding molecule has an FcRn binding activity of 15-50 nM at pH 7, a Tm greater than 60°C, and an Epibase score less than 500.

[16] The antigen-binding molecule of any one of [1]-[3] and [8]-[9], wherein the binding activity of the binding molecule to FcRn is stronger than 15 nM at pH 7, the Tm is higher than 57.5°C, and the Epibase score is less than 500.

[17] The antigen-binding molecule of any one of [1]-[3], wherein the FcRn-binding domain comprises the following amino acid substitutions:

a) amino acid substitutions at positions EU238, EU255 and/or EU258, and

b) amino acid substitution at 3 or more positions, wherein the 3 or more positions are one of the combinations provided in Tables 4-7.

[18] The antigen-binding molecule of any one of [1]-[17], wherein

a) at EU position 257 of the FcRn binding domain, the amino acid is not selected from the group consisting of alanine, valine, isoleucine, leucine and threonine, and/or

b) At EU252 position of the FcRn binding domain, the amino acid is not tryptophan.

[19] The antigen-binding molecule of any one of [1] to [18], wherein the antigen-binding molecule has binding activity to a pre-existing anti-drug antibody that is not significantly improved compared to the binding affinity of a control antibody comprising a complete FcRn-binding domain.

[20][19] The antigen-binding molecule of claim 19, wherein the FcRn-binding domain further comprises an amino acid substitution at one or more positions selected from the group consisting of: EU387, EU422, EU424, EU426, EU433, EU436, EU438, and EU440.

[21][20] The antigen-binding molecule of [21][20], wherein the FcRn-binding domain comprises one or more amino acid substitutions selected from the group consisting of:

Arginine at position EU387,

Glutamic acid, arginine, or serine, aspartic acid, lysine, threonine, or glutamine at position EU422;

Glutamic acid, arginine, lysine, or asparagine at position EU424;

Aspartic acid, glutamine, alanine, or tyrosine at position EU426;

Aspartic acid at position EU433;

Threonine at position EU436;

Glutamic acid, arginine, serine, or lysine at position EU438; and

Glutamic acid, aspartic acid or glutamine at position EU440.

[22] The antigen-binding molecule of any one of [1]-[21], wherein the modified FcRn-binding domain comprises three or more substitutions, wherein the three or more substitutions are one of the combinations provided in Tables 12-13.

[23] The antigen-binding molecule of any one of [1]-[22], wherein the modified FcRn-binding domain comprises three or more substitutions, wherein the three or more substitutions are one of the combinations provided in Tables 14-15.

[24] The antigen-binding molecule of any one of [20]-[23], wherein the FcRn-binding domain comprises:

a) tyrosine at EU position 252, arginine at EU position 387, tyrosine at EU position 434, and valine at EU position 436; or

b) tyrosine at position EU252, glutamic acid at position EU422, tyrosine at position EU434, and valine at position EU436; or

c) tyrosine at EU position 252, arginine at EU position 422, tyrosine at EU position 434, and valine at EU position 436; or

d) tyrosine at EU252, serine at EU422, tyrosine at EU434, and valine at EU436; or

e) tyrosine at EU252, glutamic acid at EU424, tyrosine at EU434, and valine at EU436; or

f) tyrosine at EU252, arginine at EU424, tyrosine at EU434, and valine at EU436; or

g) tyrosine at EU252, tyrosine at EU434, valine at EU436, and glutamic acid at EU438; or

h) tyrosine at EU position 252, tyrosine at EU position 434, valine at EU position 436, and arginine at EU position 438; or

i) tyrosine at EU position 252, tyrosine at EU position 434, valine at EU position 436, and serine at EU position 438; or

j) Tyrosine at EU252, Tyrosine at EU434, Valine at EU436 and Glutamic acid at EU440.

[25] The antigen-binding molecule of any one of [1] to [24], wherein the antigen-binding molecule is an antibody.

[26] Use of the antigen-binding molecule of any one of [1] to [25] for improving antigen-binding molecule-mediated cellular uptake of an antigen.

[27] Use of the antigen-binding molecule of any one of [1]-[25] for reducing the concentration of a specific antigen in plasma, wherein the antigen-binding molecule comprises an antigen-binding domain that can bind to the antigen.

[28] A method for improving the pharmacokinetics of an antigen-binding molecule, the method comprising the step of introducing amino acid substitutions into the FcRn-binding domain of the antigen-binding molecule at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436.

[29] A method for delayed elimination of an antigen-binding molecule in a subject, the method comprising the step of introducing amino acid substitutions into the FcRn-binding domain of the antigen-binding molecule at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436.

[30] A method for prolonging the plasma retention time of an antigen-binding molecule, the method comprising the step of introducing amino acid substitutions into the FcRn-binding domain of the antigen-binding molecule at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436.

[31] A method for increasing the plasma antigen elimination rate of an antigen-binding molecule, the method comprising the step of introducing amino acid substitutions into the FcRn-binding domain of the antigen-binding molecule at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436.

[32] A method for improving the ability of an antigen-binding molecule to eliminate plasma antigens, the method comprising the step of introducing amino acid substitutions into the FcRn-binding domain of the antigen-binding molecule at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436.

[33] The method of any one of [28]-[32], wherein an additional amino acid substitution is introduced into the FcRn binding domain at EU position 256.

[34] The method of any one of [28]-[33], wherein the method further comprises the step of introducing an amino acid substitution into the FcRn binding domain at one or more positions selected from the group consisting of: EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440.

[35] A method for producing the antigen-binding molecule of any one of [1] to [25], comprising the following steps:

(a) selecting a parent FcRn binding domain and altering the parent FcRn by introducing amino acid substitutions at one or more positions selected from the group consisting of: EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436;

(b) selecting an antigen-binding domain of an antigen-binding molecule and changing at least one amino acid in the antigen-binding domain to obtain a pH-dependent antigen-binding domain or a calcium ion-dependent antigen-binding domain;

(c) obtaining a gene encoding an antigen-binding molecule in which the human FcRn-binding domain and the antigen-binding domain prepared in (a) and (b) are linked, and

(d) Producing antigen-binding molecules using the gene prepared in (c).

[36] The method of [35], wherein in step a) an additional amino acid substitution is introduced into the FcRn binding domain at EU position 256.

[37] The method of any one of [35]-[36], wherein the method further comprises the step of introducing an amino acid substitution into the FcRn binding domain at one or more positions selected from the group consisting of: EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440.

[38] An antigen binding molecule comprising a modified FcRn binding domain, wherein the modified FcRn binding domain comprises an amino acid substitution at one or more positions selected from the group consisting of: EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440, wherein the binding affinity of the antigen binding molecule for a pre-existing anti-drug antibody (ADA) at neutral pH is not significantly increased compared to the binding affinity of the antigen binding molecule comprising the intact FcRn binding domain.

[39] The antigen-binding molecule of [38], wherein the antigen-binding molecule further has increased binding affinity for FcRn in a neutral or acidic pH range.

[40] The antigen-binding molecule of [38] or [39], wherein one or more amino acids selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440 are substituted

a) Arginine at position EU387;

b) glutamic acid, arginine, serine, aspartic acid, lysine, threonine or glutamine at position EU422;

c) glutamic acid, arginine, lysine or asparagine at position EU424;

d) Aspartic acid, glutamine, alanine or tyrosine at position EU426;

e) Aspartic acid at position EU433

f) Threonine at position EU436

g) glutamic acid, arginine, serine or lysine at position EU438; and

h) Glutamic acid, aspartic acid or glutamine at position EU440.

The antigen-binding molecule of any one of [41][38]-[40], wherein the modified FcRn-binding domain comprises an amino acid substitution at one or more positions or combinations provided in Table 10.

[42] The antigen-binding molecule of any one of [38]-[40], wherein the modified FcRn-binding domain comprises any one of the amino acid substitutions or combinations of substitutions provided in Table 11.

[43] The antigen-binding molecule of any one of [39] to [42], wherein the modified FcRn-binding domain further comprises an amino acid substitution at one or more positions of the FcRn-binding domain selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU434, and EU436, wherein the substitution increases FcRn-binding activity in the neutral pH or acidic pH range.

[44] The antigen-binding molecule of any one of [39] to [43], wherein the modified FcRn-binding domain comprises an amino acid substitution at the following FcRn-binding domain position:

i) a) EU438/EU440 or b) EU424; and

ii) a) EU434, b) EU252/EU254/EU256; c) EU428/EU434; or d) EU250/EU428.

[45] [44] The antigen-binding molecule, wherein the modified FcRn-binding domain comprises the following amino acid substitutions:

i) a) EU438R/EU440E or b) EU424N; and

ii) a) N434H; b) M252Y/S254T/T256E; c) M428L/N434S; or d) T250Q and M428L (EU numbering).

[46][45] The antigen-binding molecule of [46][45], wherein the modified FcRn-binding domain comprises 3 or more amino acid substitutions, wherein the 3 or more substitutions are one of the combinations provided in Tables 13 and 15.

[47] The antigen-binding molecule of any one of [39] to [42], wherein the modified FcRn-binding domain comprises a substitution at the following position:

a) at one or more positions selected from EU387, EU422, EU424, EU438, EU440, EU433 or two or more positions wherein two positions are one selected from the combination of EU422/EU424 and EU438/EU440; and

b) Two or more positions wherein two of the positions are one of the combinations provided in Table 9.

[48][47] The antigen-binding molecule of [48], wherein the modified FcRn-binding domain comprises 3 or more amino acid substitutions, wherein the 3 or more amino acid substitutions are one of the combinations provided in Table 12 or 14.

[49] The antigen-binding molecule of any one of [39] to [48], wherein the antigen-binding molecule comprises a pH-dependent antigen-binding domain or a calcium ion-dependent antigen-binding domain.

[50] A method for reducing the binding activity of an antigen-binding molecule comprising an FcRn-binding domain to pre-existing ADA, wherein the FcRn-binding domain has enhanced binding activity to FcRn at neutral or acidic pH and enhanced binding activity to pre-existing ADA at neutral pH, the method comprising the following steps:

a) providing an antigen-binding molecule having an FcRn-binding domain with increased binding activity to FcRn at neutral or acidic pH and increased binding activity to pre-existing ADA at neutral pH; and

b) substituting amino acids in the FcRn-binding domain at one or more positions selected from EU387, EU422, EU424, EU426, EU433, EU436, EU438, and EU440 to obtain an antigen-binding molecule with a modified FcRn-binding domain.

[51][50] The method of [51], wherein step b) comprises substituting an amino acid at 3 or more positions, wherein the 3 or more positions are one of the combinations provided in Table 10.

[52] The method of [50], wherein step b) comprises introducing three or more amino acid substitutions into the FcRn binding domain, wherein the three or more amino acid substitutions are one of the combinations provided in Table 11.

[53] A method for increasing the total number of antigens that can be bound by a single antigen-binding molecule without significantly increasing the binding activity to pre-existing ADA at neutral pH compared to the parent antibody, the method comprising the following steps:

a) providing an antigen-binding molecule comprising a parent FcRn-binding domain,

b) altering the parent FcRn binding domain of step a) by substituting an amino acid in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436; and

c) altering the modified FcRn binding domain of step b) by substituting amino acids in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440.

[54] A method for promoting the extracellular release of an antigen-free antigen-binding molecule that has been taken up into a cell in an antigen-bound form without significantly increasing the binding activity of the antigen-binding molecule to pre-existing ADA at neutral pH compared to the parent antibody, the method comprising the following steps:

a) providing an antigen-binding molecule comprising a parent FcRn-binding domain,

b) altering the parent FcRn binding domain by substituting an amino acid in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436 and EU428; and

c) altering the modified FcRn binding domain of step b) by substituting amino acids in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440.

[55] A method for improving the ability of an antigen-binding molecule to eliminate plasma antigens without significantly increasing the binding activity to pre-existing ADA at neutral pH compared to the parent antibody, the method comprising the following steps:

a) providing an antigen-binding molecule comprising a parent FcRn-binding domain,

b) altering the parent FcRn binding domain by substituting an amino acid in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436 and EU428; and

c) altering the modified FcRn binding domain of step b) by substituting amino acids in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440.

[56] A method for improving the pharmacokinetics of an antigen-binding molecule without significantly increasing the binding activity to pre-existing ADA at neutral pH compared to the parent antibody, the method comprising the following steps:

a) providing an antigen-binding molecule comprising a parent FcRn-binding domain,

b) altering the parent FcRn binding domain by substituting an amino acid in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436; and

c) altering the modified FcRn binding domain of step b) by substituting amino acids in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440.

[57] A method for reducing the total plasma antigen concentration or the plasma free antigen concentration without significantly increasing the binding activity to pre-existing ADA at neutral pH compared to the parent antibody, the method comprising the following steps:

a) providing an antigen-binding molecule comprising a parent FcRn-binding domain, wherein the antigen-binding molecule comprises an antigen-binding domain that can bind to the antigen,

b) altering the parent FcRn binding domain by substituting an amino acid in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436; and

c) altering the modified FcRn binding domain of step b) by substituting amino acids in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440.

[58] A method for producing an antigen-binding molecule comprising an FcRn-binding domain having enhanced binding activity to FcRn at neutral or acidic pH and reduced binding activity to pre-existing ADA at neutral pH, the method comprising the following steps:

(a) providing an FcRn-binding domain having enhanced binding activity to FcRn in a neutral or acidic pH range and enhanced binding activity to pre-existing ADA in a neutral pH range,

(b) substituting an amino acid at one or more positions selected from the group consisting of: EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440,

(c) selecting an antigen-binding domain of an antigen-binding molecule and changing at least one amino acid in the antigen-binding domain to obtain a pH-dependent antigen-binding domain, or selecting a calcium ion-dependent antigen-binding domain;

(d) obtaining a gene encoding an antigen-binding molecule in which the human FcRn-binding domain and the antigen-binding domain prepared in (a) and (b) are linked, and

(e) using the gene prepared in (c) to produce an antigen-binding molecule, wherein the produced antigen-binding molecule has improved binding activity to FcRn at neutral or acidic pH and reduced binding activity to endogenous ADA at neutral pH compared to a parent antigen-binding domain having an intact FcRn-binding domain.

[59] The method of [58], wherein the FcRn binding domain having improved binding activity to FcRn and pre-existing ADA in a neutral or acidic pH range and improved binding activity to pre-existing ADA in a neutral pH range comprises amino acid substitutions at one or more positions selected from the group consisting of: EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434 and EU436.

[60] The method of any one of [53]-[57], wherein the amino acid substitution introduced in step a) is located at 3 or more positions, wherein the 3 or more positions are one of the combinations provided in Tables 4-7.

The method of any one of [61][53]-[60], wherein the amino acid substitutions introduced in step b) are located at 3 or more positions, wherein the 3 or more positions are one of the combinations provided in Table 10.

BRIEF DESCRIPTION OF THE DRAWINGS

[Figure 1A] Figure 1A shows a schematic diagram of antigen elimination from plasma by an antibody of the prior art ("conventional antibody") compared to a pH-dependent antigen-binding antibody with enhanced FcRn, both of which bind to soluble antigen at neutral pH. Conventional antibodies bind to antigens in plasma and are nonspecifically taken up by cells into acidic endosomes. Under the acidic conditions of the endosomal system, conventional antibodies bind to FcRn within vesicles and are transported back to the cell surface, where the antibody is released again. During the entire internalization and recycling process, the antigen binds to the antigen-binding domain. At neutral pH, pH-dependent antigen-binding antibodies with enhanced FcRn binding bind to FcRn on the cell surface and are rapidly internalized into the cell, thus being internalized into the cell at a higher frequency than conventional antibodies. Under the acidic conditions of the endosomal system, the antigen dissociates from the modified antibody and is transported to lysosomes, where it is proteolytically degraded. Antibody still bound to FcRn is recycled back to the cell surface. Here, the recycled free antibody can bind to another antigen again. By repeating this cycle of FcRn-mediated uptake, antigen dissociation and degradation, and antibody recycling, this pH-dependent antigen-binding antibody with improved binding affinity to FcRn at neutral pH can deliver significantly larger amounts of antigen to lysosomes than conventional antibodies, thereby significantly reducing the total plasma antigen concentration compared to conventional antibodies.

[Figure 1B] Figure 1B shows a schematic representation of the dissociation of soluble antigen from an IgG antibody with a pH-dependent antigen-binding domain in endosomes. This results in increased antigen elimination and allows the antibody to bind to another antigen in plasma.

[ Fig. 2] Fig. 2 is a graph showing hFcRn binding affinity (x-axis) and Tm on the y-axis of antibodies comprising Fc variants (Fc variants F1-F599: open squares; Fc variants F600-F1052: closed squares).

[ Figure 3] Figure 3 shows a graph of hFcRn binding affinity (x-axis) and high molecular weight (HMW) fraction (expressed in %) (y-axis) of antibodies comprising Fc variants (Fc variants F1-F599: open squares, Fc variants F600-F1050: closed squares).

[ Fig. 4] Fig. 4 is a graph showing the hFcRn binding affinity (x-axis) and immunogenicity scores (Epibase scores) of antibodies comprising Fc variants (Fc variants F1-F599: open squares, Fc variants F600-F1052: closed squares).

[ Figure 5] Figure 5 is a graph showing the hFcRn-binding affinity (x-axis) and melting temperature Tm (y-axis) of antibodies comprising Fc variants having hFcRn-binding affinity stronger than 15 nM (Fc variants of F1-F599 having Kd less than or equal to 15 nM: open squares, Fc variants of F600-F1052 having Kd less than or equal to 15 nM (Group 1): closed squares).

[ Fig. 6 ] Fig. 6 is a graph showing the hFcRn-binding affinity (x-axis) and HMW (expressed in %) (y-axis) of antibodies comprising Fc variants having hFcRn-binding affinity stronger than 15 nM (Fc variants of F1-F599 having Kd less than or equal to 15 nM: open squares; Fc variants of F600-F1052 having Kd less than or equal to 15 nM (Group 1): closed squares).

[ Fig. 7 ] Fig. 7 is a graph showing the hFcRn-binding affinity and immunogenicity scores of antibodies comprising Fc variants having hFcRn-binding affinity stronger than 15 nM (Fc variants of F1-F599 with Kd less than or equal to 15 nM: open squares; Fc variants of F600-F1052 with Kd less than or equal to 15 nM (Group 1): closed squares).

FIG8 is a graph showing the hFcRn-binding affinity and Tm of antibodies comprising Fc variants having hFcRn-binding affinities between 15 nM and 50 nM (Fc variants of F1-F599 with Kd=15-50 nM, open squares; Fc variants of F600-F1052 with Kd=15-50 nM (Group 2): solid squares)

[ Figure 9] Figure 9 shows a graph of hFcRn-binding affinity and HMW (%) of antibodies comprising Fc variants whose hFcRn-binding affinity is between 15 nM and 50 nM (Fc variants of F1-F599 with Kd = 15-50 nM, open squares; Fc variants of F600-F1052 with Kd = 15-50 nM (Group 2): solid squares).

[ Figure 10] Figure 10 shows a graph of the hFcRn binding affinity and immunogenicity scores of antibodies comprising Fc variants whose hFcRn binding affinity is between 15 nM and 50 nM (Fc variants of F1-F599 with Kd = 15-50 nM, open squares; Fc variants of F600-F1052 with Kd = 15-50 nM (Group 2): solid squares).

[ Figure 11] Figure 11 shows a graph of hFcRn-binding affinity and Tm of antibodies comprising Fc variants whose hFcRn-binding affinities are between 50 nM and 150 nM (Fc variants of F1-F599 with Kd = 50-150 nM, open squares; Fc variants of F600-F1052 with Kd = 50-150 nM (Group 3): solid squares).

[ Figure 12] Figure 12 shows a graph of hFcRn-binding affinity and HMW (%) of antibodies comprising Fc variants with hFcRn-binding affinities between 50 nM and 150 nM (Fc variants of F1-F599 with Kd = 50-150 nM, open squares; Fc variants of F600-F1052 with Kd between 50-150 nM (Group 3): closed squares).

[ Fig. 13] Fig. 13 is a graph showing the hFcRn-binding affinity and immunogenicity scores of antibodies comprising Fc variants having hFcRn-binding affinities between 50 nM and 150 nM (Fc variants of F1-F599 with Kd = 50-150 nM: open squares; Fc variants of F600-F1052 with Kd = 50-150 nM (Group 3): closed squares).

[ Figure 14] Figure 14 shows a graph of hFcRn binding affinity and Tm of antibodies comprising Fc variants whose hFcRn binding affinities range from 150 nM to 700 nM (Fc variants F1-F599 with Kd = 150-700 nM, open squares; Fc variants F600-F1052 with Kd = 150-700 nM (Group 4): solid squares).

[ Fig. 15] Fig. 15 is a graph showing the hFcRn-binding affinity and HMW (%) of antibodies comprising Fc variants having hFcRn-binding affinities between 150 nM and 700 nM (Fc variants of F1-F599 with Kd = 150-700 nM: open squares; Fc variants of F600-F1052 with Kd = 150-700 nM (Group 4): closed squares).

[ Figure 16] Figure 16 shows a graph of the hFcRn binding affinity and immunogenicity scores of antibodies comprising Fc variants whose hFcRn binding affinities range from 150 nM to 700 nM (Fc variants of F1-F599 with Kd = 150-700 nM: open squares; Fc variants of F600-F1052 with Kd = 150-700 nM (Group 4): closed squares).

[ Fig. 17] Fig. 17 is a graph showing plasma antigen (hsIL-6R) concentrations over time in human FcRn transgenic mice after injection of Fv4-IgG1, Fv4-F652, Fv4-F890, and Fv4-F946 and in control mice (no antibody injection).

[ Fig. 18] Fig. 18 is a graph showing plasma antibody concentrations over time in human FcRn transgenic mice after injection of Fv4-IgG1, Fv4-F652, Fv4-F890, and Fv4-F946.

[ Fig. 19] Fig. 19 is a graph showing plasma antigen (hsIL-6R) concentrations over time in control (no antibody injection) and human FcRn transgenic mice after injection of Fv4-IgG1, Fv4-F11, and Fv4-F652.

[ Fig. 20] Fig. 20 is a graph showing plasma antibody concentrations over time in human FcRn transgenic mice after injection of Fv4-IgG1, Fv4-F11, and Fv4-F652.

[ Fig. 21] Fig. 21 is a graph showing electrochemiluminescence (ECL) reactions of sera from 30 RA patients against humanized anti-IL-6 receptor antibody Fv4-IgG1 ( Fig. 21-1 ), its YTE variant ( Fig. 21-2 ), and LS variant ( Fig. 21-3 ).

[Figure 22] Figure 22 shows graphs of electrochemiluminescence (ECL) reactions of sera from 15 RA patients against humanized anti-IL-6 receptor antibodies Fv4-IgG1 (Figure 22-1), Fv4-N434H (Figure 22-2), Fv4-F11 (Figure 22-3), Fv4-F68 (Figure 22-4), Fv4-890 (Figure 22-5) and Fv4-F947 (Figure 22-6).

[Figure 23] Figure 23 shows the mean (Figure 23-1), geometric mean (Figure 23-2) and median (Figure 23-3) of the ECL reactions of sera from 15 RA patients shown in Figure 22 against Fv4-IgG1, Fv4-F11, Fv4-F68, Fv4-F890 and Fv4-F947.

[Figure 24] Figure 24 shows graphs of the electrochemiluminescence (ECL) reactions of sera from 15 RA patients against the humanized anti-IL-6 receptor antibody Fv4-IgG1 (Figure 24-1) and variants Fv4-F890, Fv4-F1058, Fv4-F1059, Fv4-F1060, Fv4-F1061, Fv4-F1062, Fv4-F1063, Fv4-F1064, Fv4-F1065, Fv4-F1066, Fv4-F1067, Fv4-F1068, Fv4-F1069, Fv4-F1070, Fv4-F1071, Fv4-F1072, and Fv4-F1073 (Figures 24-2 to 24-18).

[Figure 25] Figure 25 shows the average (Figure 25-1), geometric mean (Figure 25-2) and median (Figure 25-3) of the ECL reactions of sera from 15 RA patients shown in Figure 24 against Fv4-IgG1, variants Fv4-F890, Fv4-F1058, Fv4-F1059, Fv4-F1060, Fv4-F1061, Fv4-F1062, Fv4-F1063, Fv4-F1064, Fv4-F1065, Fv4-F1066, Fv4-F1067, Fv4-F1068, Fv4-F1069, Fv4-F1070, Fv4-F1071, Fv4-F1072 and Fv4-F1073.

[ Fig. 26] Fig. 26 is a graph showing electrochemiluminescence (ECL) reactions of sera from 15 RA patients against variants Fv4-F1104, Fv4-F1105, and Fv4-F1106.

[Figure 27] Figure 27 shows graphs of electrochemiluminescence (ECL) reactions of sera from 15 RA patients against variants Fv4-F1107, Fv4-F1108, Fv4-F1109, Fv4-F1110, Fv4-F1111, Fv4-F1112, Fv4-F1113, and Fv4-F1114 (Figures 27-1 to 27-8).

[ Fig. 28] Fig. 28 shows graphs of electrochemiluminescence (ECL) reactions of sera from 15 RA patients against variants Fv4-F1230 ( Fig. 28-1 ), Fv4-F1231 ( Fig. 28-2 ), and Fv4-F1232 ( Fig. 28-3 ).

[ Fig. 29] Fig. 29 shows graphs of electrochemiluminescence (ECL) reactions of sera from 15 RA patients against variants Fv4-F947, Fv4-F1119, Fv4-F1120, Fv4-F1121, Fv4-F1122, Fv4-F1123, and Fv4-F1124.

[Figure 30] Figures 30-1 to 30-4 show electrochemiluminescence (ECL) reactions of serum from 15 RA patients against variants Fv4-F939, Fv4-F1291, Fv4-F1268, and Fv4-F1269. Figures 30-5 to 30-9 show electrochemiluminescence (ECL) reactions of serum from 30 RA patients against variants Fv4-F1243, Fv4-F1245, Fv4-F1321, Fv4-F1340, and Fv4-F1323.

[ Fig. 31] Fig. 31 is a graph showing electrochemiluminescence (ECL) reactions of sera from 15 RA patients against variants Fv4-F890 ( Fig. 31-1 ) and Fv4-F1115 (=F890+S424N, Fig. 31-2 ).

[Figure 32] Figure 32 shows the serum from 15 or 30 RA patients against the variants Fv4-YTE (Figure 32-1), Fv4-F1166 (=YTE+Q438R/S440E, Figure 32-2), Fv4-F1167 (=YTE+S424N, Figure 32-3), Fv4-LS (Figure 32-4), Fv4-F1170 (=LS+Q438R /S440E, Figure 32-5), Fv4-F1171 (LS+S424N, Figure 32-6), Fv4-N434H (Figure 32-7), Fv4-F1172 (＝N434H+Q438R/S440E, Figure 32-8), and Fv4-F1173 (＝N434H+S424N, Figure 32-9).

[Figure 33] Figure 33 shows graphs of electrochemiluminescence (ECL) reactions of sera from 30 RA patients against variants Fv4-LS, Fv4-F1380 (Figure 33-2), Fv4-F1384 (Figure 33-3), Fv4-F1385 (Figure 33-4), Fv4-F1386 (LS+S426Y, Figure 33-5), Fv4-F1388 (Figure 33-6) and Fv4-F1389 (LS+Y436T, Figure 33-7).

[ Fig. 34] Fig. 34 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F939.

[ Fig. 35] Fig. 35 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1378.

[ Fig. 36] Fig. 36 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1379.

[Fig. 37] Fig. 37 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1262

[Fig. 38] Fig. 38 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1138

[Figure 39] Figure 39 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1344

[Figure 40] Figure 40 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1349

[Fig. 41] Fig. 41 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1350

[Figure 42] Figure 42 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1351

[Fig. 43] Fig. 43 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1261

[Figure 44] Figure 44 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1263

[Fig. 45] Fig. 45 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1305

[Figure 46] Figure 46 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1306

[Figure 47] Figure 47 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1268

[Figure 48] Figure 48 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1269

[Figure 49] Figure 49 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1413

[Fig. 50] Fig. 50 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1416

[Fig. 51] Fig. 51 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1419

[Fig. 52] Fig. 52 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1420

[Fig. 53] Fig. 53 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1370

[Fig. 54] Fig. 54 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1371

[Fig. 55] Fig. 55 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1599

[Fig. 56] Fig. 56 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1600

[Fig. 57] Fig. 57 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1566

[Fig. 58] Fig. 58 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1448

[Figure 59] Figure 59 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1601

[Fig. 60] Figure 60 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1602

[Fig. 61] Fig. 61 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1603

[Fig. 62] Fig. 62 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1531

[Fig. 63] Fig. 63 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1604

[Fig. 64] Figure 64 shows a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1605

[Fig. 65] Figure 65 shows a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1586

[Fig. 66] Figure 66 shows a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1592

[Fig. 67] Figure 67 shows a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1610

[Fig. 68] Figure 68 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1611

[Figure 69] Figure 69 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1612

[Fig. 70] Figure 70 shows a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1613

[Fig. 71] Fig. 71 is a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1614

[Figure 72] Figure 72 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1615

[Figure 73] Figure 73 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1567

[Figure 74] Figure 74 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1572

[Figure 75] Figure 75 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1576

[Figure 76] Figure 76 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1578

[Figure 77] Figure 77 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1579

[Figure 78] Figure 78 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1641

[Fig. 79] Figure 79 shows a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1642

[Fig. 80] Figure 80 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1643

[Fig. 81] Figure 81 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1644

[Figure 82] Figure 82 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1645

[Figure 83] Figure 83 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1646

[Figure 84] Figure 84 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1647

[Figure 85] Figure 85 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1648

[Figure 86] Figure 86 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1649

[Figure 87] Figure 87 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1650

[Figure 88] Figure 88 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1651

[Figure 89] Figure 89 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1652

[Fig. 90] Figure 90 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1653

[Fig. 91] Figure 91 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1654

[Figure 92] Figure 92 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1655

[Fig. 93] Figure 93 shows a graph showing the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1329

[Figure 94] Figure 94 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1331

[Figure 95] Figure 95 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1718

[Figure 96] Figure 96 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1719

[Figure 97] Figure 97 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1720

[Figure 98] Figure 98 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1721

[Figure 99] Figure 99 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1671

[Figure 100] Figure 100 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1670

[Figure 101] Figure 101 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1711

[Figure 102] Figure 102 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1712

[Figure 103] Figure 103 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1713

[Figure 104] Figure 104 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1722

[Figure 105] Figure 105 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1723

[Figure 106] Figure 106 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1724

[Figure 107] Figure 107 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1725

[Figure 108] Figure 108 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1675

[Figure 109] Figure 109 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1714

[Figure 110] Figure 110 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1715

[Figure 111] Figure 111 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1716

[Figure 112] Figure 112 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1717

[Figure 113] Figure 113 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1683

[Figure 114] Figure 114 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1756

[Figure 115] Figure 115 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1757

[Figure 116] Figure 116 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1758

[Figure 117] Figure 117 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1759

[Figure 118] Figure 118 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1681

[Figure 119] Figure 119 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1749

[Figure 120] Figure 120 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1750

[Figure 121] Figure 121 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1751

[Figure 122] Figure 122 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1760

[Figure 123] Figure 123 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1761

[Figure 124] Figure 124 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1762

[Figure 125] Figure 125 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1763

[Figure 126] Figure 126 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1752

[Figure 127] Figure 127 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1753

[Figure 128] Figure 128 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1754

[Figure 129] Figure 129 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1755

[Figure 130] Figure 130 shows the electrochemiluminescence (ECL) reaction of sera from 30 RA patients against variant F1685

[ Fig. 131] Fig. 131 is a graph showing the time-lapse of plasma antibody concentrations in human FcRn transgenic mice after injection of Fv4-IgG1, Fv4-F1243, and Fv4-F1245.

[ Fig. 132] Fig. 132 is a graph showing plasma antigen (hsIL-6R) concentrations over time in human FcRn transgenic mice after injection of Fv4-IgG1, Fv4-F1243, and Fv4-F1245 and in control mice (no antibody injection).

[ Fig. 133] Fig. 133 is a graph showing the time-lapse of plasma antibody concentrations in human FcRn transgenic mice after injection of Fv4-IgG1 and Fv4-F1389.

Figure 134 shows the sensorgrams of SPR analysis. hIgA binding of anti-hIgA antibodies was analyzed under different conditions (pH, Ca concentration).

[ Fig. 135] Fig. 135 is a graph showing the time-lapse of plasma antibody concentrations in human FcRn transgenic mice after injection of GA2-F760 and GA2-F1331.

[ Fig. 136] Fig. 136 is a graph showing the plasma hIgA concentration over time in human FcRn transgenic mice after injection of GA2-F760 and GA2-F1331.

[ Fig. 137] Fig. 137 is a graph showing plasma antibody concentrations over time in human FcRn transgenic mice after injection of 278-F760 and 278-F1331.

[ Fig. 138] Fig. 138 is a graph showing the plasma hIgE(Asp6) concentration over time in human FcRn transgenic mice after injection of 278-F760 and 278-F1331.

Description of the implementation plan

Detailed Description of the Invention

Before describing the materials and methods of the present invention, it should be understood that these descriptions are illustrative only and are not intended to be restrictive. It should also be understood that the present invention is not limited to the specific sizes, shapes, dimensions, materials, methods, schemes, etc. described herein, as these can be changed according to routine experiments and/or optimizations. The terms used in this specification are only used to describe the purpose of a specific form or embodiment and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. In the event of a conflict, this specification (including definitions) shall prevail.

The disclosures of each publication, patent, or patent application mentioned in this specification are expressly incorporated herein by reference in their entirety. However, nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

As used herein, the terms "a," "an," and "the" mean "at least one," unless expressly stated otherwise.

Studies described in WO/2011/122011 indicate that antigen-binding molecules (e.g., anti-IL6 receptor antibodies) with improved binding to FcRn at pH 7.4 are able to eliminate antigens from plasma and reduce total plasma antigen concentrations. Thus, the efficiency of antigen elimination can be improved by pH-dependent antigen binding (binding to antigens in plasma at pH 7.4 and dissociating antigens within acidic endosomes at pH 6.0) or by ionized calcium concentration-dependent antigen binding (binding to antigens within plasma at high ionized calcium concentrations and dissociating antigens within endosomes at low ionized calcium concentrations) (see FIG1B ). FIG1A shows the mechanism by which pH-dependent antigen-binding antibodies with improved binding affinity to FcRn at neutral pH compared to conventional antibodies eliminate antigens from plasma.

The present invention provides novel amino acid substitutions in the FcRn-binding domain that increase the FcRn-binding activity of antigen-binding molecules in acidic and neutral pH ranges, wherein the FcRn-binding activity in the neutral pH range is stronger than that of intact IgG or an antigen-binding molecule comprising an intact FcRn-binding domain (e.g., stronger than 3200 nM). The modified antigen-binding molecules can reduce the total plasma antigen concentration after administration compared to a control antigen-binding molecule comprising the same antigen-binding domain except for the intact human IgG FcRn-binding domain.

Fc receptors are proteins on the surface of immune cells such as natural killer cells, macrophages, neutrophils, and mast cells. They bind to the Fc (crystallizable fragment) region of antibodies, which is linked to infected cells or invasive pathogens and stimulates phagocytes or cytotoxic cells to eliminate microorganisms or infected cells through antibody-mediated phagocytosis or antibody-dependent cell-mediated toxicity.

There are several different types of Fc receptors, which are classified according to the type of antibody they recognize. The term "FcRn" herein refers to the neonatal Fc receptor that binds IgG, is structurally similar to MHC class I proteins, and is encoded in humans by the FCGRT gene.

As used herein, the term "FcRn-binding domain" refers to a protein domain that binds directly or indirectly to FcRn. Preferably, the FcRn is a mammalian FcRn, more preferably a human FcRn. An FcRn-binding domain that directly binds to FcRn is an antibody Fc region. Meanwhile, a region capable of binding to a polypeptide with human FcRn-binding activity (e.g., albumin or IgG) may also bind indirectly to human FcRn via albumin, IgG, etc. Therefore, such a human FcRn-binding domain may be a region that binds to a polypeptide with human FcRn-binding activity.

As used herein, the term "Fc region" or "Fc region of an antigen-binding molecule" refers to an FcRn-binding domain that directly binds to FcRn, preferably a mammalian FcRn, more preferably a human FcRn. Specifically, the Fc region is the Fc region of an antibody. Preferably, the Fc region is a mammalian Fc region, more preferably a human Fc region. Specifically, the Fc region of the present invention is an Fc region comprising the second and third constant domains (CH2 and CH3) of a human immunoglobulin, more preferably an Fc region comprising the hinge, CH2, and CH3. Preferably, the immunoglobulin is IgG. Preferably, the Fc region is the Fc region of human IgG1.

The present invention provides antigen-binding molecules having a modified FcRn-binding domain, wherein the FcRn-binding activity of the antigen-binding molecule in the neutral pH range is improved compared to an antigen-binding molecule having an intact FcRn-binding domain.

Specifically, the present invention provides antigen binding molecules having a modified FcRn binding domain having an amino acid substitution in the FcRn binding domain at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436. The antigen binding molecules of the present invention may also comprise substitutions at other positions. For example, in addition to the substitutions at one or more positions mentioned above, the antigen binding molecule may comprise a substitution at position EU256. Preferably, the amino acid at position EU256 is substituted with glutamic acid.

The term "binding affinity" or "binding activity" refers to the strength of the non-covalent interaction between two substances, as measured by the dissociation constant (KD) of the complex formed by the two substances, unless otherwise specifically defined. For a specific target molecule (e.g., FcRn), a binding protein (or "ligand") may have a KD of, for example, less than 10 ^{^-5}, 10 ^{^-6} , 10^{^-7} , or 10 ^{^-8} M. A numerical KD for binding to a target in a first pH range that is less than the numerical KD for binding to a target in a second pH range may indicate that the binding affinity of the ligand to the target is higher in the first pH range relative to the binding of the ligand to the target in the second pH range. The difference in binding affinity may be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 50, 70, 80, 100, 500, or 1000-fold. Binding affinity can be determined by various methods, including surface plasmon resonance, equilibrium dialysis, equilibrium binding, gel filtration, ELISA, or spectroscopy (e.g., using fluorescence).

The improvement in the binding affinity of the FcRn binding domain for FcRn over a pH range is equivalent to an improvement in the binding affinity for FcRn measured compared to the binding affinity for FcRn measured for the intact FcRn binding domain. The difference in the binding affinity of KD(intact)/KD(variant) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 50, 70, 80, 100, 500, or 1000-fold. The improvement in the binding affinity of the FcRn binding domain for FcRn may be in an acidic or neutral pH range.

The term "antigen-binding molecule comprising an intact FcRn binding domain" refers to an antigen-binding molecule comprising an unmodified FcRn binding domain. As used herein, the term "intact IgG FcRn binding domain" refers to the unmodified FcRn binding domain of human IgG. Specifically, the FcRn binding domain is the FcRn binding domain of intact human IgG. Preferably, the intact FcRn binding domain is an intact Fc region. The term "antibody comprising an intact Fc region" refers to an antibody comprising an unmodified Fc region. The antibody from which the unmodified Fc region is derived is preferably IgG. More preferably, it is human IgG1, IgG2, IgG3, or IgG4, and even more preferably human IgG1. In particularly preferred embodiments of the present invention, the antibody comprising an intact Fc region is an antibody comprising an unmodified Fc region. The antibody comprising an intact Fc region can be an intact human IgG.

As used herein, the term "intact IgG" refers to unmodified IgG and is not limited to a specific class of IgG. This means that human IgG1, IgG2, IgG3, IgG4, or their allotypic variants can be used as "intact human IgG" as long as they can bind to human FcRn in an acidic pH range. Preferably, "intact IgG" is human IgG1. Preferably, intact IgG is IgG that contains a wild-type Fc region.

In the context of the present invention, the enhanced FcRn-binding activity of the antigen-binding molecule in the neutral pH range is preferably stronger than KD 3.2 micromolar. Preferably, the enhanced FcRn-binding activity in the neutral pH range is stronger than 700 nanomolar, more preferably stronger than 500 nanomolar, and most preferably stronger than 150 nanomolar.

The FcRn binding activity of the antigen-binding molecules of the present invention that is increased in the acidic pH range is generally about 2 to about 100 times the FcRn binding activity of intact IgG. Preferably, the FcRn binding activity of the antigen-binding molecules that is increased in the acidic pH range is at least 10 times the FcRn binding activity of intact IgG. More preferably, the FcRn binding activity of the antigen-binding molecules of the present invention that is increased in the acidic pH range is at least 20 times the FcRn binding activity of intact IgG.

As used herein, the terms "neutral pH range" and "neutral pH" generally refer to any pH value within the range of pH 6.7 to pH 10.0, preferably pH 7.0 to pH 8.0, examples of which include pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. A particularly preferred acidic pH value is pH 7.4, which is close to the plasma (blood) pH in vivo.

As used herein, the terms "acidic pH range" and "acidic pH" generally refer to any pH value within the range of pH 4.0 to pH 6.5, preferably pH 5.5 to pH 6.5, examples of which include pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. A particularly preferred acidic pH range is pH 5.8 to pH 6.0, which is close to the pH of early endosomes in vivo.

References to amino acid positions in this application, such as "EU387" or "position 387," are numbered according to the EU numbering system (Kabat, E.A., T.T. Wu, H.M. Perry, K.S. Gottesman, C. Foeler. 1991. Sequences of Proteins of Immunological Interest. No. 91-3242 U.S. Public Health Services, National Institutes of Health, Bethesda), unless otherwise indicated, and refer to positions in the FcRn binding domain, particularly in the Fc region. In a similar manner, substitutions are indicated by, for example, "EU387R" or "EU440E," where the number following "EU" indicates the position of the substitution according to EU numbering, and the letter following the number is the substituted amino acid given in a one-letter code. Substitutions can also be written as (amino acid 1)-position-(amino acid 2), where the first amino acid is the amino acid being substituted and the second amino acid is the replacing amino acid at the specified position.

As used herein, the terms "substitution" and "amino acid substitution" refer to the replacement of an amino acid in an amino acid sequence with another amino acid that is different from the replaced amino acid. Methods for replacing amino acids are well known to those skilled in the art and include, but are not limited to, mutations in the nucleotide sequence encoding the amino acid sequence.

More specifically, amino acid substitutions in an FcRn binding domain refer to amino acid replacements with respect to the amino acid sequence of a parent FcRn binding domain. The FcRn binding domains of the present invention also include modified FcRn binding domains that have been subjected to the desired substitutions.

The parent FcRn binding domain is an FcRn binding domain having the following amino acids and having no affinity or low affinity (weaker than 3200 nM) for FcRn at neutral pH: proline at EU238, threonine at EU250, methionine at EU252, serine at EU254, arginine at EU255, threonine at EU256, glutamic acid at EU258, asparagine at EU286, threonine at EU307 , valine at EU308, leucine at EU309, glutamine at EU311, asparagine at EU315, proline at EU387, valine at EU422, serine at EU424, serine at EU426, methionine at EU428, histidine at EU433, asparagine at EU434, tyrosine at EU436, glutamine at EU438, and serine at EU440. The parent FcRn binding domain may comprise substitutions at other positions, but preferably the parent FcRn binding domain is unmodified. Preferably, the parent FcRn binding domain is an Fc region (parent Fc region). Preferably, the parent Fc region is derived from a mammalian antibody; more preferably, the parent Fc region is the Fc region of a human antibody. The Fc region of a human antibody is referred to herein as a human Fc region.

The parent Fc region is preferably a complete Fc region, more preferably a human complete Fc region. Preferably, the parent Fc region is an IgG Fc region, more preferably a human IgG Fc region. Even more preferably, the parent Fc region is a human Fc region comprising a wild-type hinge, wild-type CH2, and wild-type CH3 domains. In the context of the present invention, the term parent antibody refers to an antibody comprising a parent Fc region.

Parent antigen binding molecules include, but are not limited to, receptor proteins (membrane-bound receptors and soluble receptors), antibodies that recognize membrane antigens (eg, cell surface markers), and antibodies that recognize soluble antigens (eg, cytokines).

As used herein, the term "parent antigen binding molecule" refers to an antigen binding molecule with a parent FcRn binding domain. The source of the "parent antigen binding molecule" is not limited and can be obtained from any organism of a non-human animal or human. Preferably, the organism is selected from mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, cattle, horses, camels, and non-human primates. In another embodiment, the "parent antigen binding molecule" can also be obtained from cynomolgus monkeys, marmosets, macaques, chimpanzees, or humans. The parent IgG can be a naturally occurring IgG or a variant or engineered form of a naturally occurring IgG. The parent IgG can 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 the known commercially available recombinantly produced IgGs outlined below. Preferably, "parent IgG" is obtained from human IgG1, but is not limited to the specific subclass of IgG. This means that human IgG1, IgG2, IgG3, or IgG4 can be appropriately used as "parent IgG." Similarly, any subclass of IgG from any of the above-mentioned organisms can be preferably used as "parent IgG". Examples of variants or engineered forms of naturally occurring IgG are described in Curr Opin Biotechnol. 2009 Dec; 20(6): 685-91; Curr Opin Immunol. 2008 Aug; 20(4): 460-70; Protein Eng Des Sel. 2010 Apr; 23(4): 195-202; WO2009/086320, WO2008/092117, WO2007/041635 and WO2006/105338, but are not limited thereto.

The FcRn binding domains or Fc regions of the present invention may comprise substitutions at two or more positions, which are referred to herein as a "combination" of substitutions. For example, an Fc region defined by the combination "EU424/EU434/EU436" is an Fc region comprising substitutions at positions EU424, EU434, and EU436.

The substituting amino acid (the amino acid in the parent FcRn binding domain is replaced by the amino acid) can be any amino acid, unless explicitly stated herein, including but not limited to: alanine (Ala, A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamic acid (glu, E), glutamine (gln, Q), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V). Preferably, the substituted amino acid at any of positions EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440 is selected from the group consisting of alanine (Ala, A), arginine (arg, R), glutamate (glu, E), glutamine (gln, Q), aspartic acid (asp, D), serine (ser, S), threonine (thr, T), tyrosine (tyr, Y) and lysine (lys, K).

In a preferred embodiment of the present invention, the antigen-binding molecule of the present invention has a modified FcRn-binding domain comprising a substitution at the following positions with an amino acid different from the substituted amino acid:

a) at positions EU252 and EU434, and

b) at one or more positions selected from the group consisting of: EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436.

The substituted amino acid can be any amino acid unless otherwise specified. Preferred substituted amino acids at positions EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434 and EU436 are shown in Table 1.

[Table 1]

Preferred substituted amino acids

Preferably, the modified FcRn-binding domains of the present invention comprise at least one amino acid substitution provided in Table 1. FcRn-binding domains can be used without any modification as long as they have at least one of the amino acids given above at the specified position and have human FcRn-binding activity in the acidic and neutral pH ranges, with improved FcRn-binding activity in the neutral pH range.

In a preferred embodiment, the modified antigen-binding molecule of the present invention comprises modifications at three or more positions in the FcRn-binding domain, wherein the three or more positions are one of the combinations given in Tables 2, 4-7.

[Table 2]

Preferred combinations of substitution positions in the FcRn binding domain

a) EU252/EU434/EU436, b) EU252/EU434/EU307/EU311, c) EU252/EU434/EU315, d) EU252/EU434/EU308, e) EU252/EU434/EU238, f) EU252/EU434/EU436/EU307/EU311, g) EU252/EU434/EU255 h) EU252/EU434/EU258 i) EU252/EU434/EU255/EU258 j) EU252/EU434/EU255/EU258

In a more preferred embodiment, the antigen binding molecules of the present invention comprise three or more amino acid substitutions in the FcRn binding domain, wherein the three or more substitutions are one of the combinations given in Tables 3, 12, 14, and 17-20.

[Table 3]

Preferred substitution combinations in the FcRn binding domain

1 M252Y/N434Y/Y436V 2 M252Y/N434Y/Y436T 3 M252Y/N434Y/Y436F 4 M252Y/N434Y/Y436V 5 M252Y/N434Y/Y436V 6 M252Y/N434Y/Y436T 7 M252Y/N434Y/Y436T 8 M252Y/N434Y/Y436F 9 M252Y/N434Y/Y436F 10 M252Y/N434Y/Y436V 11 M252Y/N434Y/Y436V 12 M252Y/H433D/N434Y/Y436V 13 M252Y/H433D/N434Y/Y436V 14 M252Y/H433D/N434Y/Y436V 15 M252Y/H433D/N434Y/Y436V 16 M252Y/S254T/T256E/T307Q/Q311A/H433D/N434Y/Y436V 17 M252Y/S254T/T256E/V308P/H433D/N434Y/Y436V 18 M252Y/H433D/N434W/Y436V 19 M252Y/H433D/N434W/Y436V 20 M252Y/S254T/T256E/H433D/N434Y/Y436V twenty one M252Y/S254T/T256E/H433D/N434Y/Y436V twenty two M252Y/S254T/T256E/H433D/N434Y/Y436V twenty three M252Y/S254T/T256E/H433D/N434Y/Y436V twenty four M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V 25 M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V 26 M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V 27 M252Y/S254T/T256E/N286E/H433D/N434Y/Y436V 28 M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V 29 M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V 30 M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V 31 M252Y/S254T/R255L/T256E/H433D/N434Y/Y436V 32 M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V 33 M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V

34 M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V 35 M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V 36 M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V 37 M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V 38 M252Y/S254T/R255L/T256E/E258D/H433D/N434Y/Y436V 39 M252Y/S254T/R255L/T256E/E258I/H433D/N434Y/Y436V 40 M252Y/S254T/T256E/H433A/N434Y/Y436V 41 M252Y/S254T/T256E/H433K/N434Y/Y436V 42 M252Y/S254T/T256E/H433P/N434Y/Y436V 43 M252Y/S254T/T256E/H433R/N434Y/Y436V 44 M252Y/S254T/T256E/H433S/N434Y/Y436V 45 M252Y/S254T/T256E/H433A/N434Y/Y436V 46 M252Y/S254T/T256E/H433A/N434Y/Y436V 47 M252Y/S254T/T256E/H433A/N434Y/Y436V 48 M252Y/S254T/T256E/H433K/N434Y/Y436V 49 M252Y/S254T/T256E/H433K/N434Y/Y436V 50 M252Y/S254T/T256E/H433K/N434Y/Y436V 51 M252Y/S254T/T256E/H433P/N434Y/Y436V 52 M252Y/S254T/T256E/H433P/N434Y/Y436V 53 M252Y/S254T/T256E/H433P/N434Y/Y436V 54 M252Y/S254T/T256E/H433R/N434Y/Y436V 55 M252Y/S254T/T256E/H433R/N434Y/Y436V 56 M252Y/S254T/T256E/H433R/N434Y/Y436V 57 M252Y/S254T/T256E/H433S/N434Y/Y436V 58 M252Y/S254T/T256E/H433S/N434Y/Y436V 59 M252Y/S254T/T256E/H433S/N434Y/Y436V 60 L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V 61 L235R/G236R/S239K/M252Y/S254T/T256F/N434Y/Y436V 62 P238D/M252Y/V308P/N434Y 63 P238D/M252W/N434Y 64 P238D/M252Y/M428F/N434Y

Stability, immunogenicity, and aggregate formation

Modification of the FcRn-binding domain by introducing substitutions can reduce the stability of the antigen-binding molecule (WO/2007/092772). The stability of pharmaceutical proteins is crucial for drug production, as poorly stable proteins tend to aggregate during storage. Therefore, reduced stability caused by substitutions in the Fc region can make it difficult to develop stable formulations (WO2007/092772).

Furthermore, the purity of the drug protein, both in terms of monomeric and high-molecular-weight species, is also crucial for drug development. While wild-type IgG1 does not contain significant amounts of high-molecular-weight species after protein A purification, engineering the FcRn-binding domain by introducing substitutions can result in larger amounts of these species. In such cases, the high-molecular-weight species need to be removed from the bulk drug substance through purification, which can be challenging during purification process development.

Furthermore, the immunogenicity of protein drugs is important in humans because the presence of anti-drug antibodies can lead to drug clearance from the body, thereby losing therapeutic efficacy (IDrugs 2009; 12: 233-7). When substitutions are introduced into a wild-type Fc domain (e.g., an IgG1 Fc domain), the modified sequence becomes non-human. Such modified sequences can be presented by MHC class II and therefore may be immunogenic in human patients.

If proteins contain Fc variants that show poor stability and purity, they will not be developed as drugs, and poor immunogenicity will hinder clinical development. It is therefore an object of the present invention to improve FcRn binding affinity at pH 7.4 without:

loss of significant stability;

an amount that increases the ratio of high molecular weight species, and

Increased risk of immunogenicity (risk of anti-drug antibody formation)

(Group 1)

Therefore, the present invention also provides an antigen-binding molecule comprising amino acid substitutions at positions EU252, EU434, EU307, and EU311 in the FcRn-binding domain, having an FcRn-binding activity of more than 15 nM at pH 7, a melting temperature, Tm, of 57.5°C or higher, an HMW of less than 2%, and low immunogenicity, wherein low immunogenicity corresponds to a score of less than 500 as measured by Epibase (Lonza).

Preferably, the antigen-binding molecule comprises amino acid substitutions at four or more positions in the FcRn-binding domain, wherein the four or more positions are selected from one of the following combinations:

a) EU252/EU434/EU307/EU311/EU436, and

b) a combination of EU252/EU434/EU307/EU311/EU436 with one or more positions selected from EU286, EU308 and EU428.

Preferred combinations are provided in Table 4.

[Table 4]

Particularly preferred are the combinations a), g), h) and i) of Table 4.

In an even more preferred embodiment, the modified FcRn binding domain comprises:

a) tyrosine at EU position 252, glutamic acid at EU position 286, glutamine at EU position 307, alanine at EU position 311, tyrosine at EU position 434, and valine at EU position 436; or

b) valine at EU position 250, tyrosine at EU position 252, glutamine at EU position 307, proline at EU position 308, alanine at EU position 311, tyrosine at EU position 434, and valine at EU position 436; or

c) valine at EU position 250, tyrosine at EU position 252, glutamic acid at EU position 286, glutamine at EU position 307, proline at EU position 308, alanine at EU position 311, tyrosine at EU position 434, and valine at EU position 436; or

d) Valine at EU250, tyrosine at EU252, glutamic acid at EU286, glutamine at EU307, proline at EU308, alanine at EU311, tyrosine at EU434 and valine at EU436.

(Group 2)

The present invention also provides an antigen-binding molecule comprising amino acid substitutions at three or more positions in the FcRn-binding domain, wherein the three or more positions are one of the following combinations selected from: a) EU252/EU434/EU307/EU311; and b) EU252/EU434/EU308; wherein the antigen-binding molecule has an FcRn-binding activity of 15-50 nM at neutral pH, a Tm greater than 60°C, and an HMW less than 2%, and wherein the antigen-binding molecule has low immunogenicity, wherein low immunogenicity corresponds to a score of less than 500 as determined using Epibase (Lonza).

In a preferred embodiment, the amino acid substitution is at 4 or more positions, wherein the 4 or more positions are one of the combinations provided in Table 5.

[Table 5]

Preferred combination

a) EU252/EU434/EU307/EU311/EU286 b) EU252/EU434/EU307/EU311/EU286/EU254 c) EU252/EU434/EU307/EU311/EU436 d) EU252/EU434/EU307/EU311/EU436/EU254 e) EU252/EU434/EU307/EU311/EU436/EU250 f) EU252/EU434/EU308/EU250 g) EU252/EU434/EU308/EU250/EU436/ h) EU252/EU434/EU308/EU250/EU307/EU311

More preferred are antigen binding molecules comprising 4 or more amino acid substitutions, wherein the 4 or more substitutions are selected from one of the following combinations:

a) tyrosine at EU252, glutamic acid at EU286, glutamine at EU307, alanine at EU311, and tyrosine at EU434;

b) tyrosine at EU252, threonine at EU254, glutamic acid at EU286, glutamine at EU307, alanine at EU311, and tyrosine at EU434;

c) tyrosine at EU position 252, glutamine at EU position 307, alanine at EU position 311, tyrosine at EU position 434, and isoleucine at EU position 436;

d) tyrosine at EU252, threonine at EU254, glutamic acid at EU286, glutamine at EU307, alanine at EU311, tyrosine at EU434, and isoleucine at EU436;

e) valine at EU position 250, tyrosine at EU position 252, threonine at EU position 254, proline at EU position 308, tyrosine at EU position 434, and valine at EU position 436;

f) valine at EU position 250, tyrosine at EU position 252, glutamine at EU position 307, alanine at EU position 311, tyrosine at EU position 434, and valine at EU position 436;

g) tyrosine at EU252, glutamine at EU307, alanine at EU311, tyrosine at EU434, and valine at EU436;

h) valine at EU250, tyrosine at EU252, proline at EU308, and tyrosine at EU434; and

i) Valine at EU position 250, Tyrosine at EU position 252, Glutamine at position 307, Proline at EU position 308, Alanine at EU position 311 and Tyrosine at EU position 434.

(Group 3)

The present invention also provides antigen-binding molecules comprising the following amino acid substitutions in the FcRn-binding domain:

a) at position EU252/EU434; and

b) at EU position 436 and/or at EU position 254 and/or at EU position 315;

The protein has an FcRn binding activity of 50-150 nM at pH 7, a Tm higher than 63° C., a HMW of less than 2%, and extremely low immunogenicity, wherein extremely low immunogenicity is defined as a score of less than 250 as determined by Epibase (Lonza).

Preferably, the amino acid substitution is at 3 or more positions, wherein the 3 or more positions are one of the combinations provided in Table 6.

[Table 6]

Preferred combination

a) EU252/EU315/EU434; b) EU252/EU434/EU436 c) EU252/EU254/EU434/EU436

In a more preferred embodiment, the modified antigen binding molecule comprises 3 or more amino acid substitutions, wherein the 3 or more substitutions are one of the following combinations selected:

a) tyrosine at EU252, aspartic acid at EU315, and tyrosine at EU434;

b) tyrosine at EU252, tyrosine at EU434, and isoleucine at EU436;

c) tyrosine at EU252, tyrosine at EU434, and leucine at EU436;

d) tyrosine at EU252, tyrosine at EU434, and valine at EU436; and

e) Tyrosine at EU position 252, Threonine at EU position 254, Tyrosine at EU position 434, and Isoleucine at EU position 436.

(Group 4)

The present invention also provides antigen-binding molecules comprising amino acid substitutions at three or more positions in the FcRn-binding domain, wherein the three or more positions are one of the combinations provided in Table 7. The modified antigen-binding molecules have an FcRn-binding activity of 150-700 nM at pH 7, a Tm greater than 66.5°C, an HMW of less than 2%, and very low immunogenicity, wherein very low immunogenicity is defined as a score of less than 250 as determined using Epibase (Lonza).

[Table 7]

a) EU307/EU311/EU434 b) EU307/EU309/EU311/EU434 c) EU307/EU309/EU311/EU434 d) EU250/EU252/EU434/EU436

Preferably, the modified antigen-binding molecule comprises 3 or more substitutions, wherein the 3 or more substitutions are one of the following combinations:

a) glutamine at position EU307, histidine at position EU311, and tyrosine at position EU434;

b) glutamine at EU307, glutamic acid at EU309, alanine at EU311, tyrosine at EU434;

c) glutamine at EU position 307, glutamic acid at EU position 309, histidine at EU position 311, tyrosine at EU position 434; or

d) Valine at EU position 250, tyrosine at EU position 252, tyrosine at EU position 434, valine at EU position 436.

Pre-existing antidrug antibodies

The replacement of amino acids in antibodies can produce negative results, such as the immunogenicity of therapeutic antibodies is improved, which in turn can lead to cytokine storms and/or the production of anti-drug antibodies (ADA). Because ADA can affect the efficacy and pharmacokinetics of therapeutic antibodies, sometimes causing serious side effects, the clinical utility and efficacy of therapeutic antibodies may be limited. Many factors affect the immunogenicity of therapeutic antibodies, and the presence of effector T cell epitopes is one of the factors. Similarly, the presence of pre-existing antibodies for therapeutic antibodies may also be problematic. Examples of such pre-existing antibodies are rheumatoid factor (RF), autoantibodies (antibodies to self-proteins) to the Fc portion of antibodies (i.e., IgG). Rheumatoid factor is particularly present in patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis. In arthritis patients, RF and IgG are linked together to form an immune complex that works on the disease process. Recently, a humanized anti-CD4 IgG1 antibody with an Asn434His mutation that induces significant rheumatoid factor binding was reported (Clin Pharmacol Ther. 2011 Feb;89(2):283-90(NPL9)). Detailed studies confirmed that the Asn434His mutation in human IgG1 increased the binding of rheumatoid factor to the Fc region of the antibody compared to the parental human IgG1.

RF is a polyclonal autoantibody directed against human IgG. The RF epitope within the human IgG sequence varies between clones, but the RF epitope appears to be located in the CH2/CH3 interface and the CH3 domain, potentially overlapping with the FcRn-binding epitope. Therefore, mutations that increase binding affinity to FcRn at neutral pH may also increase binding affinity to a specific RF clone.

Therefore, it is preferred to increase the FcRn binding affinity at neutral and/or acidic pH without simultaneously increasing the binding affinity of the therapeutic antibody to pre-existing antibodies in plasma at neutral pH.

Therefore, the present invention also provides antigen-binding molecules comprising a modified FcRn-binding domain (preferably a modified Fc region) whose binding activity to pre-existing ADA at neutral pH is not significantly increased compared to the binding affinity of an antigen-binding molecule comprising a wild-type Fc region. The modified FcRn-binding domain (modified Fc region) preferably comprises an amino acid substitution at one or more positions selected from the group consisting of: EU387, EU422, EU424, EU426, EU433, EU436, EU438, and EU440.

Preferably, the above substitutions are introduced into the FcRn-binding domain or Fc region of an antigen-binding molecule with improved affinity for FcRn at neutral or acidic pH, whereby the modified FcRn-binding domain or Fc region has increased binding activity for pre-existing ADA at neutral pH. The substitutions act to reduce binding activity for pre-existing ADA. Therefore, in a preferred embodiment, the modified FcRn-binding domain or modified Fc region of the present invention has reduced binding activity for pre-existing ADA compared to an FcRn-binding domain or Fc region that has increased binding activity for FcRn at neutral or acidic pH and increased binding activity for pre-existing anti-drug antibodies within the neutral pH range. Preferably, the antigen-binding molecule with enhanced binding activity to FcRn and pre-existing ADA at neutral pH comprises an amino acid substitution at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436. In addition to the substitutions at one or more of the above positions, a substitution may also be included at EU position 256. Preferably, the amino acid at EU position 256 is substituted with glutamic acid.

Therefore, the present invention also provides antigen binding molecules comprising a modified Fc region that has an improved affinity for FcRn at neutral or acidic pH and an insignificantly improved affinity for pre-existing anti-drug antibodies (ADA) at neutral pH compared to the binding affinity of an antigen binding molecule comprising a wild-type Fc region. In a preferred embodiment, the present invention provides antigen binding molecules comprising a modified Fc region with an improved affinity for FcRn at neutral or acidic pH, comprising amino acid substitutions at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438, and EU440.

Preferred are antigen-binding molecules comprising a modified Fc region that has improved affinity for FcRn at neutral or acidic pH without significantly increasing binding activity to pre-existing ADA at neutral pH compared to a control antigen-binding molecule, wherein the modified Fc region comprises amino acid substitutions at one or more positions selected from the substitutions shown in Table 8.

[Table 8]

Substituted amino acids:

As used herein, the terms "anti-drug antibodies" and "ADAs" refer to endogenous antibodies that have binding affinity for an epitope located on a therapeutic antibody and are therefore capable of binding to the therapeutic antibody. As used herein, the terms "pre-existing anti-drug antibodies" and "pre-existing ADAs" refer to anti-drug antibodies that are present and detectable in the patient's blood before the therapeutic antibody is administered to the patient. Preferably, the pre-existing ADAs are human antibodies. In particularly preferred embodiments, the pre-existing ADAs are rheumatoid factor, a polyclonal or monoclonal autoantibody directed against the Fc region of human IgG antibodies. The epitope of rheumatoid factor is located in the CH2/CH3 interface region and the CH3 domain, but can vary between clones.

An antigen binding molecule comprising an FcRn binding domain region (or Fc region) that has an improved affinity for FcRn at neutral or acidic pH and an improved affinity for pre-existing anti-drug antibodies at neutral pH is an antigen binding molecule that comprises an FcRn binding domain (or Fc region) modified to improve the binding affinity of the FcRn binding domain (or Fc region) of the antigen binding molecule to FcRn compared to an antibody comprising a complete FcRn binding domain (or complete Fc region). Expected modifications include, but are not limited to, amino acid substitutions in the amino acid sequence of the Fc portion of the antigen binding domain. Antigen binding molecules comprising an FcRn binding domain or Fc region that has improved binding activity to pre-existing ADA in the neutral pH range and improved binding activity to FcRn at neutral (in the case of a target antigen binding molecule whose FcRn binding activity is improved at neutral pH) or acidic pH (in the case of a target antigen binding molecule whose FcRn binding activity is improved at acidic pH) are referred to herein as "reference antibodies." The "reference antibody" is preferably a modified antigen-binding molecule before substitution of an amino acid at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438, and EU440, more preferably a modified antigen-binding molecule before introduction of any one of the substitutions provided in Table 8. The "reference antibody" may be an antigen-binding molecule comprising an amino acid substitution at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436 in the FcRn-binding domain.

An example of a “reference antibody” with improved FcRn-binding activity in the neutral pH range is an antigen-binding molecule comprising an Fc region with improved affinity for FcRn in the neutral pH range and improved affinity for pre-existing ADA at neutral pH, which comprises an amino acid substitution at the following positions in the Fc region:

a) at positions EU252 and EU434; and

b) one or more positions selected from EU238, EU250, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433 and EU436.

More preferably, the antigen-binding molecule comprising an Fc region with increased affinity for FcRn in the neutral pH range and increased affinity for pre-existing ADA in the neutral pH range comprises one of the combinations provided in Table 9.

[Table 9]

Preferred combinations of substitutions for reference antibodies with improved FcRn binding activity in the neutral pH range

1 M252Y/N434Y/ 2 M252Y/N434Y/Y436V 3 M252Y/N434Y/Y436F 4 M252Y/N434Y/Y436V 5 M252Y/S254T/T256E/T307Q/Q311A/N434Y/Y436V 6 M252Y/S254T/T256E/V308P/N434Y/Y436V 7 M252Y/N434W/Y436V 8 M252Y/S254T/T256F/N434Y/Y436V 9 M252Y/S254T/T256E/N286E/N434Y/Y436V 10 M252Y/S254T/R255L/T256E/N434Y/Y436V 11 M252Y/S254T/R255L/T256E/N434Y/Y436V 12 M252Y/S254T/R255L/T256E/E258D/N434Y/Y436V 13 M252Y/S254T/R255L/T256E/E258I/N434Y/Y436V 14 M252Y/S254T/T256E/H433A/N434Y/Y436V 15 M252Y/S254T/T256E/H433K/N434Y/Y436V 16 M252Y/S254T/T256E/H433P/N434Y/Y436V 17 M252Y/S254T/T256E/H433R/N434Y/Y436V 18 M252Y/S254T/T256E/H433S/N434Y/Y436V 19 M252Y/S254T/T256E/H433A/N434Y/Y436V 20 L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V twenty one L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V twenty two EU238D/EU252Y/EU434Y/EU436V twenty three EU252Y/EU434Y/EU436V twenty four EU250V/EU252Y/EU434Y/EU436V/EU3070/EU308P/EU311A 25 EU252Y/EU434Y/EU436V/EU235R/EU239K 26 EU252Y/EU434Y 27 EU252Y/EU434Y/EU436V

An example of a "reference antibody" having improved FcRn-binding activity in the acidic pH range is an antigen-binding molecule comprising an Fc region having improved affinity for FcRn in the acidic pH range and improved affinity for pre-existing ADA in the neutral pH range, which preferably comprises the following substitutions:

i) on EU434, or

ii) at two or more positions, wherein the two or more positions are one of the combinations selected from: a) EU252/EU254/EU256; b) EU428/EU434; and c) EU250/EU428.

Preferably, the antigen-binding molecule comprises an Fc region with increased affinity in the acidic pH range and increased affinity for pre-existing ADA at neutral pH.

i) replace N434H; or

ii) one of the following combinations: a) M252Y/S254T/T256E; b) M428L/N434S; and c) T250Q and M428L (EU numbering).

Preferred antigen-binding molecules comprise an Fc region comprising one of the following substitutions or combinations: a) M252Y/S254T/T256E, b) M428L/N434S, or c) T250Q and M428L, or d) N434H (EU numbering) that have improved FcRn-binding activity at acidic pH without improving binding activity in the neutral pH range.

The binding activity of the Fc region of the antigen binding molecule to pre-existing anti-drug antibodies is represented in this application by electrochemiluminescence (ECL) reaction at neutral pH; however, there are other suitable methods for determining the binding activity to pre-existing ADA known to those skilled in the art. The ECL assay is described in, for example, Moxness et al. (Clin Chem, 2005, 51: 1983-85) and the embodiments of the present invention. Those skilled in the art can appropriately select the conditions for the assay for determining the binding activity to pre-existing ADA, so the conditions are not particularly limited.

The increased or higher binding affinity to a pre-existing ADA is increased compared to the binding affinity of a control antigen binding molecule to a pre-existing ADA.

The term "control antigen-binding molecule" as used herein refers to an antigen-binding molecule comprising a complete human Fc region, preferably an antibody or antibody derivative comprising a complete human Fc region.

The binding affinity for pre-existing ADA can be evaluated at any temperature from 10°C to 50°C. Preferably, a temperature from 15°C to 40°C is used to determine the binding affinity between the human Fc region and the human pre-existing ADA. More preferably, any temperature from 20°C to 35°C, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35°C, is used to determine the binding affinity between the human Fc region and the human pre-existing ADA. The preferred temperature is between 20 and 25°C, more preferably at 25°C. In a preferred embodiment, the interaction between the human pre-existing ADA and the human Fc region is measured at pH 7.4 (or pH 7.0) and at 25°C.

In the context of the present invention, the term "increased binding affinity for pre-existing ADA" means that the measured binding affinity (i.e., KD) of the antigen-binding molecule of the present invention for pre-existing ADA is increased compared to the measured binding affinity of a control antigen-binding molecule for pre-existing ADA. This increase in binding affinity for pre-existing ADA can be observed in individual patients or in groups of patients.

As used herein, the terms "patients" and "patients" are not particularly limited and include all humans who suffer from a disease and to whom a therapeutic antigen binding molecule is administered during treatment. Preferably, the patient is a person suffering from an autoimmune disease. More preferably, the patient is a person suffering from an arthritis disease or systemic lupus erythematosus (SLE). Arthritis diseases particularly include rheumatoid arthritis.

In the context of the present invention, a significant increase in binding activity to pre-existing ADA in an individual patient corresponds to an increase in the binding activity of the therapeutic antigen-binding molecule (i.e., therapeutic antibody) comprising a modified Fc region to pre-existing ADA measured in the patient by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%, compared to the binding affinity of the control antigen-binding molecule to pre-existing ADA. Preferably, the increase in binding activity of the antigen-binding molecule comprising a modified Fc region is at least 20%, more preferably at least 30%, even more preferably at least 40%, and most preferably at least 50%, compared to the binding affinity of the control antigen-binding molecule to pre-existing ADA. Alternatively, a significant increase in the binding activity of the antigen-binding molecule to pre-existing ADA in a patient is preferably an increase in the ECL reaction of the antigen-binding molecule to an ECL of more than 250, preferably to an ECL of at least 500, more preferably to an ECL of at least 1000, and most preferably to an ECL of at least 2000. More preferably, the increase is an increase compared to an ECL reaction of less than 500 (preferably less than 250) of the control antigen-binding molecule. Preferred ranges between the binding activity of the control antigen-binding molecule to pre-existing ADA and the binding activity of the antigen-binding molecule with a modified Fc region to pre-existing ADA are, in particular, less than 250 to at least 250, less than 250 to at least 500, less than 500 to 500 or more, less than 500 to 1000 or more, and less than 500 to at least 2000 ECL reactions.

An increase in binding activity to pre-existing ADAs can also correspond to an increase in the proportion of patients in a patient population whose antigen-binding molecules have an increased ECL response of at least 500 (preferably at least 250) at neutral pH compared to the proportion of patients whose ECL response to a control antigen-binding molecule at neutral pH is at least 500 (preferably at least 250). A "significant" increase in the proportion of patients in a patient population preferably means an increase of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% in the proportion of patients whose ECL response to rheumatoid factor at neutral pH is 500 or less (preferably 250 or more) for therapeutic antigen-binding molecules comprising a modified Fc region compared to the proportion of patients whose ECL response to a control antigen-binding molecule is at least 20%, more preferably at least 30%, even more preferably at least 40%, and most preferably 50% or more.

In the context of the present invention, a reduction in binding affinity for pre-existing ADA refers to a reduction in the measured binding activity (i.e., KD or ECL reaction) compared to the binding activity measured for a reference antibody. This reduction in binding affinity for pre-existing ADA can be observed in individual patients or in patient groups. The reduction in affinity of therapeutic antigen binding molecules for pre-existing ADA at neutral pH in individual patients refers to a reduction in the measured binding activity at neutral pH compared to the binding activity for pre-existing ADA measured for a reference antibody in the patient. The significant reduction in a preferred individual patient is a reduction in the measured binding activity of the modified antigen binding molecules for pre-existing ADA at neutral pH by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the binding activity of the reference antibody for pre-existing ADA. More preferably, the reduction is at least 30%, even more preferably 40%, and most preferably 50% or higher compared to the reference antibody.

Alternatively, a significant decrease in the binding activity of the modified antigen-binding molecule for pre-existing ADA in an individual patient can be measured as a decrease in the ECL reaction of the antigen-binding molecule from an ECL reaction of 500 or greater (preferably an ECL of 1000 or greater, most preferably an ECL of 2000 or greater) to less than 500, preferably less than 250, compared to the ECL reaction of a reference antibody. A preferred decrease is from an ECL reaction of 500 or greater to an ECL reaction of less than 500, more preferably from at least 250 to less than 250, even more preferably from at least 500 to less than 250. Preferred ranges are particularly from at least 250 to less than 250, from at least 500 to less than 250, from at least 1000 to less than 250, from at least 2000 to less than 250, from at least 500 to less than 500, from at least 1000 to less than 500, and from at least 2000 to less than 500.

A reduction can also be a decrease in the percentage of patients in a patient population with increased binding of their pre-existing ADA to the modified antigen-binding molecule in the neutral pH range. In other words, a reduction can be measured as a decrease in the percentage of patients whose pre-existing ADA have an ECL reaction to the modified antigen-binding molecule, compared to the ECL reaction of a reference antibody. Preferably, the reduction can be a decrease of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% in the proportion of patients in a patient population with increased binding activity of the therapeutic antigen-binding molecule to pre-existing ADA, compared to the proportion of patients with increased binding activity of the reference antibody to pre-existing ADA, wherein increased binding is represented by an ECL reaction of 500 or greater, preferably 250 or greater. Preferably, the reduction is at least 20%, more preferably at least 30%, even more preferably 40%, and most preferably 50% or greater.

In a preferred embodiment, the therapeutic antigen binding molecules of the present invention have low binding activity to pre-stored ADA at neutral pH. Specifically, compared with the binding activity of the reference antibody to pre-stored ADA at neutral pH, the binding activity of the modified antigen binding molecules of the present invention to pre-stored ADA at neutral pH is preferably significantly reduced. More preferably, compared with the binding affinity of the control antigen binding molecules, the binding activity of the modified antigen binding molecules of the present invention to pre-stored ADA at neutral pH is not significantly improved (the binding activity to pre-stored ADA is roughly the same as that of the control antigen binding molecules). The low binding activity or baseline affinity to pre-stored ADA is preferably an ECL reaction of less than 500 in each patient. Preferably, the ECL reaction is less than 250. In a patient group, the low binding activity to pre-stored ADA is an ECL reaction of less than 500 in 90% of the patients, more preferably in 95% of the patients, and most preferably in 98% of the patients in the patient group.

In a more preferred embodiment, the antigen binding molecule comprises a modified FcRn binding domain with improved affinity for FcRn at neutral or acidic pH, wherein the binding activity to pre-existing ADA at neutral pH is not significantly improved compared to a control antigen binding molecule, whereby the modified FcRn binding domain of the invention comprises substitutions at one or more positions or combinations provided in Table 10.

[Table 10]

Positions and combinations of positions substituted in the FcRn binding domain

1) EU387 2) EU422 3) EU424 4) EU426 5) EU436 6) EU438 7) EU440 8) EU438 and EU440 9) EU422 and EU424 10) EU433

In a more preferred embodiment, the antigen binding molecules of the invention comprise a modified FcRn binding domain having one or more substitutions or combinations provided in Table 11.

[Table 11]

Substitutions and combinations of substitutions in the FcRn binding domain

1 EU387R 2 EU422E 3 EU422R 4 EU422S 5 EU424E 6 EU424R 7 EU438E 8 EU438R 9 EU438S 1O EU440E 11 EU422E/EU424R 12 EU422S/FU424R 13 EU438R/EU440E 14 EU422D 1 5 EU422K 16 EU422T 17 EU422Q 18 EU438K 19 EU440D 20 EU440Q twenty one EU438R/EU440D twenty two EU438K/EU440E twenty three EU438K/FU440D twenty four EU424N 25 EU426D 26 EU426A 27 EU426Q 28 EU426Y 29 EU436F 30 EU436T 31 EU433D

In a preferred embodiment, the antigen binding molecule comprises a modified FcRn binding domain that has a) improved affinity for FcRn at neutral or acidic pH, and b) does not significantly increase binding affinity for pre-existing ADA at neutral pH, compared to a control antigen binding molecule, said antigen binding molecule comprising any one combination of substitutions provided in Table 12.

It is also preferred that the antigen-binding molecule with improved FcRn binding activity in the neutral pH range and no significantly improved binding affinity for pre-existing ADA at neutral pH, compared to an antigen-binding molecule comprising a wild-type Fc region, comprises an amino acid substitution in the FcRn-binding domain at the following positions: a) one or more positions selected from the group consisting of EU387, EU422, EU424, EU438, EU440, and EU433, or b) two or more positions, wherein the two or more positions are a combination of EU422/EU424 or EU438/EU440. More preferably, the substitution is selected from the substitutions provided in Table 11.

Even more preferably, the FcRn-binding domain of an antigen-binding molecule that has improved FcRn-binding activity in the neutral pH range and does not significantly improve binding affinity for pre-existing ADA at neutral pH compared to an antigen-binding molecule comprising a wild-type Fc region comprises any one of the substitution combinations provided in Table 12. Specifically, a preferred modified antigen-binding molecule that has improved FcRn-binding activity in the neutral pH range and does not significantly improve binding affinity for pre-existing ADA at neutral pH comprises three or more substitutions in the FcRn-binding domain, wherein the three or more substitutions are any one of the combination numbers (2)-(26) and (28)-(59) provided in Table 12.

[Table 12]

Combinations of substitutions in the Fc region that improve FcRn binding activity in the neutral pH range without significantly improving binding activity to pre-existing ADA (positions are given according to the EU numbering system).

The present invention also provides an antigen-binding molecule having improved FcRn binding activity in the acidic pH range and no significant increase in binding affinity for pre-existing ADA at neutral pH compared to a control antigen-binding molecule, comprising an amino acid substitution at a) EU424 or b) EU438/EU440.

More preferably the substitution is selected from a) EU424N and EU438R/EU440E.

Preferably, the FcRn-binding domain of an antigen-binding molecule that has improved FcRn-binding activity in the acidic pH range and does not significantly increase binding affinity for pre-existing ADA at neutral pH compared to a control antigen-binding molecule comprises one of the substitution combinations provided in Table 13. More preferably, the antigen-binding molecule that has improved FcRn-binding activity in the acidic pH range and does not significantly increase binding affinity for pre-existing ADA at neutral pH compared to a control antigen-binding molecule comprises any one of the substitution combination numbers (13) to (28) provided in Table 13.

[Table 13]

Combinations of substitutions in the Fc region that improve FcRn binding activity in the acidic pH range without significantly improving binding activity to pre-existing ADA (positions are given according to the EU numbering system).

1 EU252Y/EU254T/EU256E/EU438R/EU440E 2 EU252Y/EU254T/EU256E/EU424N 3 EU428L/EU434S/EU438R/EU440E 4 EU424N/EU428L/EU434S 5 EU426D/EU428L/EU434S 6 EU426A/EU428L/EU434S 7 EU426Q/EU428L/EU434S 8 EU426Y/EU428L/EU434S 9 EU428L/EU434S/EU436F 10 EU428L/EU434S/EU436T 11 EU434H/FU438R/FU440E 12 EU424N/EU434H 13 N434Y/Y436V/Q438R/S440E 14 N434Y/Y436V/Q438R/S440D 15 N434Y/Y436V/Q438K/S440E 16 N434Y/Y436V/Q438K/S440D 17 H433D/N434Y/Y436V/Q438R/S440E 18 H433D/N434Y/Y436V/Q438R/S440D 19 H433D/N434Y/Y436V/Q438K/S440E 20 H433D/N434Y/Y436V/Q438K/S440D twenty one N434Y/Y436T/Q438R/S440E twenty two N434Y/Y436T/Q438R/S440D twenty three N434Y/Y436T/Q438K/S440E twenty four N434Y/Y436T/Q438K/S440D 25 H433D/N434Y/Y436T/Q438R/S440E 26 H433D/N434Y/Y436T/Q438R/S440D 27 H433D/N434Y/Y436T/Q438K/S440E 28 H433D/N434Y/Y436T/Q438K/S440D

In addition to substitutions at any of positions EU387, EU422, EU424, EU426, EU433, EU436, EU438, and EU440, the Fc regions of the invention may comprise further amino acid substitutions at one or more of the following positions:

EU248, EU249, EU250, EU251, EU252, EU253, EU254, EU255, EU256, EU257, EU305, EU306, EU307, EU308, EU309, EU310, EU311, EU312, EU313, EU314, EU342, EU343, EU344, EU345, EU346, EU347, EU348, EU349, EU350, EU351, EU352, EU380, EU381, EU382, EU383, EU384, EU385, EU386, EU388, EU414, EU415, EU416, EU417, EU418, EU419, EU420, EU421, EU423, EU425, EU427, EU428, EU429, EU430, EU431, EU432, EU433, EU434, EU435, EU436, EU437, EU441, EU442, EU443, and EU444.

Substitution of the Fc region at any of these positions can reduce binding affinity to pre-existing ADA, particularly to rheumatoid factor, without negatively affecting binding affinity to FcRn.

In addition, the method of the present invention may further comprise the step of substituting the Fc region of the antigen-binding molecule at one or more of the following positions as described above:

EU248, EU249, EU250, EU251, EU252, EU253, EU254, EU255, EU256, EU257, EU305, EU306, EU307, EU308, EU309, EU310, EU311, EU312, EU313, EU314, EU342, EU343, EU344, EU345, EU346, EU347, EU348, EU349, EU350, EU351, EU352, EU380, EU381, EU382, EU383, EU384, EU385, EU386, EU388, EU414, EU415, EU416, EU417, EU418, EU419, EU420, EU421, EU423, EU425, EU427, EU428, EU429, EU430, EU431, EU432, EU433, EU434, EU435, EU436, EU437, EU441, EU442, EU443, and EU444.

Weak or no binding activity to effector receptors or complement proteins

In combination with Fcγ receptors or complement proteins, it is also possible to cause undesirable effects (such as improper platelet activation). The modified antigen binding molecules that are not combined with effector receptors (such as FcγRIIa receptors) are safer and/or more effective. Therefore, in a preferred embodiment, the modified antigen binding molecules of the present invention also have weak binding activity to effector receptors or are not combined with effector receptors. Examples of effector receptors include but are not limited to activating Fcγ receptors, particularly Fcγ receptor I, Fcγ receptor II and Fcγ receptor III. Fcγ receptor I includes Fcγ receptor Ia, Fcγ receptor Ib and Fcγ receptor Ic and its subtypes. Fcγ receptor II includes Fcγ receptor IIa (which has two allotypes R131 and H131) and Fcγ receptor IIb. Fcγ receptor III includes Fcγ receptor IIIa (which has two allotypes: V158 and F158) and Fcγ receptor IIIb (which has two allotypes: FcγIIIb-NA1 and FcγIIIb-NA2). Antibodies that have weak binding activity or do not bind to effector receptors include, for example, antibodies containing a silent Fc region or antibodies without an Fc region (e.g., Fab, F(ab)'2, scFv, sc(Fv)2, diabodies).

Examples of Fc regions with weak or no binding activity to effector receptors are described, for example, in Strohl et al. (Current Opinion in Biotechnology (2009) 20(6), 685-691). Specifically, it describes examples such as aglycosylated Fc regions (N297A, N297Q), silent Fc regions (which are Fc regions engineered to silence effector function (or immunosuppressive)) (IgG1-L234A/L235A, IgG1-H268Q/A330S/P331S, IgG1-C226S/C229S, IgG1-C226S/C229S/E233P/L234V/L235A, IgG1-L234F/L235E/P331S, IgG2-V234A/G237A, IgG2-H268Q/V309L/A330S/A331S, IgG4-L235A/G237A/E318A, IgG4-L236E). WO2008/092117 discloses antibodies comprising silent Fc regions comprising substitutions G236R/L328R, L235G/G236R, N325A/L328R or N325L/L328R (positions according to the EU numbering system). In addition, WO2000/042072 discloses antibodies comprising silent Fc regions comprising substitutions at one or more of positions EU233, EU234, EU235 and EU237. WO2009/011941 discloses antibodies comprising silent Fc regions comprising deletions from residues EU231 to EU238. Davis et al. (Journal of Rheumatology (2007) 34(11): 2204-2210) disclose antibodies comprising a silent Fc region comprising the substitutions C220S/C226S/C229S/P238S. Shields et al. (Journal of Biological Chemistry (2001) 276(9), 6591-6604) disclose antibodies comprising a silent Fc region comprising the substitution D265A.

The term "weak binding to an effector receptor" refers to binding activity that is 95% or less, preferably 90% or less, 85% or less, 80% or less, 75% or less, more preferably 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the binding activity of an intact IgG (or an antibody comprising an intact Fc region) to an effector receptor. The binding activity of FcγRs is preferably reduced to at least about 1/10 or less, about 1/50 or less, or about 1/100 or less of the binding activity of an intact IgG (or an antibody comprising an intact Fc region) to an effector receptor.

A silent Fc region is a modified Fc region comprising one or more amino acid substitutions, insertions, additions, and/or deletions that reduce binding to effector receptors compared to an intact Fc region. Binding activity to effector receptors can be greatly reduced such that the Fc region no longer binds to the effector receptor. Examples of silent Fc regions include, but are not limited to, Fc regions comprising amino acid substitutions at one or more positions selected from the group consisting of: EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270, EU271, EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331, and EU332.

Specifically, the silent Fc region has a substitution with an amino acid selected from the following list at one or more positions selected from EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270, EU271, EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331 and EU332. Preferably, the silent Fc region has a substitution with an amino acid selected from the following list at one or more positions selected from EU235, EU237, EU238, EU239, EU270, EU298, EU325 and EU329.

The amino acid at EU position 234 is preferably substituted with one of the amino acids selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser and Thr.

The amino acid at EU position 235 is preferably substituted with one of the following amino acids: Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val and Arg.

The amino acid at EU position 236 is preferably substituted with one of the amino acids selected from the group consisting of Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro and Tyr.

The amino acid at EU position 237 is preferably substituted with one of the following amino acids: Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr and Arg.

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

The amino acid at EU position 239 is preferably substituted with one of the amino acids selected from the group consisting of Gln, His, Lys, Phe, Pro, Trp, Tyr and Arg.

The amino acid at EU position 265 is preferably substituted with one of the amino acids selected from the group consisting of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr and Val.

The amino acid at EU266 is preferably substituted with one of the following amino acids: Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp and Tyr.

The amino acid at position EU267 is preferably substituted with one of the amino acids selected from the group consisting of Arg, His, Lys, Phe, Pro, Trp and Tyr.

The amino acid at EU position 269 is preferably substituted with one of the following amino acids: Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

The amino acid at EU position 270 is preferably substituted with one of the amino acids selected from the group consisting of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

The amino acid at position EU271 is preferably substituted with one of the amino acids selected from the group consisting of Arg, His, Phe, Ser, Thr, Trp and Tyr.

The amino acid at EU position 295 is preferably substituted with one of the amino acids selected from the group consisting of Arg, Asn, Asp, Gly, His, Phe, Ser, Trp and Tyr.

The amino acid at EU position 296 is preferably substituted with one of the amino acids selected from the group consisting of Arg, Gly, Lys and Pro.

The amino acid at position EU297 is preferably substituted with Ala.

The amino acid at EU position 298 is preferably substituted with one of the amino acids selected from the group consisting of Arg, Gly, Lys, Pro, Trp and Tyr.

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

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

The amino acid at position EU325 is preferably substituted with one of the amino acids selected from the group consisting of Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr and Val.

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

The amino acid at position EU328 is preferably substituted with one of the amino acids selected from the group consisting of Arg, Asn, Gly, His, Lys and Pro.

The amino acid at EU329 is preferably substituted with one of the following amino acids: Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val and Arg.

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

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

The amino acid at position EU332 is preferably substituted with one of the amino acids selected from the group consisting of Arg, Lys and Pro.

Preferably, the silent Fc region comprises a substitution at position EU235 with Lys or Arg, a substitution at position EU237 with Lys or Arg, a substitution at position EU238 with Lys or Arg, a substitution at position EU239 with Lys or Arg, a substitution at position EU270 with Phe, a substitution at position EU298 with Gly, a substitution at position EU325 with Gly, or a substitution at position EU329 with Lys or Arg. More preferably, the silent Fc region comprises a substitution at position EU235 with Arginine and a substitution at position EU239 with Lysine. More preferably, it comprises the substitution L235R/S239K.

In addition, the modified antigen binding molecules of the present invention are preferably aglycosylated. More preferably, the modified antigen binding molecules of the present invention comprise mutations in the heavy chain glycosylation site to prevent glycosylation at the site, as described in WO2005/03175. Therefore, in a preferred embodiment of the present invention, the modified aglycosyl antigen binding molecules are prepared by modifying the heavy chain glycosylation site, i.e., introducing substitutions N297Q or N297A (positions according to the EU numbering system), and expressing the protein in a suitable host cell. For introducing substitutions, the methods described in Examples can be used.

In a specific embodiment of the present invention, the modified antigen-binding molecules of the present invention have weak binding activity to complement proteins or do not bind to complement proteins. Preferably, the complement protein is C1q. The weak binding activity to complement proteins is preferably reduced by about 10 times or more, about 50 times or more, or about 100 times or more compared to the binding activity of intact IgG or an antibody comprising an intact Fc region to complement proteins. The binding activity of the Fc region to complement proteins can be reduced by modifications of the amino acid sequence, such as amino acid substitutions, insertions, additions, and/or deletions.

In a preferred embodiment of the present invention, the antigen-binding molecule has increased FcRn binding affinity at acidic or neutral pH and has weak or no binding activity to effector receptors and/or complement proteins. Preferably, such antigen-binding molecules comprise substitutions in the FcRn-binding domain at the following positions:

a) one or more positions selected from EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436, and

b) one or more positions selected from the group consisting of: EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270, EU271 , EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331 and EU332 (according to the EU numbering system). More preferably, the modified antigen-binding molecule of the present invention having reduced or no binding activity to effector receptors and/or complement proteins comprises one or more substitutions in the Fc region selected from the group consisting of: substitution at EU position 235 with Lys or Arg, substitution at EU position 237 with Lys or Arg, substitution at EU position 238 with Lys or Arg, substitution at EU position 239 with Lys or Arg, substitution at EU position 270 with Phe, substitution at EU position 298 with Gly, substitution at EU position 325 with Gly, and substitution at EU position 329 with Lys or Arg. Even more preferably, it comprises substitution at EU position 235 with Arg and substitution at EU position 239 with Lys in the Fc region. Even more preferably, it comprises the substitution combination L235R/S239K in the Fc region.

Preferably, this type of antigen binding molecule does not significantly improve the binding activity of pre-existing ADA. Therefore, the antigen binding molecules of the present invention that reduce the binding activity of effector receptors and/or complement proteins or have no binding activity are also included in c) amino acid substitutions on one or more positions selected from EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440. In a more preferred embodiment of the present invention, the modified antigen binding molecules include 3 or more amino acid substitutions in the FcRn binding domain, wherein the 3 or more substitutions are one of the combinations provided in Tables 14 and 15.

[Table 14]

Substitution combinations that increase FcRn binding activity at neutral pH without significantly increasing binding activity to pre-existing ADA and that reduce binding activity to effector receptors and/or complement proteins

[Table 15]

Substitution combinations that increase FcRn binding activity at acidic pH without significantly increasing binding activity to pre-existing ADA and that reduce binding activity to effector receptors and/or complement proteins

1 L235R/S239K/N434Y/Y436V/Q438R/S440E 2 L235R/S239K/N434Y/Y436V/Q438R/S440D 3 L235R/S239K/N434Y/Y436V/Q438K/S440E 4 L235R/S239K/N434Y/Y436V/Q438K/S440D 5 L235R/S239K/H433D/N434Y/Y436V/Q438R/S440E 6 L235R/S239K/H433D/N434Y/Y436V/Q438R/S440D 7 L235R/S239K/H433D/N434Y/Y436V/Q438K/S440E 8 L235R/S239K/H433D/N434Y/Y436V/Q438K/S440D 9 L235R/S239K/N434Y/Y436T/Q438R/S440E 10 L235R/S239K/N434Y/Y436T/Q438R/S440D 11 L235R/S239K/N434Y/Y436T/Q438K/S440E 12 L235R/S239K/N434Y/Y436T/Q438K/S440D 13 L235R/S239K/H433D/N434Y/Y436T/Q438R/S440E 14 L235R/S239K/H433D/N434Y/Y436T/Q438R/S440D 15 L235R/S239K/H433D/N434Y/Y436T/Q438K/S440E 16 L235R/S239K/H433D/N434Y/Y436T/Q438K/S440D

Other modifications

Furthermore, in addition to the above modifications, the antigen-binding molecules of the present invention do not contain an amino acid selected from the group consisting of alanine, valine, isoleucine, leucine, and threonine at EU position 257 of the FcRn-binding domain.

and/or does not contain a tryptophan at EU position 252 of the FcRn binding domain. In other words, in addition to any of the modifications described above, preferred antigen binding molecules of the present invention comprise an alanine, valine, isoleucine, leucine, threonine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, methionine, phenylalanine, proline, serine, tryptophan or tyrosine at EU position 257, and an arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, methionine, phenylalanine, proline, serine or tyrosine at EU position 252.

Also preferred are modified FcRn binding domains which, in addition to the substitution at any of the positions or combinations of positions mentioned herein, further comprise a lysine at EU position 239 and/or a phenylalanine at EU position 270.

Antigen-binding molecules

The antigen-binding molecules of the present invention are not particularly limited, as long as they include an antigen-binding domain having specific binding activity to a target antigen and an FcRn-binding domain of the present invention. Preferred antigen-binding domains include, for example, domains having the antigen-binding region of an antibody. The antigen-binding region of an antibody includes, for example, CDRs. The antigen-binding region of an antibody may contain all six CDRs of a complete antibody, or one, two, or more CDRs. The antigen-binding region of an antibody may contain amino acid deletions, substitutions, additions, and/or insertions, or may contain portions of CDRs.

On the other hand, the antigen binding molecules of the present invention include antigen binding molecules with antagonistic activity (antagonistic antigen binding molecules), antigen binding molecules with agonistic activity (agonistic antigen binding molecules) and molecules with cytotoxicity. In a preferred embodiment, the antigen binding molecules are antagonistic antigen binding molecules, particularly antagonistic antigen binding molecules that recognize antigens (e.g., receptors or cytokines).

The antigen binding molecules of the present invention are preferably antibodies. In the case of the present invention, preferred antibodies include, for example, IgG antibodies. When the antibody to be used is an IgG antibody, the type of IgG is not particularly limited; Therefore, IgG can belong to any isotype (subclass) such as IgG1, IgG2, IgG3 or IgG4. For human IgG1, IgG2, IgG3 or IgG4 constant regions, genetic polymorphisms (allotypes) are described in "Sequences of proteins of immunological interest, NIH Publication No. 91-3242". In the present application, these allotypes can also be used for constant regions. In particular, for human IgG1, amino acids Asp-Glu-Leu (DEL) and Glu-Glu-Met (EEM) can be used for residues in 356-358 of EU numbering. Similarly, for human immunoglobulin kappa constant regions, genetic polymorphisms (allotypes) are described in "Sequences of proteins of immunological interest, NIH Publication No. 91-3242". In the present application, these allotypes can also be used for constant regions. Furthermore, the antigen-binding molecules of the present invention may include antibody constant regions, and amino acid mutations may be introduced into the constant regions. Examples of amino acid mutations to be introduced include, but are not limited to, those that enhance or weaken binding to Fcγ receptors (Proc Natl Acad Sci USA. 2006 Mar 14; 103(11): 4005-10). Alternatively, it is also possible to alter pH-dependent binding by selecting an appropriate constant region, such as that of IgG2 (WO09125825).

When the antigen binding molecules of the present invention are antibodies, the antibodies can be derived from any animal, such as mice, humans, rats, rabbits, goats or camels. Preferably, the antibodies are human antibodies. In addition, the antibodies can be modified antibodies, such as chimeric antibodies, and particularly modified antibodies etc. comprising amino acid substitutions in the sequence of humanized antibodies. The categories of antibodies considered by the present invention also include bispecific antibodies, antibody modified products connected with various molecules and polypeptides comprising antibody fragments (particularly immunogenic and/or immunoreactive antibody fragments). In preferred embodiments, the antigen binding molecules are monoclonal antibodies.

"Chimeric antibodies" are antibodies produced by combining sequences derived from different animals. Specifically, chimeric antibodies include, for example, antibodies having heavy and light chain variable (V) regions from mouse antibodies and heavy and light chain constant (C) regions from human antibodies. Methods for producing chimeric antibodies are known. For example, in the case of human-mouse chimeric antibodies, DNA encoding the antibody V regions can be linked to DNA encoding the human antibody C regions; this can be inserted into an expression vector and introduced into a host to produce a chimeric antibody.

"Humanized antibodies", also known as reconstructed human antibodies, are known in the art as antibodies in which the complementary determining regions (CDRs) of antibodies derived from non-human mammals (e.g., mice) are transplanted into the CDRs of human antibodies. 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 application EP125023 and WO96/02576). Humanized antibodies can be produced by known methods. For example, the CDRs of mouse antibodies can be determined, and DNA encoding antibodies in which the CDRs are connected to the framework regions (FRs) of human antibodies can be obtained. Humanized antibodies can then be produced using a system using conventional expression vectors. Such DNA can be synthesized by PCR using several oligonucleotides prepared having portions overlapping the terminal regions of both CDRs and FRs as primers (see the method described in WO98/13388). The human antibody FRs connected by the CDRs are selected so that the CDRs form a suitable antigen-binding site. If necessary, the amino acids in the antibody variable region FRs can be changed so that the CDRs of the reshaped human antibody can form a suitable antigen-binding site (Sato et al., Cancer Res. (1993) 53: 10.01-6). The amino acid residues in the FRs that can be changed include those that directly bind to the antigen through non-covalent bonds (Amit et al., Science (1986) 233: 747-53), those that affect or have an effect on the CDR structure (Chothia et al., J. Mol. Biol. (1987) 196: 901-17), and those that participate in VH-VL interactions (EP239400).

When the antigen-binding molecules of the present invention are chimeric antibodies or humanized antibodies, the constant regions of these antibodies are preferably derived from human antibodies. For example, C-γ1, C-γ2, C-γ3 and C-γ4 can be used for H chains, while C-κ and C-λ can be used for L chains. In addition, if necessary, amino acid mutations can be introduced into the human antibody C region to increase or decrease binding to Fc-γ receptors or to improve antibody stability or yield. The chimeric antibodies of the present invention preferably include variable regions derived from non-human mammalian antibodies and constant regions derived from human antibodies. At the same time, humanized antibodies preferably include CDRs derived from non-human mammalian antibodies and FRs and C regions derived from human antibodies. The constant region derived from human antibodies preferably includes a human FcRn binding region. Such antibodies include, for example, IgG (IgG1, IgG2, IgG3 and IgG4). The constant region used in the humanized antibodies of the present invention can be a constant region of an antibody of any isotype. Preferably, a constant region derived from human IgG1 is used, but is not limited thereto. The FRs derived from human antibodies (which are used in humanized antibodies) are not particularly limited and can be derived from antibodies of any isotype.

The term "bispecific antibody" as used herein refers to an antibody that has variable regions that recognize different epitopes in the same antibody molecule. A bispecific antibody can 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 may include, for example, scFv-Fc (WO2005/037989), dAb-Fc, and Fc fusion proteins. Antibody fragments in such polypeptides may include, for example, Fab fragments, F(ab')2 fragments, scFvs (Nat Biotechnol. 2005 Sep; 23(9): 1126-36), and domain antibodies (dAbs) (WO2004/058821, WO2003/002609). When the molecule includes an Fc region, the Fc region may be used as a human FcRn-binding domain. Alternatively, the FcRn-binding domain may be fused to these molecules.

In addition, the antigen binding molecules applicable to the present invention can be or can comprise antibody-like molecules (such as fusion proteins of the Fc region of the present invention and antibody-like molecules). Antibody-like molecules (scaffold molecules, peptide molecules) are molecules that can display function by being combined with target molecules (Current Opinion in Biotechnology (2006) 17: 653-658; Current Opinion in Biotechnology (2007) 18: 1-10; Current Opinion in Structural Biology (1997) 7: 463-469; Protein Science (2006) 15: 14-27), and include, for example, DARPin (WO2002/020565), Affibody (Affibody) (WO1995/001937), Avimer (WO2004/044011; WO2005/040229) and Adnectin (WO2002/032925). If these antibody-like molecules can bind to target molecules in a pH-dependent or calcium-dependent manner and/or have human FcRn-binding activity in the neutral pH range, it may be possible to promote cellular uptake of antigens by the antigen-binding molecules, promote reduction in plasma antigen concentrations by administration of the antigen-binding molecules, improve the pharmacokinetics of the antigen-binding molecules, and increase the number of antigens that can be bound by a single antigen-binding molecule.

In addition, the antigen-binding molecule may be a protein produced by the fusion between the FcRn-binding domain of the present invention and a receptor protein that binds to a target (including a ligand), including, for example, TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, and CTLA4-Fc fusion protein (Nat Med. 2003 Jan; 9(1):47-52; BioDrugs. (2006) 20(3):151-60). If these receptor-FcRn binding domain fusion proteins, in addition to having FcRn binding activity in the neutral pH range, also bind to the target (including a ligand) in a pH-dependent or calcium-dependent manner, it is possible to promote cellular uptake of the antigen through the antigen-binding molecule, promote the reduction of plasma antigen concentration by administering the antigen-binding molecule, improve the pharmacokinetics of the antigen-binding molecule, and increase the number of antigens that can be bound by a single antigen-binding molecule. The receptor protein is appropriately designed and modified so as to include a domain that binds to the receptor protein and the target (including a ligand). As mentioned in the examples above (i.e., TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, and CTLA4-Fc fusion protein), particularly preferred are soluble receptor molecules comprising the extracellular domains of these receptor proteins required for binding to the target (including ligands). These designed and modified receptor molecules are referred to as artificial receptors in the present invention. Methods for designing and modifying receptor molecules to construct artificial receptor molecules are known in the art and are even routine.

In addition, the antibodies of the present invention may have modified sugar chains. Antibodies with modified sugar chains include, for example, antibodies with modified glycosylation (WO99/54342), antibodies lacking fucose added to sugar chains (WO00/61739, WO02/31140, WO2006/067847, WO2006/067913), and antibodies with sugar chains bisecting GlcNAc (WO02/79255).

According to Journal of Immunology (2009) 182: 7663-7671, in the acidic pH range (pH 6.0), the human FcRn binding activity of intact human IgG1 is KD 1.7 micromolar (μM), while in the neutral pH range, almost no activity is detected. Therefore, in a preferred embodiment, the antigen-binding molecules of the present invention include such antigen-binding molecules whose human FcRn binding activity in the acidic pH range is stronger than KD 1.7 micromolar, and in the neutral pH range is the same as or stronger than the human FcRn binding activity of intact human IgG. In a more preferred embodiment, its binding activity to pre-existing ADA in the neutral pH range is not significantly improved compared to intact IgG1. The above KD value is determined by the method described in the following document: Journal of Immunology (2009) 182: 7663-7671 (by immobilizing the antigen-binding molecule on a chip and loading human FcRn as an analyte).

The dissociation constant (KD) can be used as the value of human FcRn binding activity. However, the human FcRn binding activity of intact human IgG has almost no human FcRn binding activity in a neutral pH range (pH 7.4). Therefore, it is often difficult to calculate the activity as KD. The method for evaluating whether the human FcRn binding activity is higher than that of intact human IgG at pH 7.4 includes loading analyte with the same concentration, and then comparing the evaluation method of the intensity of the Biacore reaction. Specifically, when the reaction at pH 7.4 after human FcRn is loaded onto a chip fixed with an antigen binding molecule is stronger than the reaction at pH 7.4 after human FcRn is loaded onto a chip fixed with an intact human IgG, the human FcRn binding activity of the antigen binding molecule is identified as being higher than that of intact human IgG at pH 7.4.

In the context of the present invention, pH 7.0 can also be used as a neutral pH range. Using pH 7.0 as a neutral pH can promote weak interactions between human FcRn and the FcRn-binding domain. Regarding the temperature used for the assay, binding affinity can be assessed at any temperature between 10°C and 50°C. Preferably, a temperature between 15°C and 40°C is used to assess the binding affinity between the human FcRn-binding domain and human FcRn. More preferably, a temperature between 20°C and 35°C, such as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is used to assess the binding affinity between the human FcRn-binding domain and human FcRn. The temperature of 25°C described in Example 5 of WO2011/122011 is an example of an embodiment of the present invention. In a preferred embodiment, the interaction between human FcRn and the FcRn-binding domain can be measured as described in Example 5 of WO2011/122011 at pH 7.0 and 25° C. The binding affinity of the antigen-binding molecule to human FcRn can be measured by Biacore as described in Example 5 of WO2011/122011.

Binding affinity within a neutral pH range is preferably measured at pH 7.4, which is close to the in vivo plasma (blood) pH. When the binding affinity between the human FcRn binding domain and human FcRn is difficult to evaluate due to its low affinity at pH 7.4, pH 7.0 can be used as an alternative to pH 7.4. Binding affinity within an acidic pH range is preferably measured at pH 6.0, which is close to the pH of early endosomes in vivo. As for the temperature used for the assay conditions, the binding affinity between the human FcRn binding domain and human FcRn can be evaluated at any temperature between 10°C and 50°C. Preferably, a temperature between 15°C and 40°C is used to determine the binding affinity between the human FcRn binding domain and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is used to determine the binding affinity between the human FcRn-binding domain and human FcRn. A temperature of 25°C is described, for example, in Example 5 of WO2011/122011 and in the Examples herein.

It is preferred to use intact human IgG1, IgG2, IgG3 or IgG4 as a reference intact human IgG to compare its human FcRn binding activity or in vivo activity with the antigen binding molecules. It is preferred to use an antigen binding molecule comprising the same antigen-binding domain as the target antigen binding molecule and an intact human IgG Fc domain as the human FcRn binding domain as a reference. It is more preferred to use intact human IgG1 as a reference intact human IgG to compare its human FcRn binding activity or in vivo activity with the human FcRn binding activity or in vivo activity of the antigen binding molecules of the present invention.

Those skilled in the art may appropriately select conditions other than pH for the antigen binding or human FcRn binding activity assays, and the conditions are not particularly limited. For example, the activity can be assayed using MES buffer at 37°C as described in WO2009/125825. In another embodiment, the activity can be assayed using sodium phosphate buffer at 25°C as described in Examples 4 or 5 of WO2011/122011. Meanwhile, the antigen binding activity and human FcRn binding activity of the antigen-binding molecule can be assayed using methods known to those skilled in the art, such as Biacore (GE Healthcare). When the antigen is a soluble antigen, the activity of the antigen-binding molecule binding to the soluble antigen can be assayed by adding the antigen as the analyte to a chip immobilized with the antigen-binding molecule. Alternatively, when the antigen is a membrane-bound antigen, the activity of the antigen-binding molecule binding to the membrane-bound antigen can be assayed by adding the antigen-binding molecule as the analyte to an antigen-immobilized chip. The human FcRn-binding activity of the antigen-binding molecule can be measured by adding human FcRn or the antigen-binding molecule as an analyte to a chip immobilized with the antigen-binding molecule or human FcRn, respectively.

The present invention provides antigen-binding molecules of the present invention, comprising an antigen-binding domain and a human Fc region with enhanced FcRn binding activity within a neutral pH range. Preferably, the binding activity to pre-existing ADA is not significantly enhanced within a neutral pH range. The FcRn binding activity of such antigen-binding molecules within a neutral pH range is preferably stronger than a KD of 3.2 micromolar. More preferably, the FcRn binding activity within a neutral pH range is stronger than 700 nanomolar, even more preferably stronger than 500 nanomolar, and most preferably stronger than 150 nanomolar. Preferably, the antigen-binding molecules have enhanced human FcRn binding activity within a neutral pH range and an antigen-binding activity that is lower in an acidic pH range than in a neutral pH range or lower under low calcium concentrations than under high calcium concentration conditions. Preferably, the binding activity of such antigen-binding molecules to pre-existing ADA is not significantly enhanced within a neutral pH range. The present invention also provides antigen-binding molecules of the present invention comprising an antigen-binding domain and a human FcRn-binding domain, wherein the human FcRn-binding activity is enhanced within a neutral pH range, and further wherein the human FcRn-binding activity within a neutral pH range is 28-fold that of intact human IgG, more preferably 38-fold that of intact human IgG within a neutral pH range. Preferably, such antigen-binding molecules do not significantly enhance their binding activity to pre-existing ADA within a neutral pH range. The antigen-binding molecules of the present invention have enhanced FcRn-binding activity within a neutral pH range. Preferably, the binding activity to pre-existing ADA that is not significantly enhanced within a neutral pH range preferably has a human FcRn-binding activity that is 28-fold, preferably 38-fold, that of intact human IgG at pH 7.0 and 25°C. Alternatively, the human FcRn-binding activity of the antigen-binding molecule with enhanced FcRn-binding activity at pH 7.0 and 25°C is preferably stronger than a KD of 3.2 micromolar. More preferably, the FcRn-binding activity at pH 7.0 and at 25°C is stronger than 700 nanomolar, more preferably stronger than 500 nanomolar, and most preferably stronger than 150 nanomolar.

The present invention provides an antigen binding molecule of the present invention, comprising an antigen binding domain and a human Fc region of the present invention, which has an FcRn binding activity that is increased in an acidic pH range and a binding activity to pre-existing ADA that is not significantly increased in a neutral pH range. The present invention also provides an antigen binding molecule of the present invention comprising an antigen binding domain and a human FcRn binding domain, which has a human FcRn binding activity that is increased in an acidic pH range and a binding activity to pre-existing ADA that is not significantly increased in a neutral pH range compared to the binding activity of intact IgG to pre-existing ADA, wherein the human FcRn binding activity in the acidic pH range is about 2 times to about 100 times that of the human FcRn binding activity of intact human IgG. Preferably, the human FcRn binding activity of the antigen binding molecule of the present invention in the acidic pH range is at least 10 times that of the FcRn binding activity of intact human IgG, and more preferably, the human FcRn binding activity in the acidic pH range is at least 20 times that of the human FcRn binding activity of intact human IgG. The antigen-binding molecule of the present invention, whose FcRn-binding activity is enhanced in the acidic pH range but whose binding activity to pre-existing ADA is not significantly enhanced in the neutral pH range, has a human FcRn-binding activity that is 10 times, preferably 20 times, that of intact human IgG at pH 6.0 and 25°C.

The antigen-binding molecules of the present invention may have increased FcRn-binding activity in the neutral pH range, as well as antigen-binding activity in the acidic pH range that is lower than the antigen-binding activity in the neutral pH range, or antigen-binding activity at low calcium concentrations that is lower than the antigen-binding activity under high calcium concentration conditions. Specific examples of such antigen-binding molecules include those whose human FcRn-binding activity at pH 7.4 is higher than that of intact Ig, and whose antigen-binding activity is lower at pH 5.8 than at pH 7.4, where pH 5.8 and pH 7.4 are assumed to be the pH of early endosomes and plasma in vivo, respectively. Antigen-binding molecules whose antigen-binding activity is lower at pH 5.8 than at pH 7.4 can also be referred to as antigen-binding molecules whose antigen-binding activity is stronger at pH 7.4 than at pH 5.8. The value of KD (pH 5.8) / KD (pH 7.4), which is the ratio of the dissociation constant (KD) for the antigen at pH 5.8 and pH 7.4, is 1.5, 2, 3, 4, 5, 10, 15, 20, 50, 70, 80, 100, 500, 1000 or 10,000, preferably 2 or more, more preferably 10 or more, and still more preferably 40 or more. The upper limit of the KD (pH 5.8) / 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 is possible to generate it using the techniques of those skilled in the art.

Also preferred are antigen-binding molecules of the present invention that have increased FcRn binding activity in an acidic pH range and that have lower antigen-binding activity in an acidic pH range than in a neutral pH range, or that have lower antigen-binding activity at low calcium concentrations than at high calcium concentrations. Preferably, the binding activity of such antigen-binding molecules to pre-existing ADA is not significantly increased in the neutral pH range. Specific examples of such antigen-binding molecules include those that have higher binding activity to human FcRn than IgG at pH 5.8-pH 6.0 (the pH being considered to be the pH of early endosomes in vivo), and whose antigen-binding activity is lower at pH 5.8 than at pH 7.4. Antigen-binding molecules whose antigen-binding activity is lower at pH 5.8 than at pH 7.4 can also be referred to as antigen-binding molecules whose antigen-binding activity is weaker at pH 5.8 than at pH 7.4. Preferably, antigen-binding molecules whose binding activity to FcRn is increased in the acidic pH range have FcRn binding activity that is stronger than that of intact human IgG in the neutral pH range.

The modified FcRn-binding domain of the present invention is applicable to any antigen-binding molecule, regardless of the type of target antigen.

The antigen binding molecules of the present invention may have other properties. For example, it can be an agonist or antagonist antigen binding molecule, as long as it a) has a human FcRn binding activity that is necessary to increase in a neutral pH range, or b) has a human FcRn binding activity that is increased in an acidic range and its binding activity to pre-existing ADA is not significantly improved. Preferably, the antigen binding activity of this type of antigen binding molecule is lower in the acidic pH range than in the neutral pH range. Preferred antigen binding molecules of the present invention include, for example, antagonist antigen binding molecules. This type of antagonist antigen binding molecule is generally an antigen binding molecule that inhibits receptor-mediated intracellular signal transduction by blocking the binding between the ligand (agonist) and the receptor.

At the same time, the antigen binding molecules of the present invention can recognize any antigen. The specific antigens recognized by the antigen binding molecules of the present invention include, for example, the above-mentioned receptor proteins (membrane-bound receptors and soluble receptors), membrane antigens (such as cell surface markers) and soluble antigens (such as cytokines). This type of antigen includes, for example, the following antigens.

The antigen binding molecules of the present invention comprising antigen binding domains can utilize pH differences as environmental differences between plasma and endosomes for differential binding affinity of antigen binding molecules to antigens in plasma and endosomes (strong binding in plasma and weak binding in endosomes). Because the environmental differences between plasma and endosomes are not limited to pH differences, other factors (such as ionized calcium concentration) whose concentrations are different in plasma and endosomes can be used to replace the pH-dependent binding properties of the antigen binding molecules for binding to antigens. Such factors can also be used to produce antibodies that bind to antigens in plasma but dissociate from antigens in endosomes. Therefore, the present invention also includes antigen binding molecules comprising human FcRn binding domains, whose human FcRn binding activity increases within the neutral pH range and whose antigen binding activity is lower in endosomes than in plasma. Preferably, these antigen binding molecules do not significantly increase the binding activity of pre-existing ADA within the neutral pH range. The human FcRn binding activity of such antigen binding molecules is stronger than the human FcRn binding activity of intact human IgG in plasma, and in addition, the affinity of the antigen binding domains of such antigen binding molecules for antigens within endosomes is lower than the affinity in plasma. Preferably, the antigen binding domain is an antigen binding domain (pH dependent antigen binding domain) whose antigen binding activity is lower than the antigen binding activity within the neutral pH range in the acidic pH range or an antigen binding domain (calcium concentration dependent antigen binding domain) whose antigen binding activity is lower than that under high calcium concentration conditions at low calcium concentrations. The present invention also includes antigen binding molecules with human FcRn binding domains, which have improved human FcRn binding activity within the acidic pH range, and the antigen binding molecules further include antigen binding domains whose affinity for antigen is lower in endosomes than in plasma, so that the human FcRn binding activity of antigen binding molecules in endosomes is stronger than the human FcRn binding activity of intact human IgG, and the antigen binding activity of antigen binding molecules is stronger in endosomes than in plasma. Preferably, these antigen binding molecules do not significantly improve the binding activity of pre-stored ADA within the neutral pH range. Preferred antigen-binding domains are those whose antigen-binding activity is lower in the acidic pH range than in the neutral pH range (pH-dependent antigen-binding domains) or whose antigen-binding activity is lower at low calcium concentrations than at high calcium concentrations (calcium concentration-dependent antigen-binding domains).

In particular, when the antigen-binding molecules of the present invention include an antigen-binding domain that is a pH-dependent antigen-binding domain or a calcium concentration-dependent antigen-binding domain, the antigen-binding molecules of the present invention promote the uptake of antigens by cells. Antigen-binding molecules are easily dissociated from antigens in endosomes and are then released outside cells by binding to human FcRn. It is assumed that the antigen-binding molecules of the present invention are easily combined with antigens in plasma again. Therefore, for example, when the antigen-binding molecules of the present invention neutralize the antigen-binding molecules, the reduction of plasma antigen concentration can be promoted by administering the molecules.

Antigen binding domain

Preferably, the antigen-binding domain of the antigen-binding molecule has a reduced affinity for the antigen at acidic pH or at calcium ion concentrations. More preferably, the antigen-binding domain is a pH-dependent antigen-binding domain or an ionized calcium concentration-dependent antigen-binding domain as described herein.

A) pH-dependent antigen binding domain,

In addition, the antigen binding molecules of the present invention preferably include a pH-dependent antigen binding domain whose antigen binding activity in the acidic pH range is lower than that in the neutral pH range. The antigen binding molecules preferably have an antigen binding activity lower than that in the neutral pH range in the acidic pH range. The binding activity ratio is not limited, as long as the antigen binding activity in the acidic pH range is lower than that in the neutral pH range. In a preferred embodiment, the antigen binding molecules of the present invention include 2 times or more of the antigen binding molecules whose antigen binding activity is at pH 5.8 at pH 7.4, and preferably 10 times or more of the antigen binding activity is at pH 5.8 at pH 7.4. In a further preferred embodiment, the antigen binding molecules of the present invention include 40 times or more of the antigen binding molecules whose antigen binding activity is at pH 5.8 at pH 7.4.

The specific examples of the antigen binding molecules of the present invention include the embodiments described in WO2009/125825. In preferred embodiments, the antigen binding molecules of the present invention comprising pH-dependent antigen-binding domains have an antigen-binding activity at pH 5.8 lower than that at pH 7.4, wherein the KD (pH 5.8) / KD (pH 7.4) value (which is the ratio of the KD for the antigen at pH 5.8 to the KD for the antigen at pH 7.4) is preferably 2 or greater, more preferably 10 or greater, and even more preferably 40 or greater. The upper limit of the KD (pH 5.8) / KD (pH 7.4) value is not particularly limited and can be any value, such as 400, 1,000 or 10,000, as long as it is possible to produce using the technology of those skilled in the art.

In another preferred embodiment, the KD(pH5.8)/KD(pH7.4) value of the antigen-binding molecule of the present invention whose antigen-binding activity at pH 5.8 is lower than that at pH 7.4 (which is the ratio of the KD for the antigen at pH 5.8 to the KD for the antigen at pH 7.4) is 2 or greater, more preferably 5 or greater, even more preferably 10 or greater, still more preferably 30 or greater. The upper limit of the KD(pH5.8)/KD(pH7.4) value is not particularly limited and can be any value, for example, 50, 100 or 200, as long as it can be generated using the techniques of those skilled in the art.

Conditions other than the pH at which antigen-binding activity, binding activity to pre-existing ADA, and human FcRn-binding activity are measured can be appropriately selected by those skilled in the art, and these conditions are not particularly limited; however, for example, measurements can be performed in MES buffer at 37°C as described in the Examples. Furthermore, the antigen-binding activity of antigen-binding molecules can be measured by methods known to those skilled in the art, for example, using Biacore T100 (GE Healthcare) as described in the Examples.

The method for reducing (weakening) the antigen-binding activity of an antigen-binding molecule in an acidic pH range to less than the antigen-binding activity in a neutral pH range (method for imparting pH-dependent binding ability) is not particularly limited, and suitable methods are known to those skilled in the art. For example, WO2009/125825 describes a method for reducing (weakening) the antigen-binding activity in an acidic pH range to less than the antigen-binding activity in a neutral pH range by replacing the amino acid in the antigen-binding domain with histidine or inserting histidine into the antigen-binding domain. It is also known that the pH-dependent antigen-binding activity of an antibody can be imparted by replacing the amino acid of the antibody with histidine (FEBS Letter (1992) 309 (1): 85-88). Other suitable methods include methods for replacing the amino acid in the antigen-binding domain with a non-natural amino acid or inserting a non-natural amino acid into the antigen-binding domain. It is known that pKa can be artificially adjusted by using non-natural amino acids (Angew. Chem. Int. Ed. 2005, 44, 34; Chem Soc Rev. 2004 Sep, 33(7): 422-30; Amino Acids. (1999) 16(3-4): 345-79). In the context of the present invention, any non-natural amino acid can be used. In fact, it is possible to use non-natural amino acids known to those skilled in the art.

In a preferred embodiment, the antigen binding molecules of the present invention comprising an antigen-binding domain with an antigen-binding activity lower than that in the neutral pH range in the acidic pH range include such antigen binding molecules, wherein at least one amino acid of the antigen binding molecules is replaced by histidine or non-natural amino acids and/or at least one histidine or non-natural amino acid is inserted therein. The site into which histidine or non-natural amino acid mutations are introduced is not particularly limited, and it can be any site that a person skilled in the art would consider suitable, as long as, compared to before replacement, the resulting antigen-binding activity is weaker than the antigen-binding activity in the neutral pH range in the acidic pH range (KD (in the acidic pH range)/KD (in the neutral pH range) value is larger or kd (in the acidic pH range)/kd (in the neutral pH range) value is larger). In the case where the antigen binding molecule is an antibody, examples include variable regions and CDRs of antibodies. Those skilled in the art can appropriately determine the number of amino acids to be replaced by histidine or non-natural amino acids and the number of amino acids to be inserted. One amino acid can be replaced by histidine or non-natural amino acid, or one amino acid can be inserted, or two or more amino acids can be replaced by histidine or non-natural amino acid, or two or more amino acids can be inserted. In addition, in addition to the replacement of histidine or non-natural amino acid or the insertion of histidine or non-natural amino acid, the deletion, addition, insertion and/or replacement of other amino acids can also be carried out simultaneously. The replacement of histidine or non-natural amino acid or the insertion of histidine or non-natural amino acid can be carried out randomly using methods such as histidine scanning, which uses histidine rather than alanine in alanine scanning known to those skilled in the art. Antigen binding molecules whose KD (pH 5.8)/KD (pH 7.4) or kd (pH 5.8)/kd (pH 7.4) improves compared to before the mutation can be selected from the antigen binding molecules into which histidine or non-natural amino acid mutations have been randomly introduced.

It is preferred to maintain the binding activity of the antigen-binding domain at neutral pH (i.e. pH 7.4). When the antigen-binding activity of the antigen-binding molecule before histidine or non-natural amino acid mutation is set to 100%, the antigen-binding activity of the antigen-binding molecule after histidine or non-natural amino acid mutation is at least 10% or greater at pH 7.4, preferably 50% or greater, more preferably 80% or greater, and even more preferably 90% or greater. The antigen-binding activity at pH 7.4 after histidine or non-natural amino acid mutation can be stronger than the antigen-binding activity at pH 7.4 before histidine or non-natural amino acid mutation. When the antigen-binding activity of the antigen-binding molecule is reduced due to the replacement or insertion of histidine or non-natural amino acid, the antigen-binding activity can be adjusted by introducing one or more amino acid replacements, deletions, additions and/or insertions into the antigen-binding molecule so that the antigen-binding activity is equal to the antigen-binding activity before histidine replacement or insertion.

In the context of the present invention, when the antigen-binding molecule is an antibody, possible sites for histidine or non-natural amino acid substitution include, for example, antibody CDR sequences and sequences responsible for CDR structure, including, for example, sites described in WO2009/125825.

Furthermore, the present invention provides antigen-binding molecules in which at least one amino acid is substituted with histidine or an unnatural amino acid at one of the following positions:

Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b, and H102

Light chains: L24, L27, L28, L32, L53, L54, L56, L90, L92, and L94

H32, H61, L53, L90 and L94 of these change sites are assumed to be highly common change sites. Amino acid positions are represented by Kabat numbering (Kabat et al., Sequences of Immunological Interest. 5th Edition. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to the 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. Particularly preferred combinations of histidine or non-natural amino acid substitution sites include, for example, combinations of H27, H31 and H35; combinations of H27, H31, H32, H35, H58, H62 and H102, combinations of L32 and L53; and combinations 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.

When the antigen is IL-6 receptor (e.g., human IL-6 receptor), preferred sites for alteration include, but are not particularly limited to:

Heavy chain: H27, H31, H32, H35, H50, H58, H61, H62, H63, H64, H65, H100b, and H102

Light chains: L24, L27, L28, L32, L53, L56, L90, L92, and L94

Particularly preferred combinations of sites for histidine or non-natural amino acid substitution include, for example, combinations of H27, H31, and H35; combinations of H27, H31, H32, H35, H58, H62, and H102; combinations of L32 and L53; and combinations 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.

Histidine or non-natural amino acid substitutions may be made at one or more of the above positions.

Alternatively, the antigen binding molecules of the present invention may comprise an antibody constant region that is changed so that the antigen binding activity at pH 5.8 is lower than the antigen binding activity at pH 7.4. The method for changing the antibody constant region contained by antigen binding molecules is known to those skilled in the art and is in fact conventional. The specific examples of the antibody constant region after the change include the constant region (SEQ ID NO: 11, 12, 13 and 14) described in the WO2009/125825 embodiment.

Meanwhile, methods for altering antibody constant regions are known, including, 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). These methods also include methods for reducing antigen-binding activity in an acidic pH range (increases the off-rate in an acidic pH range) by introducing amino acid substitutions into the amino acid sequence of a wild-type isotype (the amino acid sequence of a wild-type IgG1, IgG2, IgG3, or IgG4). The hinge region sequences in antibody constant regions vary considerably among isotypes (IgG1, IgG2, IgG3, and IgG4), and the differences in hinge region amino acid sequences have a significant impact on antigen-binding activity. Therefore, it is possible to select an appropriate isotype to reduce antigen-binding activity in an acidic pH range (increase the off-rate in an acidic pH range), depending on the type of antigen or epitope. Furthermore, since differences in the amino acid sequence of the hinge region greatly affect antigen-binding activity, it is assumed that preferred amino acid substitution sites in the amino acid sequence of the wild-type isoform are within the hinge region.

The above method can be used to produce antigen binding molecules whose antigen binding activity is reduced (weakened) in the acidic pH range to less than the antigen binding activity in the neutral pH range from antigen binding molecules that do not have the properties by amino acid substitution or insertion (antigen binding molecules that bind in a pH-dependent manner). Other methods include methods for directly obtaining antigen binding molecules with the above properties. For example, pH-dependent antigen binding can be used as an indicator of antibodies obtained from animals (mice, rats, hamsters, rabbits, human immunoglobulin transgenic mice, human immunoglobulin transgenic rats, human immunoglobulin transgenic rabbits, llamas, camels, etc.) immunized with antigens, and antibodies with the desired target properties can be directly selected by screening. Antibodies can be produced by hybridoma technology or B cell cloning technology (Bernasconi et al., Science (2002) 298, 2199-2202; WO2008/081008), which are methods known to those skilled in the art, but are not limited to these. Alternatively, using pH-dependent antigen binding as an indicator, antibodies with target properties can be directly selected from libraries providing antigen-binding domains by in vitro screening. Such libraries include human naive libraries, immune libraries from non-human animals and humans, semisynthetic libraries, and synthetic libraries, which are known to those skilled in the art (Methods Mol Biol. 2002; 178: 87-100; J Immunol Methods. 2004 Jun; 289(1-2): 65-80; and Expert Opin Biol Ther. 2007 May; 7(5): 763-79), but are not limited thereto. However, the method is not particularly limited to these examples.

B) Ionized calcium-dependent antigen binding domain

In another preferred embodiment, the antigen-binding molecule of the present invention comprises a calcium-dependent antigen-binding domain. The antigen-binding activity of such an antigen-binding molecule depends on calcium concentration, whereby the antigen-binding activity at low calcium concentrations is lower than that at high calcium concentrations.

Preferably, the antigen-binding activity includes the antigen-binding activity under the ionized calcium concentration of 0.5-10 micromolar. More preferably, the ionized calcium concentration includes the ionized calcium concentration of the early endosome in vivo. Specifically, the antigen-binding activity includes the activity under 1-5 micromolar. Meanwhile, the antigen-binding activity of the antigen-binding molecule under high calcium concentration is not particularly limited, as long as it is the antigen-binding activity under the ionized calcium concentration of 100 micromolar-10mM. Preferably, the antigen-binding activity includes the antigen-binding activity under the ionized calcium concentration of 200 micromolar-5mM. Preferably, the low calcium concentration is the ionized calcium concentration of 0.1-30 micromolar, and the high calcium concentration is the ionized calcium concentration of 100 micromolar-10mM.

Preferably, the low calcium concentration is the concentration of ionized calcium in endosomes, and the high calcium concentration is the concentration of ionized calcium in plasma. More specifically, antigen-binding molecules comprising such calcium-dependent antigen-binding domains include those whose antigen-binding activity at ionized calcium concentrations in early endosomes in vivo (e.g., low calcium concentrations of 1-5 micromolar) is lower than their antigen-binding activity at ionized calcium concentrations in plasma in vivo (e.g., high calcium concentrations of 0.5-2.5 mM).

As for the antigen-binding activity of an antigen-binding molecule whose antigen-binding activity at low calcium concentration is lower than that at high calcium concentration, there is no limitation in this difference in antigen-binding activity as long as the antigen-binding activity at low calcium concentration is lower than that at high calcium concentration. It is even acceptable that the antigen-binding activity of the antigen-binding molecule is only slightly lower under low calcium concentration conditions.

In a preferred embodiment, for antigen-binding molecules of the present invention whose antigen-binding activity at low calcium concentrations (low Ca) is lower than that at high calcium concentrations (high Ca), the value of KD(low Ca)/KD(high Ca) (which is the ratio of KD between low and high calcium concentrations) is 2 or greater, preferably the value of KD(low Ca)/KD(high Ca) is 10 or greater, and more preferably the value of KD(low Ca)/KD(high Ca) is 40 or greater. The upper limit of the KD(low Ca)/KD(high Ca) value is not particularly limited and can be any value, for example, 400, 1,000, and 10,000, as long as it can be generated by techniques known to those skilled in the art.

In another preferred embodiment, for an antigen-binding molecule comprising a calcium-dependent antigen-binding domain whose antigen-binding activity at low calcium concentrations is lower than that at high calcium concentrations, the kd(low Ca)/kd(high Ca) value (which is the ratio of the kd of the antigen between low calcium concentration conditions and pH 7.4) is 2 or greater, preferably the kd(low Ca)/kd(high Ca) value is 5 or greater, more preferably the kd(low Ca)/kd(high Ca) value is 10 or greater, and even more preferably the kd(low Ca)/kd(high Ca) value is 30 or greater. The upper limit of the kd(low Ca)/kd(high Ca) value is not particularly limited and can be any value, for example, 50, 100, and 200, as long as it can be generated by techniques known to those skilled in the art.

The antigen-binding activity of antigen binding molecules can be measured by methods known to those skilled in the art. Those skilled in the art can select suitable conditions beyond ionized calcium concentration. The antigen-binding activity of antigen binding molecules can be evaluated by using KD (dissociation constant), apparent KD (apparent dissociation constant), dissociation rate kd (dissociation rate), apparent kd (apparent dissociation: apparent dissociation rate) etc. They can be measured by methods known to those skilled in the art, for example, using Biacore (GE Healthcare), Scatchard diagram, FACS etc.

The antigen binding molecules to be screened by the screening method of the present invention can be any antigen binding molecules.For example, the antigen binding molecules with native sequence or the antigen binding molecules with substituted amino acid sequence can be screened.The antigen binding molecules comprising calcium ion dependent antigen binding domains to be screened by the screening method of the present invention can be prepared by any method. Antibodies and libraries prepared by hybridomas such as pre-stored antibodies, pre-stored libraries (phage libraries, etc.) and the B cells of autoimmune animals or by immune animals can be used, by introducing the amino acid (such as aspartic acid or glutamic acid) that can chelate calcium or non-natural amino acid mutations into such antibodies or libraries obtained (library with high levels of non-natural amino acids or amino acids (such as aspartic acid or glutamic acid) that can chelate calcium, introducing non-natural amino acid mutations or amino acids (such as aspartic acid or glutamic acid) that can chelate calcium at specific sites, etc.) etc.

Conventional methods in the art can be used to easily screen, identify, and isolate antigen-binding molecules whose antigen-binding activity under low calcium concentration conditions is lower than that under high calcium concentration conditions (see, for example, PCT application number PCT/JP2011/077619). Examples of such screening methods include the step of determining an antigen-binding molecule having at least one function selected from the following:

(i) Promote the uptake of antigens by cells;

(ii) the ability to bind to an antigen two or more times;

(iii) a function of promoting a decrease in plasma antigen concentration; and

(iv) Excellent plasma retention function.

Specifically, the present invention provides a method for screening an antigen-binding molecule comprising a calcium-dependent antigen-binding domain, the method comprising the following steps:

(a) determining the antigen-binding activity of the antigen-binding molecule under low calcium concentration conditions;

b) measuring the antigen-binding activity of the antigen-binding molecule under high calcium concentration conditions; and

(c) Selecting an antigen-binding molecule whose antigen-binding activity under low calcium concentration conditions is lower than its antigen-binding activity under high calcium concentration conditions.

A method for producing an antigen-binding molecule having a calcium ion-dependent antigen-binding domain includes, for example, the following steps:

(a) determining the antigen-binding activity of the antigen-binding molecule under low calcium concentration conditions;

(b) measuring the antigen-binding activity of the antigen-binding molecule under high calcium concentration conditions; and

(c) Selecting an antigen-binding molecule whose antigen-binding activity under low calcium concentration conditions is lower than its antigen-binding activity under high calcium concentration conditions.

Another method for producing an antigen-binding molecule having a calcium-dependent antigen-binding domain is a method comprising the following steps:

(a) contacting an antigen with an antigen-binding molecule or a library of antigen-binding molecules under conditions of high calcium concentration;

(b) obtaining an antigen-binding molecule that binds to the antigen in step (a);

(c) placing the antigen-binding molecule obtained in step (b) under low calcium concentration conditions;

(d) obtaining an antigen-binding molecule whose antigen-binding activity in step (c) is lower than the activity selected in step (b);

(e) obtaining a gene encoding the antigen-binding molecule obtained in step (d); and

(f) producing an antigen-binding molecule using the gene obtained in step (e).

Steps (a) to (e) may be repeated two or more times. Therefore, the present invention provides a method further comprising repeating steps (a) to (e) in the above method two or more times. The number of times steps (a) to (e) are repeated is not particularly limited; however, the number is generally 10 times or less.

Antigen binding molecules for production method of the present invention can be prepared by any conventional method.For example, pre-stored antibodies, pre-stored libraries (phage libraries etc.), antibodies and libraries prepared from hybridomas obtained by immune animals or the B cells of autoimmune animals, antibodies and libraries prepared by introducing histidine or non-natural amino acid mutations into the above-mentioned antibodies and libraries (libraries with high levels of histidine or non-natural amino acids, libraries introducing histidine or non-natural amino acids at specific sites etc.) etc. can be used.

Other methods for screening such calcium ion-dependent antigen-binding molecules or calcium ion-dependent antigen-binding domains are described in PCT Application No. PCT/JP2011/077619.

antigen

The antigens recognized by the antigen-binding molecules of the present invention, such as the antibodies of the present invention, are not particularly limited. Such antigen-binding molecules of the present invention can recognize any antigen. Specific examples of antigens recognized by the antigen-binding molecules of the present invention include, but are not limited to, 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, adiponectin, ADP-ribosyl cyclase-1, aFGF, AGE, ALCAM, ALK, ALK-1, ALK-7, allergens, α1-antichymotrypsin (an tichemotrypsin), alpha-1 antitrypsin, alpha-synuclein, alpha-V/beta-1 antagonist, aminin, amylin, amyloid 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, apoA1, apo serum amyloid A, Apo-SAA, APP, APRIL, AR, ARC, ART, Artemin, ASPARTIC, atrial natriuretic factor, atrial natriuretic peptide, atrial natriuretic peptide A, atrial natriuretic peptide B, atrial natriuretic peptide C, av/b3 integrin, Axl, B7-1, B7-2, B7-H, BACE, BACE-1, Bacillus anthracis anthracis) protective antigen, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, β-2-microglobulin, β-lactamase, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, B-lymphocyte stimulating factor (BIyS), BMP, BMP-2 (BMP-2a), BMP-3 (osteoblast), BMP-4 (BMP-2b), BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8 (BMP-8a), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3), BMPs, BOK, bombesin, bone-derived neurotrophic factor, bovine growth hormone, BPDE, BPDE-DNA, BRK-2, BTC, B lymphocyte cell adhesion molecule, C10, C1-inhibitor, C1q, C3, C3a, C4, C5, C5a (complement 5a), CA125, CAD-8, cadherin-3, calcitonin, cAMP, carbonic anhydrase-IX, carcinoembryonic antigen (CEA), cancer-associated antigen, cardiotrophin-1, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, tissue Proteinase V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1/I-309, CCL11/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, CCL20/MIP-3-α, CCL21/SLC, CCL22/MDC, CCL23/MPIF- 1. CCL24/eotaxin-2, CCL25/TECK, CCL26/eotaxin-3, CCL27/CTACK, CCL28/MEC, CCL3/M1P-1-α, CCL3Ll/LD-78-β, CCL4/MIP-1-β, CCL5/RANTES, CCL6/C10, CCL7/MCP-3, CCL8/MCP-2, CCL9/10/MTP-1-γ, CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7 , CCR8, CCR9, CD1, CD10, CD105, CD11a, CD11b, CD11c, CD123, CD13, CD137, CD138, CD14, CD140a, CD146, CD147, CD148, CD15, CD152, CD16, CD164, CD18, CD19, CD2, CD20, CD21, CD22, CD23, CD25, CD26, CD27L, CD28, CD29, CD3, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD37 , CD38, CD3E, CD4, CD40, CD40L, CD44, CD45, CD46, CD49a, CD49b, CD5, CD51, CD52, CD54, CD55, CD56, CD6, CD61, CD64, CD66e, CD7, CD70, CD74, CD8, CD80(B7-1), CD89, CD95, CD105, CD158a, CEA, CEACAM5, CFTR, cGMP, CGRP receptor, CINC, CKb8-1, claudin-18, CLC, Clostridium botulinum toxin, Clostridium difficile toxin, Clostridium perfringens toxin perfringens) toxin, c-Met, CMV, CMVUL, CNTF, CNTN-1, complement factor 3 (C3), complement factor D, corticosteroid binding globulin, colony stimulating factor-1 receptor, COX, C-Ret, CRG-2, CRTH2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1/Fractalkine, CX3CR1, CXCL, CXCL1/Gro-α, CXCL10, CXCL11/I-TAC, CXCL12/SDF-l-α/β, CXCL13/BCA-1, CXCL14/BRAK, CXCL15/Lungkine, CXCL16, CXCL16, CXCL2/Gro-βCXCL3/Gro-γ, CXCL3, CXCL4/PF4, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, CXCL8/IL-8, CXCL9/Mig, CXCL10/IP-10, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cystatin C, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, delta-like protein ligand 4, des(1-3)-IGF-1 (brain IGF-1), Dhh, DHICA oxidase, Dickkopf-1, digoxin, dipeptidyl peptidase IV, DK1, DNAM-1, DNase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), containing EGF-like Domain-containing protein 7, elastase, elastin, EMA, EMMPRIN, ENA, ENA-78, endosialin, endothelin receptor, endotoxin, neprilysin, eNOS, Eot, eotaxin, eotaxin-2, eotaxini, EpCAM, EphrinB2/EphB4, Epha2 tyrosine kinase receptor, epidermal growth factor receptor (EGFR), ErbB2 receptor, ErbB3 tyrosine kinase receptor, ERCC, erythropoietin (EPO), erythropoietin receptor, E-selectin, ET-1, Exodus-2, RSV F protein, F10, F11, F12, F13, F5, F9, factor Ia, factor IX, factor Xa, factor VII, factor VIII, factor VII Ic, Fas, FcαR, FcεRI, FcγIIb, FcγRI, FcγRIIa, FcγRIIIa, FcγRIIIb, FcRn, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8, FGF-acidic, FGF-basic, FGFR, FGFR-3, fibrin, fibroblast activation protein (FAP), fibroblast growth factor, 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, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, F ZD9, G250, Gas6, GCP-2, GCSF, G-CSF, G-CSF receptor, GD2, GD3, 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-α1, GFR-α2, GFR-α3, GF-β1, gH envelope glycoprotein, GITR, Glucagon, Glucagon receptor, Glucagon-like peptide 1 receptor, Glut4, Glutamate carboxypeptidase II, Glycoprotein hormone receptor, Glycoprotein Gram-negative protein IIb/IIIa (GPIIb/IIIa), glypican-3, GM-CSF, GM-CSF receptor, gp130, gp140, gp72, granulocyte-CSF (G-CSF), GRO/MGSA, growth hormone-releasing factor, GRO-β, GRO-γ, Helicobacter pylori (H. pylori) hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCC1, HCMV gB envelope glycoprotein, HCMVUL, hematopoietic growth factor (HGF), HepBgp120, heparinase, heparin cofactor II, liver 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 MIBgp120V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HMGB-1, HRG, Hrk, HSP47, Hsp90, HSVgD glycoprotein, human myocardial globulin, human cytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin, human tissue-type plasminogen activator (t-PA), huntingtin, HVEM, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN-α, IFN-β, IFN-γ, IgA, IgA receptor, IgE, IGF, IG F binding protein, IGF-1, IGF-1R, IGF-2, IGFBP, IGFR, IL, IL-1, IL-10, IL-10 receptor, IL-11, IL-11 receptor, IL-12, IL-12 receptor, IL-13, IL-13 receptor, IL-15, IL-15 receptor, IL-16, IL-16 receptor, IL-17, IL-17 receptor, IL-18 (IGIF), IL-18 receptor, IL-1α, IL-1β, IL-1 receptor, IL-2, IL-2 receptor, IL-20, IL-20 receptor, IL-21, IL-21 receptor, IL-23, IL-23 receptor, IL-2 receptor, IL-3, IL-3 receptor, IL-31, IL-31 receptor, IL-3 receptor, IL-4, IL-4 receptor IL-5, IL-5 receptor , IL-6, IL-6 receptor, IL-7, IL-7 receptor, IL-8, IL-8 receptor, IL-9, IL-9 receptor, immunoglobulin immune complex, immunoglobulin, INF-α, INF-α receptor, INF-β, INF-β receptor, INF-γ, INF-γ receptor, IFN-1 type, IFN-1 type receptor, influenza virus (influenza), inhibin, inhibin α, inhibin β, iNOS, insulin, insulin A-chain, insulin B-chain, insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein, integrin, integrin α2, integrin α3, integrin α4, integrin α4/β1, integrin α-V/β-3, integrin α-V/β-6, integrin α4/β7, integrin α5/β1, integrin α5/β3, Integrin α5/β6, integrin α-δ (αV), integrin α-θ, integrin β1, integrin β2, integrin β3 (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, LAMP, LAPP (amyloid peptide, islet amyloid polypeptide), LAP (TGF-1), latency-associated peptide, latent TGF-1, latent TGF-1bp1, LBP, LDGF, LDL, LDL receptor, LECT2, Lefty, leptin, luteinizing hormone (leutinizing hormone) hormone) (LH), Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, LFA-3 receptor, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphocyte chemotactic peptide, lymphotoxin beta receptor, lysosphingolipid receptor receptor), Mac-1, macrophage-CSF (M-CSF), MAdCAM, MAG, MAP2, MARC, maspin, MCAM, MCK-2, MCP, MCP-1, MCP-2, MCP-3, MCP-4, MCP-I (MCAF), M-CSF, MDC, MDC (67a.a.), MDC (69a.a.), megsin, Mer, MET tyrosine kinase receptor family, metalloproteinase, membrane glycoprotein OX2, Mesothelin , MGDF receptor, MGMT, MHC (HLA-DR), microbial proteins, MIF, MIG, MIP, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, monocyte chemoattractant protein (monocyte chemoattractant protein attractant protein), monocyte colony inhibitory factor, mouse gonadotropin-related peptide, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, mucin (Mud), Muellerian inhibitory substance, Mug, MuSK, myelin-associated glycoprotein, myeloid progenitor inhibitory factor-1 (MPIF-I), NAIP, nanobody, NAP, NAP-2, NCA90, NCAD, N-cadherin, NCAM, neprilysin, neural cell adhesion molecule, Neroserpin, neuron growth factor (NGF), neurotrophin-3, neurotrophin-4, neurotrophin-6, neuropilin 1, neurotrophin, NGF-β, NGFR, NKG20, N-methionyl human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, hepatitis C virus nonstructural protein type 3 (NS3), NOS, Npn, NRG-3, NT, NT-3, NT-4, NTN, OB, OGG1, oncostatin M, OP-2, OPG, OPN, OSM, OSM receptor, osteoinductive factor, osteopontin, OX40L, OX40R, oxidized LDL, p150, p95, PADPr, parathyroid hormone, PARC, PAR P, PBR, PBSF, PCAD, P-cadherin, PCNA, PCSK9, PDGF, 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 various sizes (e.g., PEG-20, PEG-30, PEG40), PP14, prekallikrein, prion protein, procalcitonin, programmed cell death protein 1, proinsulin, prolactin, proprotein Converting enzyme PC9, prorelaxin, prostate-specific membrane antigen (PSMA), protein A, 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, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, Ret, reticulon4, rheumatoid factor, RLIP76, RPA2, RPK-1, RSK, RSV Fgp, S100, RON-8, SCF/KL, SCGF, sclerostin, SDF-1, SDF1α, SDF1β, SER INE, serum amyloid P, serum albumin, sFRP-3, Shh, Shiga-like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, sphingosine 1-phosphate receptor 1, Staphylococcal lipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF), streptokinase, superoxide dismutase, syndecan-1, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TB, TCA-3, T-cell receptor α/β, TdT, TECK, TEM1, TEM5, TEM7, TEM8, tenascin, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β Pan specific, TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-βRl (ALK-5), TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TGF-I, thrombin, thrombopoietin (TPO), thymic stromal lymphopoietin (lymphoprotein) receptor, thymic Ck-1, thyroid-stimulating hormone (TSH), thyroxine, thyroxine-binding globulin, Tie, TIMP , TIQ, tissue factor, tissue factor protease inhibitor, tissue factor protein, TMEFF2, Tmpo, TMPRSS2, TNF receptor I, TNF receptor II, TNF-α, TNF-β, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAILR1Apo-2/DR4), TNFRSF10B (TRAILR2DR5/KILLER/TRICK-2A/TRICK-B), TNFRSF10C (TRAIL R3DcR1/LIT/TRID), TNFRSF10D (TRAIL R4DcR2/TRUNDD), TNFRSF11A (RANK ODF R/TRANCE R), TNFRSF11B (OPG OCIF/TR1), TNFRSF12 (TWEAKRFN14), TNFRSF12A, TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14(HVEM ATAR/HveA/LIGHTR/TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITRAITR), TNFRSF19(TROYTAJ/TRADE), TNFRSF19L(RELT), TNFRSF1A(TNF Rl CD120a/p55-60), TNFRSF1B (TNF RIICD120b/p75-80), TNFRSF21(DR6), TNFRSF22(DcTRAILR2TNFRH2), TNFRSF25(DR3Apo-3/LARD/TR-3/TRAMP/WSL-1), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII/TNFC R), TNFRSF4(OX40ACT35/TXGP1R), TNFRSF5(CD40p50), TNFRSF6(Fas Apo-1/APT1/CD95), TNFRSF6B(DcR3M68/TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137/ILA), TNFRST23 (DcTRAIL R1TNFRH1), TNFSF10 (TRAIL Apo-2 ligand/TL2), TNFSF11 (TRANCE/RANK ligand ODF/OPG ligand), TNFSF12 (TWEAKApo-3 ligand/DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFFBLYS/TALL1/THANK/TNFSF20), TNFSF14 (LIGHT HVEM ligand/LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand AITR ligand/TL6), TNFSF1A (TNF-a Conectin/DIF/TNFSF2), TNFSF1B (TNF-b LTa/TNFSF1), TNFSF3 (LTb TNFC/p33), TNFSF4 (OX40 ligand gp34/TXGP1), TNFSF5 (CD40 ligand CD154/gp39/HIGM1/IMD3/TRAP), TNFSF6 (Fas ligand Apo-1 ligand/APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TNF-α, TNF-β, TNIL-I, toxin metabolites, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, transforming growth factors (TGF) such as TGF-α and TGF-β, transmembrane glycoprotein NMB, transthyretin, TRF, Trk, TROP-2, trophoblast glycoprotein, TSG, TSLP, tumor necrosis factor (TNF), tumor-associated antigen CA125, tumor-associated antigen expressing Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VAP-1, vascular endothelial growth factor (VEGF), vaspin, VCAM, VCAM-1, VECAD, VE-cadherin , 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, VLA-1, VLA-4, VNR integrin, von Willebrand factor (vWF), WIF-1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8 A, WNT8B, WNT9A, WNT9B, XCL1, XCL2/SCM-1-β, XCL1/lymphocyte chemoattractant peptide, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aa, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, chromogranin A, chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1. 1. Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, Factor B, Factor D, Factor H, Properdin, Chitosan, Fibrinogen, Fibrin, Prothrombin, Thrombin, Tissue Factor, Factor V, Factor Va, Factor VII, Factor VIIa, Factor VIII, Factor VIIIa, Factor IX, Factor IXa, Factor X, Factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA, S1P.

The antigen binding molecules of the present invention can reduce the total plasma antigen concentration of the above-mentioned antigens. The antigen binding molecules of the present invention can also eliminate viruses, bacteria and fungi from the plasma by binding to the structural components of viruses, bacteria and fungi. In particular, RSV F protein, Staphylococcus lipoteichoic acid, Clostridium difficile toxin, Shiga-like toxin II, Bacillus anthracis protective antigen and Hepatitis C virus E2 glycoprotein can be used as structural components of viruses, bacteria and fungi.

use

The present invention also provides various uses of the antigen-binding molecules of the present invention.

Therefore, the present invention provides the use of the modified antigen-binding molecules of the present invention for improving antigen-binding molecule-mediated cell uptake of antigens. In addition, the present invention also provides a method for improving antigen-binding molecule-mediated cell uptake of antigens, the method comprising modifying the antigen-binding molecule comprising a parent FcRn-binding domain by replacing an amino acid at one or more positions selected from the following, thereby increasing the FcRn binding activity at neutral pH compared to an antigen-binding molecule with a complete FcRn-binding domain: EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436.

The term "cell uptake of antigen" herein mediated by antigen binding molecules means that the antigen is taken into cells by endocytosis. Meanwhile, the term "promoting uptake of cells" herein means improving the intracellular uptake rate of the antigen binding molecules bound to the antigen in plasma, and/or reducing the amount of the taken-in antigen that is recirculated to plasma. This means that before the FcRn binding domain modification and therefore before the human FcRn binding activity of the antigen binding molecules is improved in the neutral pH range, or before the antigen binding activity (binding capacity) of the antigen binding molecules is improved and reduced in the acidic pH range to less than its antigen binding activity in the neutral pH range, the rate of uptake of cells is promoted. Preferably compared with intact IgG, more preferably compared with intact human IgG, the rate is improved. Therefore, in the present invention, it is possible to evaluate whether antigen binding molecules promote cell uptake of antigens based on the increase in the rate of cell uptake of antigens. The rate of antigen uptake by cells can be calculated by, for example, monitoring the decrease in antigen concentration over time in the culture medium containing cells expressing human FcRn, or monitoring the amount of antigen taken into cells expressing human FcRn over time after adding the antigen and the antigen-binding molecule to the culture medium. For example, the method of the present invention that promotes the rate of antigen uptake by cells mediated by antigen-binding molecules can be used to increase the rate of antigen elimination from plasma by administering the antigen-binding molecules of the present invention. Therefore, whether the rate of antigen elimination from plasma can be accelerated or the total plasma antigen concentration is reduced by, for example, administering the antigen-binding molecules of the present invention, to evaluate whether the antigen-binding molecule-mediated cell uptake of antigen is promoted.

The term "total plasma antigen concentration" herein means the sum of the antigen concentration to which the antigen binding molecules are bound and the unbound antigen concentration or "plasma free antigen concentration," which is the concentration of antigen to which the antigen binding molecules are unbound. Various methods for measuring "total plasma antigen concentration" and "plasma free antigen concentration" are well known in the art as described below.

The present invention also provides the purposes of the antigen binding molecules of the present invention for increasing the total number of antigens that can be combined with a single antigen binding molecule before its degradation. The present invention also provides a method for increasing the number of antigens that can be combined with a single antigen binding molecule by using the antigen binding molecules of the present invention. Specifically, the present invention provides a method for increasing the number of antigens that can be combined with a single antigen binding molecule by replacing an amino acid in one or more positions selected from the following in the parent FcRn binding domains of the antigen binding molecule, thereby improving the FcRn binding activity at neutral pH compared to the antigen binding molecules with complete FcRn binding domains, increasing the method for the total number of antigens that can be combined with a single antigen binding molecule: EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434 and EU436.

"Conventional antibodies" can usually only bind to one or two antigens before they degrade in the endosome. The antigen binding molecules of the present invention can increase the cycle number achieved before the antigen binding molecules are degraded, wherein each cycle is composed of the following: antigen binds to the antigen binding molecules in the plasma, intracellular uptake of the antigen binding molecules bound to the antigen, dissociation with the antigen in the endosome, and then the antigen binding molecules return to the plasma. This means that before the FcRn binding domain is modified and therefore before the human FcRn binding activity of the antigen binding molecules is improved in the neutral pH or acidic range, or before the antigen binding activity (binding capacity) of the antigen binding molecules is improved and reduced in the acidic pH range to less than the antigen binding activity in the neutral pH range, the cycle number increases. Therefore, it is possible to evaluate whether the cycle number increases by testing whether the above-mentioned "intracellular uptake is promoted" or the following "pharmacokinetics are improved."

The present invention also provides the antigen binding molecules of the present invention for improving the purposes of antigen elimination from mammal (i.e. people) blood. Specifically, the present invention provides the antigen binding molecules of the present invention for reducing the purposes of plasma specific antigen concentration, wherein the antigen binding molecules include the antigen binding domains that can be combined with the antigen. The present invention also provides a method for reducing plasma specific antigen concentration, wherein the antigen binding molecules include the antigen binding domains that can be combined with the antigen, and it is by replacing amino acid in parent FcRn binding domains at one or more positions selected from the following compared with the antigen binding molecules with complete FcRn binding domains. Improve the FcRn binding activity at neutral pH: EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434 and EU436.

The present invention also provides the use of the antigen-binding molecules of the present invention for promoting the extracellular release of antigen-free antigen-binding molecules taken into cells in the form of antigen binding. More specifically, the present invention provides a method for promoting the extracellular release of antigen-free antigen-binding molecules taken into cells in the form of antigen binding without significantly improving the binding activity to pre-existing ADA at neutral pH compared to the parent antibody by replacing amino acids at one or more positions selected from the following in the parent FcRn binding domain, thereby improving the FcRn binding activity at neutral pH compared to the antigen-binding molecules with complete FcRn binding domains: EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434 and EU436.

Herein, "antigen-free antigen-binding molecules taken into cells in an antigen-bound form are released extracellularly" does not necessarily mean that all antigen-binding molecules taken into cells and bound to antigens are released outside the cells in an antigen-free form. It is acceptable that the proportion of antigen-binding molecules released outside the cells in an antigen-free form increases compared to before modification of the FcRn-binding domain, thereby reducing the antigen-binding activity of the antigen-binding molecule in the acidic pH range to less than that in the neutral pH range and improving human FcRn binding activity in the neutral pH range. Antigen-binding molecules released outside the cells preferably maintain antigen-binding activity.

The present invention also provides the use of the FcRn binding domains of the present invention for improving the ability of antigen binding molecules to eliminate plasma antigens. In the present invention, "a method for improving the ability to eliminate plasma antigens" is synonymous with "a method for improving the ability of antigen binding molecules to eliminate antigens from plasma". More specifically, the present invention provides a method for improving the ability of antigen binding molecules to clear plasma antigens by replacing amino acids at one or more positions selected from the following in the parent FcRn binding domain, thereby improving the FcRn binding activity at neutral and/or acidic pH compared to antigen binding molecules with complete FcRn binding domains: EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434 and EU436.

The term "ability to eliminate plasma antigens" herein refers to the ability of an antigen-binding molecule to eliminate antigens from plasma when administered or secreted in vivo. Therefore, "increasing the ability of an antigen-binding molecule to eliminate plasma antigens" herein means accelerating the rate of antigen elimination from plasma when administered compared to before modification of the FcRn-binding domain, thereby increasing the human FcRn binding activity of the antigen-binding molecule in the neutral pH range, or before increasing human FcRn binding activity and simultaneously reducing its antigen-binding activity in the acidic pH range to less than that in the neutral pH range. The improvement in the activity of an antigen-binding molecule to eliminate antigens from plasma can be evaluated, for example, by administering a soluble antigen and an antigen-binding molecule in vivo and measuring the concentration of the soluble antigen in plasma after administration. When the concentration of the soluble antigen in plasma decreases after administration of the soluble antigen and the modified antigen-binding molecule, it can be determined that the ability of the antigen-binding molecule to eliminate plasma antigens has increased. Soluble antigen can be in the form of antigen bound to or unbound by the antigen-binding molecules, and its concentration can be measured as "the concentration of antigen bound to the antigen-binding molecules in plasma" and "the concentration of antigen unbound to the antigen-binding molecules in plasma" (the latter being synonymous with "plasma free antigen concentration"), respectively. Since "total plasma antigen concentration" refers to the sum of the concentration of antigen bound to and unbound by the antigen-binding molecules, or "plasma free antigen concentration," the latter being the concentration of antigen unbound by the antigen-binding molecules, the concentration of soluble antigen can be measured as "total plasma antigen concentration." Various methods for measuring "total plasma antigen concentration" or "plasma free antigen concentration" are well known in the art, as described below.

The present invention also provides the use of the FcRn binding domain of the present invention for improving the pharmacokinetics of antigen binding molecules. More specifically, the present invention provides a method for improving the pharmacokinetics of antigen binding molecules by replacing amino acids in a parent FcRn binding domain at one or more positions selected from the following: EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434 and EU436, thereby increasing the FcRn binding activity at neutral and/or acidic pH compared to an antigen binding molecule with a complete FcRn binding domain.

Herein, "improvement of pharmacokinetics", "improvement of pharmacokinetics" and "excellent pharmacokinetics" can be described in another way as "improvement of plasma (blood) retention", "improvement of plasma (blood) retention", "excellent plasma (blood) retention" and "extended plasma (blood) retention". These terms are used herein as synonyms.

Improved pharmacokinetics include, inter alia:

(1) Delayed elimination compared to a control antigen-binding molecule (e.g., an antigen-binding molecule with an intact FcRn-binding domain): Prolonged time between administration and elimination of the antigen-binding molecule from plasma; and/or

(2) prolonging the plasma retention time of an antigen-binding molecule (preferably in a form in which an antibody or antibody derivative can bind to its antigen upon administration of the antigen-binding molecule) compared to the plasma retention time of a control antigen-binding molecule (e.g., an antigen-binding molecule with an intact FcRn-binding domain); and/or

(3) shortening the time during which the antigen is free (not bound to the antigen-binding molecule in vivo) between administration and elimination of the antigen-binding molecule compared to a control antigen-binding molecule (prolonging the time during which the antigen-binding molecule is bound to its antigen in the subject's body between administration and elimination compared to a control antigen-binding molecule (e.g., an antigen-binding molecule with an intact FcRn-binding domain); and/or

(4) increasing the ratio of antigen bound to the antigen-binding molecule to total antigen in the body before antibody degradation compared to the ratio of antigen bound to a control antigen-binding molecule (e.g., an antigen-binding molecule with an intact FcRn-binding domain) (increasing the number of binding events between the administration of the antibody or antibody derivative and its antigen compared to the number of binding events to the control antigen-binding molecule between administration and degradation).

(5) Reduction of the total plasma antigen concentration or plasma free antigen concentration after administration of the antigen-binding molecule compared to the total plasma antigen concentration or plasma free antigen concentration after administration of a control antigen-binding molecule (e.g., an antigen-binding molecule having an intact FcRn-binding domain).

The present invention also provides a method for delayed elimination of an antigen-binding molecule in a subject, comprising the step of introducing a modification into the FcRn-binding domain of the antigen-binding molecule at one or more positions selected from the group consisting of: EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436.

The term "improvement in pharmacokinetics" as used herein refers not only to a prolongation of the time between administration of the antigen-binding molecule to a subject (human or non-human animal such as mouse, rat, monkey, rabbit and dog) and its elimination from the plasma (e.g., until the antigen-binding molecule is degraded intracellularly, etc., and cannot return to the plasma), but also to a prolongation of the plasma retention of the antigen-binding molecule in a form that permits antigen binding (e.g., in an antigen-free form of the antigen-binding molecule) from administration until the antigen-binding molecule is degraded.

Therefore, the present invention also provides a method for prolonging the plasma retention time of an antigen-binding molecule, the method comprising the step of introducing a modification into the FcRn-binding domain of the antigen-binding molecule at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436. Intact human IgG can bind to FcRn of non-human animals. For example, mice are preferably used for administration to confirm the properties of the antigen-binding molecules of the present invention, because intact human IgG can bind more firmly to mouse FcRn than to human FcRn (Int Immunol. 2001 Dec; 13(12): 1551-9). As another example, mice in which the native FcRn gene is disrupted and carries a transgenic human FcRn gene to be expressed (Methods Mol Biol. 2010; 602: 93-104) can also be preferably used for administration to confirm the properties of the antigen-binding molecules of the present invention described below.

Specifically, " improvement of pharmacokinetics " also includes the time extension between the degradation of the antigen binding molecules (antigen-free form of antigen binding molecules) that are not bound to the antigen and are given during the period. If the antigen binding molecules have been bound to the antigen, the antigen binding molecules in the plasma cannot be bound to the new antigen. Therefore, the longer the time during which the antigen binding molecules are not bound to the antigen, the longer the time during which there is the potential for being bound to the new antigen (the higher the chance of being bound to another antigen). In other words, more antigens are combined in a shorter period of time. Therefore, the antigen binding molecules of modification can be given to accelerate the elimination of antigen from plasma, and increase the plasma concentration of the antigen-free form of antigen binding molecules, and extend the total time that antigen is combined with antigen binding molecules.

Specifically, herein, "improvement in the pharmacokinetics of an antigen-binding molecule" includes improvement in the pharmacokinetic parameters of the antigen-free form of the antigen-binding molecule (any of extended plasma half-life, extended mean plasma residence time, and reduced plasma clearance), prolonged binding of the antigen to the antigen-binding molecule after administration of the modified antigen-binding molecule, and accelerated elimination of the antigen from the plasma mediated by the antigen-binding molecule.

The improvement of the pharmacokinetics of the antigen-binding molecule can be evaluated by measuring any one of the following parameters: plasma half-life, mean plasma residence time, and plasma clearance of the antigen-binding molecule or its antigen-free form ("Pharmacokinetics: Enshu-niyoru Rikai (Understanding through practice)" Nanzando). For example, after the antigen-binding molecule is administered to a mouse, rat, monkey, rabbit, dog or human, the plasma concentration of the antigen-binding molecule or its antigen-free form is measured. Then, each parameter is measured. When the plasma half-life or mean plasma residence time is prolonged, it can be determined that the pharmacokinetics of the antigen-binding molecule is improved. The parameters can be measured by methods known to those skilled in the art. The parameters can be appropriately evaluated by, for example, non-compartmental analysis using the pharmacokinetic analysis software WinNonlin (Pharsight) according to the accompanying instruction manual. The plasma concentration of the antigen-free antigen-binding molecule can be measured by methods known to those skilled in the art, for example, using the assay method described in Clin Pharmacol. 2008 Apr; 48(4): 406-17.

The term "improvement in pharmacokinetics" herein also includes an extension of the time that the antigen binds to the antigen-binding molecule after administration of the antigen-binding molecule. Whether the time that the antigen binds to the antigen-binding molecule is extended after administration of the antigen-binding molecule can be assessed by measuring the plasma concentration of free antigen. Extension can be determined based on the measured plasma concentration of free antigen or the time required to increase the ratio of free antigen concentration to total antigen concentration.

The present invention also provides the use of an antigen-binding molecule of the present invention for reducing the total plasma antigen concentration or plasma free antigen concentration of a specific antigen, wherein the antigen-binding molecule comprises an antigen-binding domain that binds to the antigen. More specifically, the present invention provides a method for reducing the total plasma antigen concentration or plasma free antigen concentration, the method comprising the steps of:

a) providing an antigen-binding molecule comprising a parent FcRn-binding domain, wherein the antigen-binding molecule comprises an antigen-binding domain that can bind to the antigen,

b) substituting amino acids in the parent FcRn-binding domain at one or more positions selected from EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434 and EU436, thereby improving the FcRn-binding activity at neutral pH compared to an antigen-binding molecule with an intact FcRn-binding domain.

In addition, the present invention provides a method comprising the step of introducing modifications into the FcRn binding domain of the antigen binding molecule at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436. As used herein, the term "antigen elimination rate" refers to the amount of antigen that an antigen binding molecule can remove from plasma during the time between administration and elimination (i.e., degradation) of an antibody or antibody derivative.

Can be by methods known to those skilled in the art, for example, by Pharm Res.2006 January; 23 (1): the method described in 95-103, to measure the plasma concentration of free antigen not combined with antigen binding molecules or the ratio of free antigen concentration to total concentration. Alternatively, when the antigen has a specific function in vivo, it can be evaluated whether the antigen is combined with the antigen binding molecules (antagonistic molecules) that neutralize the antigen function by testing whether the antigen function is neutralized. It can be evaluated whether the antigen function is neutralized by measuring the in vivo marker that reflects the antigen function. It can be evaluated whether the antigen is combined with the antigen binding molecules (agonistic molecules) that activate the antigen function by measuring the in vivo marker that reflects the antigen function.

The determination of the ratio of the amount of plasma free antigen and the total amount of antigen in plasma, in vivo marker determination and other such measurements are not particularly limited; However, it is preferably implemented after a certain time after giving antigen binding molecules. In the present invention, the time after giving antigen binding molecules is not particularly limited; Those skilled in the art can determine the appropriate time according to the properties of the antigen binding molecules given. Such time includes, for example, 1 day after giving antigen binding molecules, 3 days after giving antigen binding molecules, 7 days after giving antigen binding molecules, 14 days after giving antigen binding molecules, and 28 days after giving antigen binding molecules. In this article, the term "plasma antigen concentration" means "total plasma antigen concentration", which is the sum of the antigen bound by antigen binding molecules and the unbound antigen concentration; Or it means "plasma free antigen concentration", which is the unbound antigen concentration of antigen binding molecules.

By administering the antigen binding molecules of the present invention, the total plasma antigen concentration may be reduced by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold or even higher compared to administration of a reference antigen binding molecule comprising an intact human IgG Fc region as the human FcRn binding domain, or compared to when no antigen binding domain molecule of the present invention is 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 molecules at each time point

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

C=A/B.

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

Compared to administration of a reference antigen-binding molecule comprising an intact human IgG Fc region as the human FcRn-binding domain, the antigen/antigen-binding molecule molar ratio 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 higher by administration of the antigen-binding molecules of the present invention.

In this article, intact human IgG1, IgG2, IgG3 or IgG4 are preferably used as intact human IgG for the purpose of comparing a reference intact human IgG with 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 target antigen-binding molecule and an intact human IgG Fc region as the human FcRn binding domain can be appropriately used. More preferably, intact human IgG1 is used for the purpose of comparing a reference intact human IgG with an antigen-binding molecule for its human FcRn binding activity or in vivo activity.

Reduction in total plasma antigen concentration or antigen/antibody molar ratio can be evaluated as described in Examples 6, 8, and 13 of WO2011/122011. More specifically, when the antigen-binding molecule of interest does not cross-react with the mouse counterpart, it can be evaluated using an antigen-antibody co-injection model or a steady-state antigen infusion model using human FcRn transgenic mice strain 32 or strain 276 (Jackson Laboratories, Methods Mol Biol. 2010; 602: 93-104). When the antigen-binding molecule cross-reacts with its mouse counterpart, it can be evaluated by injecting only the antigen-binding molecule into human FcRn transgenic mice strain 32 or strain 276 (Jackson Laboratories). In the co-injection model, a mixture of the antigen-binding molecule and antigen is administered to the mouse. In the steady-state antigen infusion model, an infusion pump filled with antigen solution is implanted in the mouse to achieve a constant plasma antigen concentration, and then the mouse is injected with the antigen-binding molecule. The same dose is used for all administered test antigen-binding molecules. The total plasma 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.

Can measure total plasma antigen concentration or free antigen concentration and antigen/antigen binding molecules molar ratio to evaluate long-term effect of the present invention after giving 2,4,7,14,28,56 or 84 days.In other words, after giving antigen binding molecules 2,4,7,14,28,56 or 84 days, measure long-term plasma antigen concentration by measuring total plasma antigen concentration or free antigen concentration and antigen/antigen binding molecules molar ratio, to evaluate the character of antigen binding molecules of the present invention.Can determine whether antigen binding molecules of the present invention realize the reduction of plasma antigen concentration or antigen/antigen binding molecules molar ratio by the reduction of evaluating any one or more above-mentioned time points.

The total plasma antigen concentration or free antigen concentration and antigen/antigen binding molecules molar ratio can be measured 15 minutes, 1, 2, 4, 8, 12 or 24 hours after administration to evaluate the short-term effect of the present invention. In other words, short-term plasma antigen concentration can be measured by measuring the total plasma antigen concentration or free antigen concentration and antigen/antigen binding molecules molar ratio 15 minutes, 1, 2, 4, 8, 12 or 24 hours after administration of the antigen binding molecules to evaluate the properties of the antigen binding molecules of the present invention.

More specifically, the antigen-binding molecules described herein that have a long-term effect on the activity of eliminating plasma antigens have human FcRn binding activity at pH 7.0 and 25°C that is 28-440 times that of intact human IgG1 or a KD in the range of 3.0 micromolar to 0.2 micromolar. Preferably, the KD is in the range of 700 nanomolar to 0.2 nanomolar, more preferably in the range of 500 nanomolar to 3.5 nanomolar, and more preferably in the range of 150 nanomolar to 3.5 nanomolar. Long-term plasma antigen concentration is determined by measuring the total plasma antigen concentration or free antigen concentration 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 long-term effect of the antigen-binding molecule of the present 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 molecule of the present invention can be determined by evaluating the reduction at any one or more of the above time points.

More specifically, the antigen-binding molecules of the present invention that have a short-term effect on plasma antigen elimination have human FcRn-binding activity at pH 7.0 and 25°C that is 440-fold stronger than that of intact human IgG, or a KD greater than 0.2 micromolar, preferably greater than 700 nanomolar, more preferably greater than 500 nanomolar, and most preferably greater than 150 nanomolar. The short-term effect of the antigen-binding molecules of the present invention on plasma antigen elimination can be assessed by measuring the total plasma antigen concentration or free antigen concentration and the antigen/antigen-binding molecule molar ratio 15 minutes, 1, 2, 4, 8, 12, or 24 hours after administration of the antigen-binding molecules to determine the short-term effect.

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

In the context of the present invention, improvement of pharmacokinetics in humans is preferred. When plasma retention in humans is difficult to determine, it can be predicted based on 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 article, the term "reduce 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" means that the antigen-binding activity of the antigen-binding molecule at pH 4.0-pH 6.5 is weakened compared to its antigen-binding activity at pH 6.7-pH 10.0. Preferably, the above statement means that the antigen-binding activity of the antigen-binding molecule at pH 5.5-pH 6.5 is weakened compared to the antigen-binding activity at pH 7.0-pH 8.0, and more preferably means that the antigen-binding activity at early endosomal pH is weakened compared to its antigen-binding activity at plasma pH in vivo. Specifically, the antigen-binding activity of the antigen-binding molecule is weakened at pH 5.8-pH 6.0 compared to the antigen-binding activity of the antigen-binding molecule at pH 7.4.

In this article, the term "reduce the antigen-binding activity of the antigen-binding molecule in the neutral pH range to less than the antigen-binding activity in the acidic pH range" means that the antigen-binding activity of the antigen-binding molecule at pH 6.7-pH 10.0 is weakened compared to its antigen-binding activity at pH 4.0-pH 6.5. Preferably, the above statement means that the antigen-binding activity of the antigen-binding molecule at pH 7.0-pH 8.0 is weakened compared to the antigen-binding activity at pH 5.5-pH 6.5, and more preferably means that the antigen-binding activity at plasma pH in vivo is weakened compared to its antigen-binding activity at early endosomal pH. Specifically, the antigen-binding activity of the antigen-binding molecule at pH 7.4 is weakened compared to the antigen-binding activity of the antigen-binding molecule at pH 5.8-pH 6.0.

At the same time, herein, 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" can also be expressed as "increasing the antigen-binding activity of the antigen-binding molecule in the neutral pH range to greater than the antigen-binding activity in the acidic pH range." Specifically, in the present invention, the ratio of the antigen-binding activity of the antigen-binding molecule between the acidic and neutral pH ranges can be increased. For example, in the following embodiment, the value of KD (pH 5.8) / KD (pH 7.4) is increased. The ratio of the antigen-binding activity of the antigen-binding molecule between the acidic and neutral pH ranges can be increased by, for example, reducing its antigen-binding activity in the acidic pH range, increasing its antigen-binding activity in the neutral pH range, or both.

The expression "reducing the antigen-binding activity of the antigen-binding molecule in the neutral pH range to less than the antigen-binding activity in the acidic pH range" can also be expressed as "increasing the antigen-binding activity of the antigen-binding molecule in the acidic pH range to greater than the antigen-binding activity in the neutral pH range". Specifically, in the present invention, the ratio of the antigen-binding activity of the antigen-binding molecule between the acidic and neutral pH ranges can be increased. For example, in the following embodiment, the value of KD(pH7.4)/KD(pH5.8) is increased. The ratio of the antigen-binding activity of the antigen-binding molecule between the acidic and neutral pH ranges can be increased by, for example, reducing its antigen-binding activity in the neutral pH range, increasing its antigen-binding activity in the acidic pH range, or both.

As used herein, the term "reducing the antigen-binding activity (binding capacity) at a low calcium ion concentration to less than that at a high calcium ion concentration" means reducing the binding affinity of the antigen-binding domain for the antigen at a low calcium ion concentration compared to the binding affinity of the antigen-binding domain for the antigen at a high calcium ion concentration. The low calcium concentration is preferably 0.5-10 micromolar, more preferably 0.1-30 micromolar, ionized calcium, and the high calcium concentration is 100 micromolar to 10 mM, more preferably 200 micromolar to 5 mM ionized calcium.

Herein, the expression “the antigen-binding activity in the acidic pH range is weakened compared to the antigen-binding activity in the neutral pH range” is sometimes replaced by “the antigen-binding activity in the acidic pH range is reduced to be less than that in the neutral pH range”.

Herein, human FcRn-binding activity in the acidic pH range refers to human FcRn-binding activity at pH 4.0 to pH 6.5, preferably at pH 5.5 to pH 6.5, and particularly preferably at pH 5.8 to pH 6.0, which corresponds to the pH of early endosomes in vivo. Meanwhile, herein, human FcRn-binding activity in the neutral pH range refers to human FcRn-binding activity at pH 6.7 to pH 10.0, preferably at pH 7.0 to pH 8.0, and particularly preferably at pH 7.4, which corresponds to the pH of plasma in vivo.

Although the antigen-binding molecules and uses of the present invention are not limited to any particular theory, the relationship between reducing (weakening) the antigen-binding ability of the antigen-binding molecule in the acidic pH range to less than that in the neutral pH range and/or improving (increasing) the human FcRn-binding activity in the neutral pH range and increasing the number of antigens that can be bound by a single antigen-binding molecule can be explained as follows, as it is due to promoting the uptake of the antigen-binding molecule into cells and increasing the elimination of the antigen from plasma.

For example, if the antigen-binding molecule is an antibody that binds to a membrane antigen, the antibody given to the body binds to the antigen and is then taken into the endosome of the cell together with the antigen through internalization, during which the antibody remains bound to the antigen. The antibody is then transferred to the lysosome, during which the antibody remains bound to the antigen and the antibody is degraded by the lysosome together with the antigen. Internalization-mediated self-plasma elimination is called antigen-dependent elimination, and this type of elimination has been reported in many antibody molecules (Drug Discov Today. 2006 January; 11 (1-2): 81-8). When a single molecule of an IgG antibody binds to an antigen in a bivalent manner, a single antibody molecule is internalized, during which the antibody remains bound to two antigen molecules and is degraded in the lysosome. Therefore, in the case of a typical antibody, one IgG antibody molecule cannot bind to three or more antigen molecules. For example, a single IgG antibody molecule with neutralizing activity cannot neutralize three or more antigen molecules.

The relatively prolonged retention (slow elimination) of IgG molecules in plasma is due to the function of human FcRn, a salvage receptor for IgG molecules. When taken into endosomes via pinocytosis, IgG molecules bind to human FcRn expressed there under the acidic conditions of the endosomes. While IgG molecules not bound to human FcRn are transferred to lysosomes for degradation, IgG molecules bound to human FcRn are transferred to the cell surface and, under the neutral conditions of plasma, dissociate from human FcRn and return to the plasma.

Alternatively, when the antigen-binding molecule is an antibody that binds to a soluble antigen, the antibody administered to the body binds to the antigen and is then taken up into cells, where it remains bound to the antigen. Much of the antibody taken up into cells is released outside the cells via FcRn. However, since the antibody is released outside the cells and remains bound to the antigen, it cannot bind to the antigen again. Therefore, similar to antibodies that bind to membrane antigens, in the case of typical antibodies, a single IgG antibody molecule cannot bind to three or more antigen molecules.

pH-dependent antigen-binding antibodies that strongly bind to antigens under neutral conditions in plasma but dissociate from antigens under acidic conditions in the endosome (i.e., antibodies that bind under neutral conditions but dissociate under acidic conditions) can dissociate from antigens in the endosome. When recycled to the plasma via FcRn after antigen dissociation, such pH-dependent antigen-binding antibodies can bind to antigens again; therefore, each antibody can repeatedly bind to many antigens. In addition, the antigen bound to the antigen-binding molecule dissociates in the endosome and no longer circulates to the plasma. This promotes the uptake of antigens by cells mediated by the antigen-binding molecule. Therefore, administering antigen-binding molecules can promote antigen elimination, thereby reducing plasma antigen concentrations.

Calcium concentration-dependent antigen-binding antibodies strongly bind to antigens under high calcium concentration conditions in plasma and dissociate from antigens under low calcium concentration conditions in endosomes, and can dissociate from antigens in endosomes. When the antigen is recirculated to plasma through FcRn after dissociation, the calcium concentration-dependent antigen-binding antibodies can bind to the antigen again. Therefore, this single antibody can repeatedly bind to multiple antigens. At the same time, because the antigen dissociates in the endosome, the antigen bound to the antigen-binding molecule no longer circulates to the plasma, thereby, the antigen-binding molecule promotes the uptake of the antigen by cells. Administration of the antigen-binding molecule promotes antigen elimination, which reduces the plasma antigen concentration.

By making the antibody that 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), the cell intake of the antigen mediated by the antigen binding molecule can be further promoted. Therefore, the administration of antigen binding molecules can promote antigen elimination, thereby reducing plasma antigen concentration. Generally, both the antibody and the antigen-antibody complex are taken into the cell by nonspecific endocytosis, and then transported to the cell surface by binding to FcRn under the acidic conditions of the endosomal body. The antibody and the antigen-antibody complex are recycled to the plasma by dissociating from FcRn under neutral conditions on the cell surface. Therefore, when an antibody that shows sufficient pH dependence in antigen binding (binding under neutral conditions but dissociating under acidic conditions) binds to the antigen in the plasma and then dissociates from the bound antigen in the endosome, it is assumed that the antigen elimination rate is equal to the rate at which the antigen is taken into the cell by nonspecific endocytosis. On the other hand, when the pH dependence is insufficient, the antigen that does not dissociate in the endosome is also recycled to the plasma. At the same time, when pH dependence is sufficient, the rate-determining step in antigen elimination is uptake into cells via nonspecific endocytosis. It is assumed that some FcRn is located on the cell surface because FcRn transports antibodies from endosomes to the cell surface.

The present inventors assume that IgG type immunoglobulin, which is one of the antigen binding molecules, usually has almost no FcRn binding ability in the neutral pH range, but those that show FcRn binding ability in the neutral pH range can bind to FcRn on the cell surface, and therefore are taken into the cell in an FcRn-dependent manner by binding to cell surface FcRn. The rate of FcRn-mediated uptake of cells is faster than the rate of uptake of cells by nonspecific endocytosis. Therefore, the rate of antigen elimination can be further accelerated by imparting FcRn binding ability in the neutral pH range. Specifically, antigen binding molecules with FcRn binding ability in the neutral pH range transport antigens to cells faster than typical (complete human) IgG type immunoglobulins, and then the antigen binding molecules dissociate from the antigen in the endosome. The antigen binding molecules are recycled to the cell surface or in the plasma, then bind to another antigen, and are taken into the cell by FcRn. The rate of this circulation can be further accelerated by improving the FcRn binding ability in the neutral pH range, thereby accelerating the rate of antigen elimination from the plasma. In addition, efficiency can be further improved by reducing the antigen-binding activity of antigen binding molecules in the acidic pH range to the binding activity less than that in the neutral pH range. In addition, it is assumed that the number of antigens that a single antigen binding molecule can bind increases with the increase of the cycle number reached by a single antigen binding molecule. The antigen binding molecules of the present invention comprise antigen-binding domains and FcRn binding domains. Because the FcRn binding domains do not affect antigen binding, or in view of the above-mentioned mechanism, it can be expected that no matter how the antigen type promotes the intake of antigen by the cells mediated by antigen binding molecules, therefore by reducing the antigen-binding activity (binding capacity) of antigen binding molecules in the acidic pH range to the antigen-binding activity less than that in the neutral pH range and/or increase its FcRn binding activity under plasma pH to increase antigen elimination rate.

In all of the above uses, in addition to the substitutions at one or more positions described above, the antigen-binding molecules of the present invention may further comprise a substitution at EU256. Preferably, the amino acid at EU256 is substituted with glutamic acid. Furthermore, in addition to substitutions at one or more positions selected from EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436, all of the above use methods may further comprise a substitution at EU256, wherein the amino acid at EU256 is preferably substituted with glutamic acid.

In preferred embodiments of all of the above uses and methods, the FcRn binding region is an Fc region, more preferably a human Fc region.

Furthermore, substitutions in the amino acid sequence of the parent FcRn binding domain for improving FcRn binding activity at neutral or acidic pH are preferably located at positions EU252 and EU434 and at one or more positions selected from the group consisting of EU238, EU250, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, and EU436. More preferably, the substitutions are located at three or more positions, wherein the three or more positions are one of the combinations provided in Tables 2 and 4-7. Even more preferably, the substitutions are one of the combinations provided in Table 3.

In addition, the above-mentioned application method may further comprise the step of introducing amino acid substitutions at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438, and EU440, thereby reducing the enhanced binding activity to pre-existing ADA. Preferably, the substitutions are combinations selected from Table 11.

In addition, the above-mentioned use method may further comprise the step of introducing an amino acid substitution in the FcRn binding domain at one or more positions selected from the group consisting of: EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270, EU271, EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331 and EU332 (according to the EU numbering system). Preferably, the substitution L235R/S239K is introduced. Preferably, the substitution is a combination selected from Table 14.

The antigen binding molecules of all above-mentioned purposes and application methods can include pH-dependent antigen-binding domains or calcium ion-dependent antigen-binding domains.By reducing the antigen-binding activity (binding capacity) of the above-mentioned antigen binding molecules in the acidic pH range to less than its antigen-binding activity in the neutral pH range, further promote the cell to antigen intake mediated by the antigen binding molecules of the present invention.Also preferably by reducing the antigen-binding activity (binding capacity) of the antigen binding molecules of the present invention under low calcium ion concentration (i.e. under 0.5-10 micromoles) to less than its antigen-binding activity under high calcium ion concentration (i.e. 100 micromoles-10mM), such as by replacing the antigen-binding domains of antigen binding molecules with ionized calcium concentration-dependent antigen-binding domains, further promote the cell to antigen intake.Or, parent antigen binding molecules include ionized calcium concentration-dependent antigen-binding domains.Described above is the method for reducing the antigen-binding activity (binding capacity) in the acidic pH range by changing at least one amino acid in the antigen-binding domains of the above-mentioned antigen binding molecules. It is preferred to alter the antigen-binding domain by replacing at least one amino acid with histidine or introducing at least one histidine into the antigen-binding domain of the above-mentioned antigen-binding molecule, which promotes cellular uptake of the antigen.

When these modifications increase the affinity of the antigen-binding molecule for pre-existing ADA (e.g., rheumatoid factor), the clearance of the modified antigen-binding molecule with increased FcRn-binding activity at neutral or acidic pH can be reduced. This means that by further modifying such antibodies to reduce affinity for pre-existing ADA, the number of cycles can be increased compared to the antigen-binding molecule before the second modification, and therefore before the reduction in affinity for pre-existing ADA.

These antigen binding molecules of the present invention, whose affinity for pre-existing anti-drug antibodies at neutral pH is not significantly improved compared to a wild-type Fc region, in particular compared to those comprising an amino acid substitution in the FcRn binding domain at one or more positions selected from EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440, are particularly useful as therapeutic antibodies for treating human patients suffering from autoimmune diseases, transplant rejection (graft-versus-host disease), other inflammatory diseases and allergic diseases.

Autoimmune diseases occur when the body's tissues are attacked by its own immune system. Examples of autoimmune diseases contemplated herein include systemic lupus erythematosus, lupus nephritis, pemphigoid, pemphigus, dermatomyositis, autoimmune hepatitis, Sjogren syndrome, Hashimoto's thyroiditis, rheumatoid arthritis, juvenile (type 1) diabetes, polymyositis, scleroderma, Addison disease, celiac disease, Guillain-Barre syndrome, dilated cardiomyopathy, mixed connective tissue disease, Wegener's granulomatosis, antiphospholipid antibody syndrome, vitiligo, pernicious anemia, glomerulonephritis and pulmonary fibrosis, myasthenia gravis, Graves' disease, idiopathic thrombocytopenic purpura, hemolytic anemia, diabetes, inflammatory bowel disease, Crohn's disease, ulcerative colitis, multiple sclerosis, psoriasis, and drug-induced autoimmune diseases, such as drug-induced lupus. Preferred autoimmune diseases are systemic lupus erythematosus or lupus nephritis. Transplant rejection includes graft-versus-host disease, a process in which the transplant recipient's immune system attacks the transplanted organ or tissue. Other inflammatory and allergic diseases include atherosclerosis and hay fever.

Increasing the binding affinity for pre-existing ADA can reduce the clinical utility and efficacy of therapeutic antibodies. Therefore, the effectiveness of therapeutic antibodies may be limited by pre-existing ADA because these ADA can affect their efficacy and pharmacokinetics (e.g., degradation rate). Sometimes this binding can lead to serious side effects. In addition, the present invention provides methods for reducing the binding activity of antigen-binding domains comprising an Fc region (which has increased binding activity to FcRn at neutral or acidic pH and increased binding activity to pre-existing ADA at neutral pH) to pre-existing ADA at neutral pH.

The present invention also provides use of an antigen-binding molecule of the present invention comprising a modified FcRn-binding domain for reducing the binding activity of an antigen-binding molecule having increased affinity for FcRn at neutral or acidic pH and increased binding activity to pre-existing ADA at neutral pH to pre-existing ADA.

Specifically, the present invention provides a method for reducing the binding activity of the Fc region of an antigen-binding molecule having enhanced FcRn binding activity at neutral or acidic pH to pre-existing ADA at neutral pH, the method comprising:

a) providing an Fc region having increased binding activity to FcRn at neutral or acidic pH and increased binding activity to pre-existing ADA at neutral pH, and

b) substituting an amino acid at one or more positions selected from the group consisting of: EU387, EU422, EU424, EU426, EU433, EU436, EU438, and EU440.

The preferred Fc region in step a) is a human Fc region. Preferably, the Fc region having improved binding activity to FcRn in a neutral or acidic pH range and improved binding activity to pre-existing ADA in a neutral pH range comprises amino acid substitutions at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436. More preferably, it comprises a substitution at any position selected from the group consisting of positions listed in Tables 2 and 4-7. Even more preferably, it comprises any of the substitutions or substitution combinations provided in any of Tables 3 and 17-20.

Preferably, step b) comprises substituting an amino acid at any position in Table 8. More preferably, step b) comprises introducing one of the substitutions or combinations selected from Table 11.

It is also preferred that the antigen-binding molecule further comprises a pH-dependent antigen-binding domain or a calcium ion-dependent antigen-binding domain.

Furthermore, the present invention provides a method for reducing the binding activity of an antigen-binding molecule comprising an Fc region with enhanced FcRn-binding activity at neutral pH to pre-existing ADA, the method comprising the steps of:

a) providing an antigen-binding molecule comprising an Fc region having increased binding activity to FcRn at acidic pH and increased binding activity to pre-existing ADA at neutral pH, and

b) substituting amino acids in the Fc region at one or more positions selected from the group consisting of: EU387, EU422, EU424, EU426, EU433, EU436, EU438, and EU440.

The preferred Fc region in step a) is a human Fc region. Preferably, the Fc region having improved binding activity to FcRn in the neutral pH range and improved binding activity to pre-existing ADA in the neutral pH range comprises amino acid substitutions at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436. In addition to the substitutions at one or more of the above positions, a substitution at position EU256 may also be included, wherein the amino acid at position EU256 is preferably substituted with glutamic acid. More preferably, a substitution is included at any position selected from the combination of positions listed in Tables 2 and 4-7. Even more preferably, a substitution or combination of substitutions is included from any one of the substitutions or combinations of substitutions listed in Tables 3 and 17-20.

Preferably, step b) comprises substituting an amino acid at any position in Table 10. More preferably, the position is selected from a) EU387, b) EU422, c) EU424, d) EU438, e) EU440, f) EU422/EU424 and g) EU438/EU440. Even more preferably, step b) comprises introducing one of the substitutions or combinations selected from Table 11.

Furthermore, the present invention provides a method for reducing the binding activity of an antigen-binding molecule comprising an Fc region with enhanced FcRn-binding activity at acidic pH to pre-existing ADA, the method comprising the steps of:

a) providing an antigen-binding molecule comprising an Fc region having increased binding activity to FcRn at acidic pH and increased binding activity to pre-existing ADA at neutral pH, and

b) substituting amino acids in the Fc region at one or more positions selected from the group consisting of: EU387, EU422, EU424, EU426, EU433, EU436, EU438, and EU440.

The preferred Fc region in step a) is a human Fc region. Preferably, the Fc region having improved binding activity to FcRn at acidic pH and improved binding activity to pre-existing ADA in the neutral pH range comprises the following amino acid substitutions:

i) at position EU434, or

ii) at two or more positions, wherein the two or more positions are one of the following combinations: a) EU252/EU254/EU256; b) EU428/EU434; and c) EU250/EU428. Preferably, the Fc region comprises i) the substitution N434H; or ii) one of the following combinations: a) M252Y/S254T/T256E; b) M428L/N434S; and c) T250Q and M428L (EU numbering).

In a preferred embodiment, in step b), the amino acid is substituted at a) EU424, or b) EU438/EU440. More preferably, the substitution is a) EU424N or b) the combination EU438R/EU440E.

In another preferred embodiment, the method for reducing the binding activity to pre-existing ADA further comprises step c): confirming that the binding activity of the antigen-binding molecule with a modified Fc domain to endogenous ADA is reduced compared to the binding activity of the original antigen-binding molecule comprising the complete Fc domain described in step a).

It is also preferred that the antigen-binding molecule further comprises a pH-dependent antigen-binding domain or a calcium ion-dependent antigen-binding domain.

The present invention provides use of the antigen-binding molecules of the present invention for increasing the elimination of antigens from the blood of a mammal, preferably a human patient suffering from an autoimmune disease.

The present invention also provides a method for increasing the total number of antigens that can be bound by a single antigen-binding molecule without significantly increasing the binding activity to pre-existing ADA at neutral pH compared to the parent antibody, the method comprising the following steps:

a) providing an antigen-binding molecule comprising a parent FcRn-binding domain,

b) altering the parent FcRn binding domain of step a) by substituting an amino acid in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436; and

c) altering the modified FcRn binding domain of step b) by substituting amino acids in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440.

The present invention also provides a method for promoting the extracellular release of antigen-free antigen-binding molecules that have been taken up into cells in an antigen-bound form without significantly increasing the binding activity of the antigen-binding molecules to pre-existing ADA at neutral pH compared to the parent antibody, the method comprising the following steps:

a) providing an antigen-binding molecule comprising a parent FcRn-binding domain,

b) altering the parent FcRn binding domain by substituting an amino acid in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436 and EU428; and

c) altering the modified FcRn binding domain of step b) by substituting amino acids in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440.

The present invention also provides a method for improving the ability of an antigen-binding molecule to eliminate plasma antigens without significantly increasing its binding activity to pre-existing ADA at neutral pH compared to the parent antibody, the method comprising the following steps:

a) providing an antigen-binding molecule comprising a parent FcRn-binding domain,

b) altering the parent FcRn binding domain by substituting an amino acid in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436 and EU428; and

c) altering the modified FcRn binding domain of step b) by substituting amino acids in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440.

The present invention also provides a method for improving the pharmacokinetics of an antigen-binding molecule without significantly increasing the binding activity to pre-existing ADA at neutral pH compared to the parent antibody, the method comprising the following steps:

a) providing an antigen-binding molecule comprising a parent FcRn-binding domain,

b) altering the parent FcRn binding domain by substituting an amino acid in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436; and

c) altering the modified FcRn binding domain of step b) by substituting amino acids in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440.

The present invention also provides a method for reducing the total plasma antigen concentration or the plasma free antigen concentration without significantly increasing the binding activity to pre-existing ADA at neutral pH compared to the parent antibody, the method comprising the following steps:

a) providing an antigen-binding molecule comprising a parent FcRn-binding domain, wherein the antigen-binding molecule comprises an antigen-binding domain that can bind to the antigen,

b) altering the parent FcRn binding domain by substituting an amino acid in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436; and

c) altering the modified FcRn binding domain of step b) by substituting amino acids in the amino acid sequence of the parent FcRn binding domain at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440.

The preferred Fc region in step a) of the above-mentioned application method is a human Fc region. In a preferred embodiment, the amino acid substitution at one or more positions in step b) is at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436, thereby enhancing the binding activity of the Fc region in step b) to FcRn and pre-existing ADA at a neutral pH range. In addition to the substitutions at one or more positions described above, a substitution at position EU256 may also be included, wherein the amino acid at position EU256 is preferably substituted with glutamic acid. More preferably, a substitution is included at any position selected from the combination of positions listed in Tables 2 and 4-7. Even more preferably, a substitution or combination of substitutions selected from any one of Tables 3 and 17-20 is included.

Preferably, step c) comprises substituting an amino acid at any position in Table 10. More preferably, the position is selected from the group consisting of: a) EU387, b) EU422, c) EU424, d) EU438, e) EU440, f) EU422/EU424, and g) EU438/EU440. Even more preferably, step c) comprises introducing one of the substitutions or combinations selected from Table 11.

In another preferred embodiment, the amino acid substitution at one or more positions in step b) is the following substitution:

i) at position EU434, or

ii) at two or more positions, wherein the two or more positions are selected from one of the following combinations: a) EU252/EU254/EU256; b) EU428/EU434; and c) EU250/EU428, wherein the Fc region of step b) has improved FcRn binding activity in the acidic range and improved binding activity to pre-existing ADA in the neutral pH range. Preferably, the Fc region comprises i) the substitution N434H; or ii) one of the following combinations: a) M252Y/S254T/T256E; b) M428L/N434S; and c) T250Q and M428L (EU numbering). In a preferred embodiment, in step c), the amino acid is substituted at a) EU424 or b) EU438/EU440. More preferably, the substitution is a) EU424N or b) the combination EU438R/EU440E.

Pharmaceutical composition

The present invention further relates to pharmaceutical compositions comprising antigen binding molecules of the present invention or antigen binding molecules produced by the production method of the present invention. Compared with typical antigen binding molecules, the antigen binding molecules of the present invention and the antigen binding molecules produced by the production method of the present invention have a larger activity of reducing plasma antigen concentration by administration, and therefore can be used as pharmaceutical compositions. The pharmaceutical compositions of the present invention may include a pharmaceutically acceptable carrier. In the present invention, pharmaceutical compositions generally refer to medicaments for treating or preventing or detecting and diagnosing diseases.

The pharmaceutical compositions of the present invention can be prepared by methods known to those skilled in the art. For example, they can be used parenterally in the form of injections of sterile solutions or suspensions comprising water or other pharmaceutically acceptable liquids. For example, such compositions can be prepared as follows: by appropriately combining with pharmaceutically acceptable carriers or media, particularly with sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, solvents, preservatives, adhesives, etc., in a unit dosage form required for generally recognized pharmaceutical manufacturing practices. In such preparations, the amount of active ingredient can be easily adjusted routinely to obtain the appropriate amount of a predetermined range.

Sterile compositions for injection can be prepared according to standard formulation practices using a solvent such as distilled water for injection. Aqueous solutions for injection include, for example, normal saline and isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, and sodium chloride). Suitable solubilizing agents such as alcohols (e.g., ethanol), polyols (propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80™, HCO-50) can also be used.

Oils include sesame oil and soybean oil. Benzyl benzoate and/or benzyl alcohol can be used in combination as solubilizers. Buffers (e.g., phosphate buffer and sodium acetate buffer), demulcents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and/or antioxidants can also be combined. A suitable ampoule is filled with the prepared injection.

The pharmaceutical compositions of the present invention are preferably administered parenterally. For example, the compositions may be in a dosage form for injection, nasal administration, transpulmonary administration, or transdermal administration. Such compositions may be administered systemically or topically by intravenous, intramuscular, intraperitoneal, or subcutaneous injection.

The method of administration can be appropriately selected taking into account the patient's age and symptoms. For each administration, the dosage of the pharmaceutical composition containing the antigen binding molecule can be, for example, 0.0001-1,000 mg/kg. Alternatively, the dosage can be, for example, 0.001-100,000 mg/patient. However, the present invention is not limited to the above numerical values. The dosage and method of administration can vary according to the patient's weight, age, symptoms, etc. Taking the above factors into consideration, those skilled in the art can set an appropriate dosage and method of administration.

The amino acids contained in the amino acid sequences of the present invention may be post-translationally modified. For example, modification of an N-terminal glutamine to pyroglutamic acid by pyroglutamylation is well known to those skilled in the art. Such post-translationally modified amino acids are undoubtedly included in the amino acid sequences of the present invention.

Production method

The present invention provides methods for producing the antigen-binding molecules of the present invention. Specifically, the present invention provides methods for producing antigen-binding molecules having an FcRn-binding domain with enhanced FcRn-binding activity at neutral pH compared to antigen-binding molecules comprising a wild-type Fc region.

The present invention provides a method for producing an antigen-binding molecule, comprising the following steps:

(a) selecting a parent FcRn binding domain and altering the parent FcRn by substituting an amino acid in the amino acid sequence with another amino acid at one or more positions selected from the group consisting of: EU252, EU434, EU436, EU315, EU311, EU308, EU307, EU286, EU254, EU250, EU238, EU387, EU422, EU424, EU428, EU438, and EU440;

(b) selecting an antigen-binding domain of an antigen-binding molecule and changing at least one amino acid in the antigen-binding domain to obtain a pH-dependent antigen-binding domain or a calcium ion-dependent antigen-binding domain;

(c) obtaining a gene encoding an antigen-binding molecule in which the human FcRn-binding domain and the antigen-binding domain prepared in (a) and (b) are linked; and

(d) Producing antigen-binding molecules using the gene prepared in (c).

Preferably, the selected antigen-binding molecule comprises an antigen-binding domain whose antigen-binding activity at pH 5.5-6.5 is lower than that at pH 7-8, or whose antigen-binding activity is calcium-dependent. Preferably, the FcRn-binding domain of step a) is an FcRn-binding domain of the present invention. More preferably, the FcRn-binding domain comprises amino acid substitutions at three or more positions, wherein the three or more positions are one of the combinations provided in Tables 2 and 4-7. Even more preferably, the FcRn-binding domain comprises three or more substitutions, wherein the three or more substitutions are one of the combinations provided in Tables 3, 17-20.

Step (a) may comprise making amino acid substitutions at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436, and selecting an FcRn-binding domain having a human FcRn-binding activity stronger than KD 3.2 micromolar in the neutral pH range.

Step (b) may include selecting an antigen-binding domain and changing at least one amino acid in the antigen-binding domain to obtain a pH-dependent antigen-binding domain as described above, or selecting a calcium ion-dependent antigen-binding domain. Changing the amino acid is preferably replacing at least one amino acid with histidine or inserting at least one histidine. At the same time, the site where at least one histidine mutation is introduced is not particularly limited, so it can be introduced at any position, as long as the histidine mutation reduces the antigen-binding activity in the acidic pH range to less than the antigen-binding activity in the neutral pH range. Such histidine mutations can be introduced at a single site or at two or more sites. Steps a) and b) can be repeated twice or more. The number of times steps a) and b) are repeated is not particularly limited; however, the number of times is generally 10 times or less.

The linker that effectively connects the FcRn binding domain and antigen-binding domain prepared in (a) and (b) is not limited to any form. The human FcRn binding domain and antigen-binding domain can be connected by covalent or non-covalent forces. Specifically, the linker can be a peptide linker or a chemical linker or a binding pair, such as a combination of biotin and streptavidin. Modification of the polypeptide comprising the human FcRn binding domain and the antigen-binding domain is known in the art. In another embodiment, the human FcRn binding domain of the present invention can be connected to the antigen-binding domain by forming a fusion protein between the human FcRn binding domain and the antigen-binding domain. In order to construct a fusion protein between the human FcRn binding domain and the antigen-binding domain, the genes encoding the human FcRn binding domain and the antigen-binding domain can be operably connected to form a fusion polypeptide that meets the reading frame. A linker comprising a peptide consisting of several amino acids can be appropriately inserted between the human FcRn binding domain and the antigen-binding domain. Various flexible linkers, such as a linker whose sequence consists of (GGGGS) _n (SEQ ID NO: 11), are known in the art.

The present invention also provides methods for producing antigen-binding molecules comprising an FcRn-binding domain that has improved binding activity to FcRn at neutral or acidic pH, but does not significantly improve binding activity to pre-existing ADA at neutral pH, compared to antigen-binding molecules comprising a wild-type Fc region.

Preferably, a method for producing an antigen-binding molecule comprising an Fc region with enhanced binding activity to FcRn at neutral or acidic pH and reduced binding activity to pre-existing ADA at neutral pH comprises the following steps:

(a) providing an Fc region having increased binding activity to FcRn in a neutral or acidic pH range and increased binding activity to pre-existing ADA in a neutral pH range,

b) substituting an amino acid at one or more positions selected from the group consisting of: EU387, EU422, EU424, EU426, EU433, EU436, EU438, and EU440 in the amino acid sequence of the Fc region.

(c) changing at least one amino acid in the antigen-binding domain of the antigen-binding molecule, and selecting an antigen-binding molecule whose antigen-binding activity in the neutral pH range is stronger than that in the acidic pH range;

(d) obtaining a gene encoding an antigen-binding molecule in which the human FcRn-binding domain prepared in (b) is linked to the antigen-binding domain prepared in (c), and

(e) Antigen-binding molecules are produced using the gene prepared in (d).

The preferred Fc region in step a) is a human Fc region. Preferably, the Fc region that has improved binding activity to FcRn and pre-existing ADAs in a neutral or acidic pH range, and improved binding activity to pre-existing ADAs in a neutral pH range, comprises amino acid substitutions at one or more positions selected from the group consisting of EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436. More preferably, the Fc region comprises a substitution at any position selected from the group consisting of positions listed in Tables 2 and 4-7. Even more preferably, the Fc region comprises any substitution or combination of substitutions listed in Tables 3 and 17-20. Preferably, step a) comprises providing a nucleotide sequence encoding an Fc region that has improved binding activity to FcRn and pre-existing ADAs in a neutral or acidic pH range.

Preferably, the substitution in step b) is an amino acid substitution at one or more positions or a combination of positions provided in Table 10. More preferably, the substitution in step b) is one of the substitution combinations provided in Table 11. Preferably, the amino acid at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440 is substituted in step b) by replacing one or more nucleotides in the nucleotide sequence.

Steps (b) and (c) may be performed in either order. In addition, step c) may include changing at least one amino acid in the antigen-binding domain to obtain a pH-dependent antigen-binding domain as described above, or selecting a calcium ion-dependent antigen-binding domain. In step (c), the amino acid change is preferably to replace at least one amino acid with histidine or to insert at least one histidine. At the same time, the site where at least one histidine mutation is introduced is not particularly limited, and therefore can be introduced at any position, as long as the histidine mutation reduces the antigen-binding activity in the acidic pH range to less than the antigen-binding activity in the neutral pH range. Such histidine mutations can be introduced at a single site or at two or more sites. Steps b) and c) may be repeated two or more times. The number of times steps (b) and (c) are repeated is not particularly limited; however, the number of times is typically 10 times or less.

The linker effectively connecting the FcRn binding domain prepared in (b) and (c) to the antigen-binding domain is not limited to any form. The human FcRn binding domain and the antigen-binding domain can be connected by covalent or non-covalent forces. Specifically, the linker can be a peptide linker or a chemical linker or a binding pair, such as a combination of biotin and streptavidin. Modification of the polypeptide comprising the human FcRn binding domain and the antigen-binding domain is known in the art. In another embodiment, the human FcRn binding domain of the present invention can be connected to the antigen-binding domain by forming a fusion protein between the human FcRn binding domain and the antigen-binding domain. In order to construct a fusion protein between the human FcRn binding domain and the antigen-binding domain, the genes encoding the human FcRn binding domain and the antigen-binding domain can be operably connected to form a fusion polypeptide that meets the reading frame. A linker comprising a peptide consisting of several amino acids can be appropriately inserted between the human FcRn binding domain and the antigen-binding domain. Various flexible linkers, such as a linker whose sequence consists of (GGGGS) _n (SEQ ID NO: 11), are known in the art.

Therefore, the production method of the present invention may also include the steps of changing the above amino acids and replacing or inserting histidine. In the production method of the present invention, non-natural amino acids can be used to replace histidine. Therefore, the present invention can also be understood as replacing the above histidine with non-natural amino acids.

In addition to the substitutions at one or more positions described above, step a) of the production method of the present invention may further comprise a substitution at EU position 256, wherein the amino acid at EU position 256 is preferably substituted by glutamic acid.

In addition, the production method of the present invention may also include a step of replacing an amino acid in the amino acid sequence of the Fc region at a position selected from one or more of the following: EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270, EU271, EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331 and EU332 (according to the EU numbering system). Preferably, the substitution L235R/S239K is introduced.

The parent FcRn binding domains used in the production methods of the present invention and antigen binding molecules comprising the parent FcRn binding domains can be prepared by any method. For example, pre-existing antibodies, pre-existing libraries (phage libraries, etc.), antibodies and libraries prepared from hybridomas obtained from immunized animals or B cells from immunized animals, antibodies and libraries prepared by introducing random amino acid changes into the above antibodies and libraries, antibodies and libraries prepared by introducing histidine or non-natural amino acid mutations into the above antibodies and libraries (libraries with high levels of histidine or non-natural amino acids, libraries introducing histidine or non-natural amino acids at specific sites, etc.), etc. can be used.

The antigen-binding activity and human FcRn-binding activity of the antigen-binding molecule can be measured by methods known to those skilled in the art. Conditions other than pH can be appropriately determined by those skilled in the art.

In the above production methods, the antigen and the antigen-binding molecule can bind to each other in any state, and the human FcRn and the antigen-binding molecule can bind to each other in any state. The state is not particularly limited; for example, the antigen or human FcRn can be brought into contact with an immobilized antigen-binding molecule to bind to the antigen-binding molecule. Alternatively, the antigen-binding molecule can be brought into contact with an immobilized antigen or human FcRn to bind to the antigen-binding molecule. Alternatively, the antigen-binding molecule can be brought into contact with an antigen or human FcRn in a solution to bind to the antigen-binding molecule.

The antigen binding molecules produced by the above method can be any antigen binding molecules of the present invention; preferred antigen binding molecules include, for example, those with such an antigen binding domain, wherein the antigen binding domain is an ionized calcium concentration-dependent antigen binding domain or an antigen binding domain in which an amino acid is substituted with histidine or at least one histidine is inserted, wherein the antigen binding molecule further comprises a human FcRn binding domain, wherein the human FcRn binding domain comprises an amino acid substitution at one or more positions selected from the group consisting of: EU238, EU250, EU252, EU254, EU255, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436 (EU numbering). In addition to the substitution at one or more positions, the antigen binding molecules of the present invention may also comprise a substitution at position EU256. Preferably, the amino acid at position EU256 is substituted with glutamic acid. More preferably, the FcRn binding domain comprises amino acid substitutions at three or more positions, wherein the three or more positions are one of the combinations provided in Tables 2 and 4 to 7. Even more preferably, the FcRn binding domain comprises three or more substitutions, wherein the three or more substitutions are one of the combinations provided in Tables 3, 17 to 20.

Preferred antigen-binding molecules include, for example, those having an ionized calcium concentration-dependent antigen-binding domain or an antigen-binding domain in which amino acids are substituted with histidine or at least one histidine is inserted, and further comprising a human Fc region having an amino acid substitution at one or more positions selected from the group consisting of EU387, EU422, EU424, EU426, EU433, EU436, EU438, and EU440. More preferably, the FcRn-binding domain contains amino acid substitutions at three or more positions in the human FcRn-binding domain, wherein the three or more positions are one of the combinations provided in Tables 9 and 10.

More preferred antigen-binding molecules include those having an ionized calcium concentration-dependent antigen-binding domain or an antigen-binding domain in which amino acids are substituted with histidine or at least one histidine is inserted, and further comprising a human Fc region having the following amino acid substitutions:

a) at one or more positions selected from the group consisting of: EU238, EU250, EU252, EU254, EU255, EU256, EU258, EU286, EU307, EU308, EU309, EU311, EU315, EU428, EU433, EU434, and EU436, and

b) at one or more positions selected from the group consisting of: EU387, EU422, EU424, EU426, EU433, EU436, EU438 and EU440 (EU numbering).

Preferably, the amino acid at EU position 256 is substituted with glutamic acid.

More preferably, the antigen binding molecules comprise the substitution combinations provided in Tables 11-13.

Antibodies having the desired activity can be selected by screening a variety of antibodies obtained from the following antibody libraries or hybridomas.

When changing the amino acids in an antigen-binding molecule, it is possible to use a known sequence of the amino acid sequence of the antigen-binding molecule before the change or an amino acid sequence of an 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 can be obtained from an antibody library or by cloning an antibody encoding gene 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; therefore, those skilled in the art can appropriately obtain antibody libraries. For example, regarding phage libraries, references such as 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 an unexamined Japanese national phase publication number for a non-Japanese international publication). In addition, known methods may be used, such as methods using eukaryotic cells as libraries (WO95/15393) and ribosome display methods. In addition, the technology of obtaining human antibodies by panning using human antibody libraries is also known. For example, the variable regions of human antibodies can be expressed as single-chain antibodies (scFv) on the phage surface using phage display methods, and phages that bind to the antigen can be selected. Genetic analysis of the selected phages can determine the DNA sequence encoding the variable regions of human antibodies that bind to the antigen. Once the DNA sequence of the scFv that binds to the antigen is determined, suitable expression vectors can be produced based on these sequences to obtain human antibodies. These methods are well known and can be found in WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438 and WO95/15388.

As for the method for obtaining antibody-encoding genes from hybridomas, basically known techniques can be used, which include using a desired antigen or cells expressing a desired antigen as a sensitizing antigen, immunizing with these according to a conventional immunization method, fusing the obtained immune cells with known parent cells by a conventional cell fusion method, screening for monoclonal antibody-producing cells (hybridomas) by a conventional screening method, synthesizing cDNA of the antibody variable region (V region) from the mRNA of the obtained hybridoma using a reverse transcriptase, and ligating it with a DNA encoding the desired antibody constant region (C region).

More specifically, the sensitizing antigens for obtaining the above-mentioned antigen-binding molecule genes encoding the H chain and the L chain may include, for example, both complete antigens with immunogenicity and incomplete antigens (including haptens, etc.) without 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. In addition, it is known that substances including polysaccharides, nucleic acids, lipids, etc. can be antigens. Therefore, the antigens of the antigen-binding molecules of the present invention are not particularly limited. Antigens can be prepared by methods known to those skilled in the art, such as by baculovirus-based methods (e.g., WO98/46777). Hybridomas can be produced by, for example, the method of Milstein et al. (G. Kohler and C. Milstein, Methods Enzymol. (1981) 73: 3-46). If the immunogenicity of the antigen is low, immunization can be performed after linking the antigen to an immunogenic macromolecule (e.g., albumin). Alternatively, if necessary, the antigen can be converted into a soluble antigen by linking it to other molecules. If a transmembrane molecule such as a membrane antigen (eg, a receptor) is used as an antigen, a portion of the extracellular region of the membrane antigen can be used as a fragment, or a cell expressing a transmembrane molecule on its cell surface can be used as an immunogen.

Cells producing antigen-binding molecules can be obtained by immunizing animals with the above-mentioned suitable sensitizing antigens. Alternatively, cells producing antigen-binding molecules can be prepared by in vitro immunization of lymphocytes that can produce antigen-binding molecules. Various mammals can be used 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 libraries are also known, and human antibodies can be obtained using these animals (see WO96/34096; Mendez et al., Nat. Genet. (1997) 15: 146-56). As an alternative to using such transgenic animals, for example, human lymphocytes can be sensitized in vitro with a desired antigen or cells expressing a desired antigen, and then the sensitized lymphocytes can be fused with human myeloma cells (e.g., U266) to obtain desired human antibodies having binding activity against the antigen (see Japanese Patent Application Kokoku Publication No. (JP-B) H01-59878 (published as a Japanese patent application approved for opposition). Furthermore, desired human antibodies can be obtained by immunizing transgenic animals carrying a complete human antibody gene library with a desired antigen (see WO93/12227, WO92/03918, WO94/02602, WO96/34096, and WO96/33735).

Immunization of animals can be performed 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 for emulsification. The antigen is then administered intraperitoneally or subcutaneously to the animal. Subsequently, the sensitizing antigen, mixed with Freund's incomplete adjuvant, is preferably administered several times every 4-21 days. Antibody production can be confirmed by measuring the titer of the target antibody in the animal's serum using conventional methods.

Conventional fusing agents (e.g., polyethylene glycol) can be used to fuse cells producing antigen-binding molecules obtained from lymphocytes or animals immunized with the desired antigen with myeloma cells to produce hybridomas (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) 59-103). If necessary, hybridoma cells can be cultured and allowed to grow, and known analytical methods, such as immunoprecipitation, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA), can be used to measure the binding specificity of the antigen-binding molecules produced by these hybridomas. Thereafter, if necessary, hybridomas producing target antigen-binding molecules (whose specificity, affinity, or activity has been determined) can be subcloned by methods such as limiting dilution.

Next, a probe that can be specifically bound to an antigen binding molecule (for example, an oligonucleotide complementary to the sequence encoding the constant region of an antibody) can be used to clone the gene encoding the selected antigen binding molecule from a hybridoma or a cell (sensitized lymphocyte, etc.) producing an antigen binding molecule. RT-PCR can also be used to clone the gene from mRNA. Immunoglobulin is divided into 5 different categories, IgA, IgD, IgE, IgG and IgM. These categories are further divided into several subclasses (isotypes) (for example, IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2, etc.). The H chain and L chain used to produce antigen binding molecules in the present invention are not particularly limited, and can be derived from antibodies belonging to any of these categories or subclasses; however, IgG is particularly preferred.

In this article, genetic engineering techniques can be used to change H chain encoding genes and L chain encoding genes. For antibodies such as mouse antibodies, rat antibodies, rabbit antibodies, hamster antibodies, sheep antibodies and camel antibodies, it is possible to appropriately produce genetically altered antibodies, such as chimeric antibodies and humanized antibodies, which are artificially altered for purposes such as reducing heterologous immunogenicity for people. Chimeric antibodies are antibodies comprising H chains and L chain variable regions of non-human mammalian antibodies (such as mouse antibodies) and H chains and L chain constant regions of human antibodies. Chimeric antibodies can be obtained by connecting the DNA encoding mouse antibody variable regions to the DNA encoding human antibody constant regions, inserting them into expression vectors, and importing the vectors into the host producing antibodies. Several oligonucleotides that overlap at the ends of the DNA sequences of the complementary determining regions (CDRs) of antibodies designed to connect non-human mammals (such as mice) can be used, and humanized antibodies are synthesized by PCR, which is also known as reconstructed human antibodies. The resulting DNA can be connected to the DNA encoding human antibody constant regions. The linked DNA can be inserted into an expression vector, and the vector can be introduced into a host to produce antibodies (see EP239400 and WO96/02576). When the CDRs form a favorable antigen-binding site, human antibody FRs connected by the CDRs are selected. If necessary, amino acids in the antibody variable region framework region can be replaced so that the CDRs of the reconstructed 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-mentioned humanization, antibodies can be altered to improve their biological properties, such as binding to an antigen. In the present invention, such changes can be achieved by methods such as site-directed mutagenesis (see, for example, Kunkel (1910) Proc. Natl. Acad. Sci. USA 82: 488), PCR mutagenesis, and cassette mutagenesis. In general, when compared to the amino acid sequence of the original antibody variable region, mutant antibodies with improved biological properties show an amino acid sequence homology and/or similarity of 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95% or more, 97%, 98% or 99%). In this article, sequence homology and/or similarity is defined as the ratio of amino acid residues that are homologous (identical residues) or similar (amino acid residues that are grouped together based on the overall properties of the amino acid side chains) to the residues of the original antibody after maximizing the sequence homology value by sequence alignment and gap introduction (if necessary). In general, natural amino acid residues are classified into the following groups according to the properties of their side chains:

(1) Hydrophobicity: alanine, isoleucine, valine, methionine and leucine;

(2) Neutral hydrophilicity: asparagine, glutamine, cysteine, threonine and serine;

(3) Acidic: aspartic acid and glutamic acid;

(4) Basic: arginine, histidine and lysine;

(5) residues that affect chain orientation: glycine and proline; and

(6) Aromatic: tyrosine, tryptophan and phenylalanine.

Furthermore, the present invention provides genes encoding the FcRn-binding domains and antigen-binding molecules of the present invention. The genes encoding the antigen-binding molecules of the present invention may be any gene, and may be DNA, RNA, nucleic acid analogs, etc.

In addition, the present invention also provides host cells carrying the above-mentioned genes. Host cells are not particularly limited and include, for example, Escherichia coli (E. coli) and various animal cells. Host cells can be used, for example, as production systems for producing and expressing antibodies of the present invention. In vitro and in vivo production systems can be used for polypeptide production systems. Such in vitro production systems include, for example, production systems using eukaryotic cells or prokaryotic cells.

Eukaryotic cells that can be used as host cells include, for example, animal cells, plant cells, and fungal cells. Animal cells include: mammalian cells, such as CHO (Chinese hamster ovary cell line), COS (monkey kidney cell line), myeloma (Sp2/O, NS0, etc.), BHK (baby hamster kidney cell line), Hela, Vero, HEK293 (human embryonic kidney cell line with sheared adenovirus (Ad) 5 DNA), PER.C6 cells (human embryonic retinal cell line transformed with adenovirus type 5 (Ad5) E1A and E1B genes) 293, etc. (see Current Protocols in Protein Science (May 2001, Unit 5.9, Table 5.9.1)); amphibian cells such as Xenopus laevis oocytes (Valle et al., Nature (1981) 291: 338-340); and insect cells such as Sf9, Sf21, and Tn5. CHO-DG44, CHO-DX11B, COS7 cells, HEK293 cells and BHK cells are preferably used to express the antibodies of the present invention. Among animal cells, CHO cells are particularly preferred for large-scale expression. Vectors can be introduced into host cells by, for example, the calcium phosphate method, the DEAE-dextran method, the method using the cationic liposome DOTAP (Boehringer-Mannheim), electroporation, and lipofection methods.

As for plant cells, for example, cells in tobacco (Nicotiana tabacum) source and duckweed (Lemnaminor) are known protein production systems. These cell culture calli can be used to produce the antigen binding molecules of the present invention. As for fungal cells, known protein expression systems are systems using yeast cells, such as cells of yeast genus (Saccharomyces) (such as saccharomyces cerevisiae and fission yeast (Saccharomyces pombe)); and cells of filamentous fungi such as Aspergillus (Aspergillus) (such as aspergillus niger). These cells can be used as hosts to produce the antigen binding molecules of the present invention.

Bacterial cells can be used for prokaryotic production systems. As for bacterial cells, in addition to the production systems using the above-mentioned Escherichia coli, production systems using Bacillus subtilis are known. Such systems can be used to produce the antigen-binding molecules of the present invention.

The gene obtained by the production method of the present invention is usually carried by a suitable vector (inserted into a suitable 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 a host, preferred cloning vectors include pBluescript vectors (Stratagene); however, various commercially available vectors can be used. When using a vector to produce the antigen-binding molecules of the present invention, an expression vector is particularly useful. The expression vector is not particularly limited as long as the vector expresses the antigen-binding molecules in vitro, in Escherichia coli, in cultured cells, or in vivo. For example, the pBEST vector (Promega) is preferably used for in vitro expression; the pET vector (Invitrogen) is preferably used for Escherichia coli; the pME18S-FL3 vector (GenBank accession number AB009864) is preferably used for cultured cells; the pME18S vector (Mol Cell Biol. (1988) 8: 466-472) is preferably used in vivo. In addition, the EBNA1 protein can be co-expressed to increase the copy number of the target gene. In this case, a vector containing OriP as a replication origin is used (Biotechnol Bioeng. 2001 Oct 20; 75(2): 197-203, Biotechnol Bioeng. 2005 Sep 20; 91(6): 670-7). The DNA of the present invention can be inserted into the vector by conventional methods, for example, by ligation using restriction endonuclease sites (Current protocols in Molecular Biology, ed. Ausubel et al. (1987), Published by John Wiley & Sons, Sections 11.4-11.11).

Above-mentioned host cell is not particularly limited, can use various host cells according to purpose.The example of the cell for expressing antigen binding molecules includes bacterial cell (such as Streptococcus (Streptococcus), Staphylococcus (Staphylococcus), Escherichia coli, Streptomyces (Streptomyces) and subtilis cell), eukaryotic cell (such as yeast and Aspergillus (Aspergillus) cell), insect cell (such as fruit fly S2 and SpodopteraSF9), animal cell (such as CHO, COS, HeLa, C127, 3T3, BHK, HEK293 and Bowes melanoma cell) and plant cell.Can be by known method, such as calcium phosphate precipitation method, electroporation method (Current protocols in Molecular Biology editor Ausubel etc. (1987), Publish.John Wiley＆Sons, section 9.1-9.9), lipofection method and microinjection method, vector is imported into host cell.

Host cells can be cultured by known methods. For example, when animal cells are used as hosts, DMEM, MEM, RPMI1640 or IMDM can be used as culture medium. These can be used with serum supplements such as FBS or fetal calf serum (FCS). Cells can be cultured in serum-free culture. During the culture process, the preferred pH is about 6-8. Incubation is generally carried out at 30-40°C for about 15-200 hours. When necessary, the culture medium is replaced, aerated or stirred.

Appropriate secretion signals can be incorporated into the polypeptide of interest so that the antigen-binding molecule expressed in the host cell is secreted into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment. These signals may be endogenous to the antigen-binding molecule of interest, or may be heterologous signals.

On the other hand, production systems using animals or plants, for example, can be used as systems for in vivo polypeptide production. A target polynucleotide is introduced into the animal or plant, the polypeptide is produced in the animal or plant, and then collected. "Hosts" of the present invention include 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 SPECTRUM Biotechnology Applications (1993)). Mammals can be transgenic animals.

For example, a polynucleotide encoding an antigen-binding molecule of the present invention is prepared as a fusion gene with a gene encoding a polypeptide specifically produced in milk (e.g., goat β-casein). Next, a goat embryo is injected with a polynucleotide fragment containing the fusion gene and then implanted into a female goat. The desired antigen-binding molecule can be obtained from milk produced by a transgenic goat (born from a goat that received the embryo) or its offspring. Where appropriate, hormones can be administered to increase the amount of milk containing the antigen-binding molecule produced by the transgenic goat (Ebert et al., Bio/Technology (1994) 12: 699-702).

Insects (e.g., silkworms) can be used to produce the antigen-binding molecules of the present invention. When silkworms are used, baculovirus carrying a polynucleotide encoding the target antigen-binding molecule can be used to infect the silkworms and obtain the target antigen-binding molecule from their body fluids.

In addition, for example, when plants are used to produce the antigen binding molecules of the present invention, tobacco can be used. When tobacco is used, the polynucleotide encoding the target antigen binding molecule is inserted into a plant expression vector, such as pMON530, and the vector is then introduced into bacteria, such as Agrobacterium tumefaciens. The bacteria are then infected with tobacco, such as Nicotiana tabacum, and the desired antigen binding molecules can be collected from its leaves (Ma et al., Eur. J. Immunol. (1994) 24: 131-138). Alternatively, similar bacteria can be used to infect duckweed (Lemna minor). After cloning, the desired antigen binding molecules can be obtained from duckweed cells (Cox KM et al., Nat. Biotechnol. December 2006; 24 (12): 1591-1597).

Can separate the antigen binding molecules thus obtained from inside and outside the host cell (such as culture medium and milk), and be purified into substantially pure and homogeneous antigen binding molecules.The method for separation and purification of antigen binding molecules is not particularly limited, can adopt the separation and purification method that is usually used in polypeptide purification.Can be by suitably selecting and combining, such as chromatography column, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis and recrystallization, separate and purify antigen binding molecules.

Examples of chromatographic techniques include, but are not limited to, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Edited by Daniel R. Marshak et al. (1996) Cold Spring Harbor Laboratory Press). Such chromatographic methods can be performed 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 necessary, the antigen-binding molecule can be modified arbitrarily, and peptide moieties can 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.

Example

Although the present invention has been described in detail herein with reference to specific embodiments thereof, it is to be understood that the foregoing description is exemplary and illustrative in nature and is intended to illustrate the present invention and its preferred embodiments. Through routine experimentation, those skilled in the art will readily recognize that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which is within the limits defined by the appended claims.

[Example 1] Construction of novel neutral pH FcRn binding affinity improved Fc variants

The Fc region of antigen-binding molecules (antibodies) that interact with FcRn (Nat Rev Immunol. 2007 Sep;7(9):715-25) is engineered to improve binding affinity to FcRn at neutral pH to enhance antigen elimination from plasma. FIG1A shows the mechanism by which a pH-dependent antigen-binding antibody with improved binding affinity to FcRn at neutral pH, compared to conventional antibodies, eliminates antigens from plasma.

Examples 1-17 of WO2011/122011 disclose mutations (amino acid substitutions) that improve FcRn binding affinity at neutral pH and describe the generation of Fc variants F1-F599 (Table 16) focused on improving antibody FcRn binding affinity at neutral pH. However, drug development for antibodies containing such Fc variants should consider not only their pharmacological properties (i.e., improved FcRn binding), but also stability, purity, and immunogenicity. Antibodies exhibiting poor stability and purity are unsuitable for use as drugs, and poor immunogenicity may hinder their clinical development.

1-1. Design and production of Fc variants with improved binding affinity to hFcRn at neutral pH

Various Fc variants were designed with improved binding affinity for hFcRn at neutral pH while maintaining high stability, high purity, and low immunogenicity risk. Table 16 shows mutations (amino acid substitutions) introduced into the Fc region of wild-type IgG1 (IgG1-F1-F1434) for various Fc variants. Amino acid substitutions were introduced into VH3-IgG1 (SEQ ID NO: 1) to generate Fc variants by methods known to those skilled in the art as described in Reference Example 1 of WO2011/122011.

[Table 16]

Fc variants

Variants (IgG1-F600-IgG-F1434) each comprising the heavy chain prepared as described above and L(WT)-CK (SEQ ID NO: 2) were expressed and purified by the method known to those skilled in the art described in Reference Example 2 of WO2011/122011.

1-2. Evaluation of FcRn Binding Affinity of Fc Variants Using Biacore

The hFcRn binding affinity of the new Fc variants (F600-F1434) prepared in Example 1 and the previous Fc variants (F1-F599) prepared in Example 1 of WO2011/122011 was evaluated using a Biacore T100 (GE Healthcare). To this end, human FcRn was prepared as described in Reference Example A2. An appropriate amount of Protein L (ACTIGEN) was immobilized on a Sensor Chip CM4 (GE Healthcare) via amino coupling, and the target antibody was captured on the chip. A diluted FcRn solution and running buffer (as a reference solution) were then injected to allow human FcRn to interact with the captured antibody on the sensor chip. The running buffer used contained 50 mmol/l sodium phosphate, 150 mmol/l NaCl, and 0.05% (w/v) Tween 20 (pH 7.0). FcRn was diluted in each buffer. The chip was regenerated using 10 mmol/l glycine-HCl (pH 1.5). All measurements were performed at 25°C. The association rate constant ka (1/Ms) and dissociation rate constant _kd (1/s), both kinetic parameters, were calculated from the sensorgrams obtained in the measurements. The KD (M) of each antibody for human FcRn was determined from these values. Each parameter was calculated using Biacore T100 evaluation software (GE Healthcare). Table 16 shows the binding affinities of all Fc variants.

1-3. Evaluation of the stability of Fc variants using differential scanning fluorimetry (DSF)

Differential scanning fluorimetry (DSF) was used to evaluate the stability of the new Fc variants (F600-F1434) prepared in Example 1 and the previous Fc variants (F1-F599) prepared in Example 1 of WO2011/122011. This method involves measuring the fluorescence intensity of a polarity-sensitive probe at increasing temperatures and obtaining transition temperatures that expose hydrophobic regions of the protein. Transition temperatures obtained using DSF have been reported to correlate well with melting temperatures obtained using differential scanning calorimetry (Journal of Pharmaceutical Science 2010;4:1707-1720). SYPRO Orange dye (Molecular Probes) was diluted in PBS (Sigma) and added to the protein solution. Each sample was treated with 20 μL of the dye solution. Fluorescence emission was collected at 555 nm with a fixed excitation wavelength of 470 nm. During the DSF experiment, the temperature was increased from 30°C to 99°C in 0.4°C increments, with a 6-second equilibration time at each temperature before measurement. Data were analyzed using Rotor-Gene Q Series software (QIAGEN). The temperature of the fluorescence transition was defined as the melting temperature (Tm). Table 16 shows the Tm values of Fc variants F1-F1434.

1-4. Evaluation of the purity of Fc variants using size exclusion chromatography (SEC)

Size exclusion chromatography (SEC) was used to evaluate the percentage of high molecular weight species (HMW (%)) of the new Fc variants (F600-F1434) prepared in Example 1 and the previous Fc variant (F1-F599) prepared in Example 1 of WO2011/122011. SEC was performed on an ACQUITY UPLC H-Class system (Waters). The antibodies were injected into a BEH200SEC column (1.7 microns, 4.6x150 mm, Waters). The mobile phase was 0.05 M sodium phosphate, 0.3 M sodium chloride (pH 7.0, Isekyu) and the column was run isocratically at a flow rate of 0.3 mL/min. The eluted protein was detected by UV absorbance at 215 nm. Data were analyzed using Empower2 (Waters). Peaks eluting earlier than the antibody monomer peak were recorded as HMW component percentiles. Table 16 shows the HMW (%) of all Fc variants (F1-F1434).

1-5. Evaluation of the immunogenicity of Fc variants using the in silico immunogenicity prediction tool Epibase Original risk

The clinical utility and efficacy of therapeutic antibodies can be limited by the production of anti-drug antibodies (ADAs), which can affect their efficacy and pharmacokinetics and sometimes lead to serious side effects. Although many factors influence the immunogenicity of therapeutic antibodies, multiple reports have described the importance of effector T cell epitopes present on therapeutic proteins.

Computer simulation tools for predicting T cell epitopes have been developed, such as Epibase (Lonza), iTope/TCED (Antitope), and EpiMatrix (EpiVax). By using these computer simulation tools, T cell epitopes present in each amino acid sequence can be predicted (Expert Opin Biol Ther. 2007 March; 7(3): 405-18.), for evaluating the potential immunogenicity of Fc variants. Epibase Light (Lonza) was used to evaluate the potential immunogenicity of Fc variants.

Epibase Light (Lonza) is an in silico tool that uses the FASTER algorithm (Expert Opin Biol Ther. 2007 Mar;7(3):405-18) to calculate the binding affinity of 9-mer peptides for major DRB1 alleles. Epibase Light (Lonza) identifies T cell epitopes with strong and moderate binding to MHC class II. The following in silico immunogenicity score was calculated for each Fc variant using the following formula incorporated into the Epibase Light (Lonza) system: Immunogenicity score = sum((frequency of each DRB1 allotype group) x (number of key epitopes)).

For the DRB1 allotype group frequencies used in this formula, the following DRB1 allotype group frequencies based on the Caucasian population were used.

DRB1*0701 (25.3%), DRB1*1501 (23.1%), DRB1*0301 (21.7%), DRB1*0101 (15.3%), DRB1*04 01(13.8%), DRB1*1101(11.8%), DRB1*1302(8.0%), DRB1*1401(4.9%), DRB1*0403(2.3%), DRB1*0901 (1.8%).

The total number of any strong binding and moderate binding epitopes to the variant constant region (CH1-hinge-CH2-CH3) identified by the FASTER algorithm is used as the number of key epitopes in the formula. Filtered epitopes are epitopes with human antibody germline sequences or the connecting region between the variable region and the constant region, and only non-filtered epitopes (counted as key epitopes) are considered in the immunogenicity score calculation.

The above-mentioned Epibase Light (Lonza) system was used to calculate the immunogenicity scores of the amino acid sequences of the new Fc variants (F600-F1434) described in Example 1 and the previous Fc variants (F1-F599) described in Example 1 of WO2011/122011. The immunogenicity scores of all Fc variants (F1-F1434) are shown in Table 16.

[Example 2] Identification of Fc variants with improved FcRn binding with high stability, low high molecular weight species and low immunogenicity risk

2-1. Plotting Tm, HMW (%) and immunogenicity scores against hFcRn binding affinity for previous and new Fc Variant analysis

The hFcRn binding affinity and Tm of the previous Fc variants (F1-F599) described in Example 1 of WO2011/122011 and the new Fc variants (F600-F1052) generated and evaluated in Example 1 were plotted and shown in Figure 2. The hFcRn binding affinity and HMW (%) of the previous and new Fc variants were plotted and shown in Figure 3. The hFcRn binding affinity and immunogenicity scores of the Fc variants F1-F599 and the new Fc variants (F600-F1052) were plotted and shown in Figure 4.

New Fc variants (F600-F1052) and previous Fc variants (F1-F599) with Ser239Lys or Asp270Phe mutations were deleted from the graph. Because, in the detailed analysis of Groups 1-4 below, Ser239Lys and Asp270Phe mutations improved stability (Tm), however, they did not improve FcRn binding affinity and reduced binding affinity to all human Fcγ receptors, the stability of the Fc variants should be compared within variants without either Ser239Lys or Asp270Phe mutations.

In addition, new Fc variants (F600-F1052) and previous Fc variants (F1-F599) with Pro257Xxx (Xxx is Ala, Val, Ile, Leu, or Thr) or Met252Trp mutations were deleted from the graph, despite these variants improving FcRn binding affinity. Pro257Xxx and Met252Trp mutations did not show a significant decrease in Tm, indicating that variants with Pro257Xxx and Met252Trp mutations have high stability. However, these variants with Pro257Xxx or Met252Trp mutations showed significant aggregation and precipitation during accelerated stability studies or when stored refrigerated. Due to their unfavorable stability, Fc variants with Pro257Xxx and Met252Trp mutations are unacceptable for drug development and, therefore, were deleted from the graph in the detailed analysis of Groups 1-4 below.

2-2. Detailed analysis of Group 1 (binding affinity to hFcRn stronger than 15 nM)

The novel Fc variants (F600-F1052) generated and evaluated in Example 1 with stronger than 15 nM binding affinity to hFcRn and the previous Fc variants (F1-F599) described in Example 1 of WO2011/122011 (hereinafter referred to as Group 1) were analyzed in detail by plotting hFcRn binding affinity on the X-axis against Tm, HMW (%) and immunogenicity score on the Y-axis.

Detailed analysis of Group 1 by plotting hFcRn binding affinity (KD stronger than 15 nM) on the X-axis and Tm, HMW (%) and immunogenicity score on the Y-axis are shown in Figures 5, 6 and 7, respectively.

As for the developability criteria for Fc variants in Group 1, the Tm criterion was set to be higher than 57.5°C, the HMW (%) criterion was set to be lower than 2%, and the immunogenicity score was set to be lower than 500.

Fc variants from Group 1 that met all developability criteria (Tm above 57.5°C, HMW (%) below 2% and immunogenicity score below 500) are shown in Table 17.

[Table 17]

None of the previous Fc variants (F1-F599) had an affinity stronger than 15 nM, whereas several of the new Fc variants generated in Example 1 were stronger than 15 nM and met all developability criteria. Especially when used in conjunction with pH-dependent antigen binding domains, this first group of new Fc variants described in Table 17 are extremely useful for enabling Fc domains that can extremely rapidly and massively eliminate antigens from plasma.

2-3. Detailed analysis of Group 2 (binding affinity to hFcRn between 15 nM and 50 nM)

The new Fc variants (F600-F1052) generated and evaluated in Example 1 with binding affinities to hFcRn between 15 nM and 50 nM and the previous Fc variants (F1-F599) described in Example 1 of WO2011/122011 (hereinafter referred to as "Group 2") were analyzed in detail by plotting hFcRn binding affinity on the X-axis and Tm, HMW (%) and immunogenicity score on the Y-axis.

Detailed analysis of Group 2 by plotting hFcRn binding affinity on the x-axis (KD between 15 nM and 50 nM) and Tm, HMW (%) or immunogenicity score on the Y-axis are shown in Figures 8, 9 and 10, respectively.

As for the developability criteria for Fc variants in Group 2, the Tm criterion was set to be above 60°C, the HMW (%) criterion was set to be below 2%, and the immunogenicity score was set to be below 500.

Fc variants in Group 2 that met all developability criteria (Tm above 60°C, HMW (%) below 2% and immunogenicity score below 500) are shown in Table 18.

[Table 18]

None of the previous Fc variants (F1-F599) met all developability criteria, but several of the new Fc variants generated in Example 1 did. Such Fc variants meeting Group 2 of the developability criteria are extremely useful for enabling rapid and large-scale elimination of antigens from plasma, especially when used in conjunction with pH-dependent antigen binding domains.

2-4. Detailed analysis of Group 3 (binding affinity to hFcRn between 50 nM and 150 nM)

The new Fc variants (F600-F1052) generated and evaluated in Example 1 with binding affinities to hFcRn between 50 nM and 150 nM and the previous Fc variants (F1-F599) described in Example 1 of WO2011/122011 (hereinafter referred to as "Group 3") were analyzed in detail by plotting hFcRn binding affinity on the X-axis and Tm, HMW (%) and immunogenicity score on the Y-axis.

Detailed analysis of Group 3 by plotting hFcRn binding affinity on the X-axis (KD between 50 nM and 150 nM) and Tm, HMW (%) or immunogenicity score on the Y-axis are shown in Figures 11, 12 and 13, respectively.

As for the developability criteria for Fc variants in Group 3, the Tm criterion was set to be higher than 63.0°C, the HMW (%) criterion was set to be lower than 2%, and the immunogenicity score was set to be lower than 250.

Fc variants in Group 3 that met all developability criteria (Tm above 63.0°C, HMW (%) below 2%, and immunogenicity score below 250) are shown in Table 19.

[Table 19]

None of the previous Fc variants (F1-F599) met all developability criteria, whereas several of the new Fc variants generated in Example 1 met all criteria. These new Fc variants, which meet all Group 3 developability criteria, are extremely useful for enabling moderate and sustained elimination of antigens from plasma, especially when used in conjunction with pH-dependent antigen binding domains.

2-5. Detailed analysis of Group 4 (binding affinity to hFcRn between 150 nM and 700 nM)

The new Fc variants (F600-F1052) generated and evaluated in Example 1 with binding affinities to hFcRn between 150 nM and 700 nM and the previous Fc variants (F1-F599) described in Example 1 of WO2011/122011 (hereinafter referred to as "Group 4") were analyzed in detail by plotting hFcRn binding affinity on the X-axis and Tm, HMW (%) and immunogenicity score on the Y-axis.

Detailed analysis of Group 4 by hFcRn binding affinity on the X-axis (KD between 150 nM and 700 nM) and Tm, HMW (%) or immunogenicity score on the Y-axis are shown in Figures 14, 15 and 16, respectively.

As for the developability criteria for Fc variants in Group 4, the Tm criterion was set to be higher than 66.5°C, the HMW (%) criterion was set to be lower than 2%, and the immunogenicity score was set to be lower than 250.

Fc variants in Group 4 that met all developability criteria (Tm above 66.5°C, HMW (%) below 2% and immunogenicity score below 250) are shown in Table 20.

[Table 20]

None of the previous Fc variants (F1-F599) met all developability criteria, whereas several of the new Fc variants generated in Example 1 met all criteria. These new Fc variants, which meet all Group 4 developability criteria, are extremely useful for enabling moderate and sustained elimination of antigens from plasma, especially when used in conjunction with pH-dependent antigen binding domains.

In summary, the novel Fc variants described in Tables 17-20 have high Tm, low HMW (%), and low immunogenicity scores, which are suitable for drug development of antigen-binding molecules capable of eliminating antigens from plasma.

[Example 3] In vivo antigen elimination study of novel Fc variants using human FcRn transgenes in a human IL-6 receptor steady-state infusion model

3-1. Preparation of antibodies for in vivo studies

pH-dependent anti-human IL6 receptor IgG1 antibodies: Fv4-IgG1 comprising VH3-IgG1 (SEQ ID NO: 1) and VL3-CK (SEQ ID NO: 3), the previous Fc variant Fv4-F11 comprising VH3-F11 (SEQ ID NO: 4) and VL3-CK (SEQ ID NO: 3), new Fc variants: Fv4-F652 comprising VH3-F652 (SEQ ID NO: 5) and VL3-CK (SEQ ID NO: 3), Fv4-F890 comprising VH3-F890 (SEQ ID NO: 6) and VL3-CK (SEQ ID NO: 3), and Fv4-F946 comprising VH3-F946 (SEQ ID NO: 7) and VL3-CK (SEQ ID NO: 3) were expressed and purified by the method known to those skilled in the art described in Reference Example 2 of WO2011/122011.

In vivo antigen elimination studies of Fv4-IgG1, Fv4-F11, Fv4-F652, Fv4-F890, and Fv4-F946 were performed in a human IL-6 receptor steady-state infusion model using a human FcRn transgene.

3-2. In vivo antibody studies using a steady-state infusion model using human FcRn transgenic mouse line 32

In vivo experiments were conducted using a steady-state infusion model using the human FcRn transgenic mouse line 32. An infusion pump (MINI-OSMOTIC PUMP MODEL 2004; alzet) loaded with soluble human IL-6 receptor was implanted under the skin of the back of human FcRn transgenic mouse line 32 (B6.mFcRn-/-.hFcRn Tg line32+/+ mice (B6.mFcRn-/-hFCRN Tg32B6.Cg-Fcgrt<tm1Dcr>Tg(FCGRT)32Dcr), Jackson Laboratories; Methods Mol Biol. (2010) 602:93-104) to create a model animal in which the plasma concentration of soluble human IL-6 receptor is maintained constant. Anti-human IL-6 receptor antibodies were administered to the model animals to evaluate the in vivo kinetics of soluble human IL-6 receptor administration. Before implantation of the infusion pump and 7 and 17 days after the antibody was administered into the tail vein, a monoclonal anti-mouse CD4 antibody (in-house) was administered at 20 mg/kg to inhibit the production of neutralizing antibodies against the soluble human IL-6 receptor. An infusion pump containing 92.8 micrograms/ml of soluble human IL-6 receptor was then implanted under the skin on the back of the mouse. Three days after implantation of the infusion pump, the anti-human IL-6 receptor antibody was administered once into the tail vein. In Study 1, Fv4-IgG1, Fv4-F652, Fv4-F890, and Fv4-F946 were administered at a 1 mg/kg dose together with approximately 1 g/kg Sanglopor (CSL Behring), while in Study 2, Fv4-IgG1, Fv4-F11, and Fv4-F652 were administered at 1 mg/kg. In both studies, no antibody was administered to the control group (no antibody injection). Blood was collected at the appropriate time point after administration of the anti-human IL-6 receptor antibody. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 15 minutes to separate the plasma. The separated plasma was stored in a freezer at -20°C or lower until measurement.

3-3. Measurement of anti-human IL-6 receptor antibody plasma concentration by ELISA

The concentration of anti-human IL-6 receptor antibodies in mouse plasma was measured by ELISA. Anti-human IgG (γ chain-specific) F(ab')2 antibody fragments (Sigma) were dispensed onto a Nunc-ImmunoPlate MaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4°C to prepare an anti-human IgG-immobilized plate. Calibration curve samples with plasma concentrations of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, and 0.0125 μg/ml and mouse plasma samples diluted 100-fold or more were prepared. 200 μl of 20 ng/ml hsIL-6R was added to 100 μl of the calibration curve samples and plasma samples, and the samples were allowed to stand at room temperature for 1 hour. Subsequently, the samples were dispensed onto an anti-human IgG-immobilized plate and allowed to stand at room temperature for 1 hour. Then, biotinylated anti-human IL-6R antibody (R&D) was added and reacted at room temperature for 1 hour. Subsequently, streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was added and allowed to react at room temperature for 1 hour. A colorimetric reaction was then performed using TMP One Component HRP Microwell Substrate (BioFX Laboratories) as a substrate. After terminating the reaction with 1N sulfuric acid (Showa Chemical), the absorbance at 450 nm was measured using a microplate reader. The concentration in mouse plasma was calculated from the absorbance of the calibration curve using SOFTmax PRO (Molecular Devices) analysis software.

3-4. Measurement of hsIL-6R Plasma Concentration by Electrochemiluminescence Assay

hsIL-6R concentrations in mouse plasma were measured by electrochemiluminescence. hsIL-6R calibration curve samples were prepared, adjusted to concentrations of 2,000, 1,000, 500, 250, 125, 62.5, and 31.25 pg/ml, and mouse plasma samples were diluted 50-fold or more. The samples were mixed with solutions of a monoclonal anti-human IL-6R antibody (R&D) labeled with ruthenium using Sulfo-Tag NHS Ester (Meso Scale Discovery), a biotinylated anti-human IL-6R antibody (R&D, Systems Inc., USA), and tocilizumab (Chugai Pharmaceutical Co., Ltd.), and then reacted overnight at 37°C. The final concentration of tocilizumab, an anti-human IL-6 receptor antibody, was 333 μg/ml, exceeding the concentration of anti-human IL-6 receptor antibody in the sample. This ensured that nearly all hsIL-6R molecules in the sample were bound to tocilizumab. The sample was then dispensed onto a MA400PR streptavidin plate (Meso Scale Discovery), allowed to react at room temperature for 1 hour, and washed. Immediately after dispensing Read Buffer T (x4) (Meso Scale Discovery), measurements were performed using a SectorPR400 reader (Meso Scale Discovery). The hsIL-6R concentration was calculated based on the response of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices).

3-5. Results of Study 1: In vivo Antigen Elimination of Novel Fc Variants

Figure 17 shows the plasma hsIL-6R concentration time profile, and Figure 18 shows the plasma antibody concentration time profile after injection of Fv4-IgG1, Fv4-F652, Fv4-F890, and Fv4-F946. Compared to Fv4-IgG1 and the control (no antibody injection), the novel Fc variants Fv4-F652, Fv4-F890, and Fv4-F946 with improved binding to FcRn at neutral pH showed significantly reduced plasma hsIL-6R concentrations, demonstrating in vivo antigen elimination of pH-dependent antigen-binding antibodies with improved binding to FcRn at neutral pH. Although Fv4-F652 and Fv4-F890 showed 30-fold and 10-fold greater antigen elimination, respectively, on day 7 compared to Fv4-IgG1, the plasma antibody concentration time profiles of Fv4-F652 and Fv4-F890 were comparable to those of Fv4-IgG1.

Therefore, this study shows that Fv4-F652 and Fv4-F890 are able to selectively eliminate soluble antigens from plasma while maintaining antibody pharmacokinetics comparable to Fv4-IgG1. Fv4-F890 belongs to Group 3, and this study shows that Group 3 Fc variants can therefore reduce plasma antigen concentrations by approximately 10-fold while maintaining antibody pharmacokinetics comparable to IgG1. This means that applying Group 3 Fc variants to pH-dependent antigen-binding IgG1 antibodies can reduce antibody dose by up to 10-fold. This reduction in antibody dose achieved through Group 3 Fc variants is particularly meaningful when antibody dose reduction is required and infrequent dosing is required.

On the other hand, Fv4-F946 showed a 100-fold reduction in plasma hsIL-6R concentration compared to Fv4-IgG1, and Fv4-F946 had greater antibody clearance than Fv4-IgG1. Fv4-F946 belongs to Group 2, and this study shows that Group 2 Fc variants can reduce plasma antigen concentration by approximately 100-fold, but have greater antibody clearance than IgG1. This means that applying Group 2 Fc variants to pH-dependent antigen-binding IgG1 antibodies can reduce total plasma antigen concentration by approximately 100-fold. In cases where the plasma target antigen concentration is too high to be neutralized by a practical antibody dose (i.e., 100 mg/kg), regardless of whether the antigen clearance of the Group 2 Fc variants is increased, the 100-fold reduction in total antigen concentration means that the target antigen can be neutralized by a dose of less than 10 mg/kg, which is a practical antibody dose.

The hFcRn binding affinity of Fv4-F652 and Fv4-F890 was measured in duplicate, with a affinity for hFcRn of 2.4E-07M (n=7) for F652 and 1.1E-07M (n=12) for F890. Previous studies described in Example 1 of WO2011/122011 showed that the extent of antigen elimination and antibody clearance correlated with binding affinity to FcRn at neutral pH. As shown in Figure 17, Fv4-F652 showed a greater degree of antigen elimination than Fv4-F890, but the antibody pharmacokinetics were comparable to those of Fv4-F890. Therefore, it was shown that the specific mutations in F652 contribute to the enhanced antigen sweeping effect.

To identify which residues contribute to the improved antigen clearance of F652, studies were performed using F11 (Met252Tyr, Asn434Tyr double mutant) and F652 (Pro238Asp, Met252Tyr, Asn434Tyr triple mutant). 2 The affinity for hFcRn was 3.1E-07 M for F11 (n=12), which is comparable to the affinity measured for F652.

3-6. Results of Study 2: In vivo Antigen Elimination Effect of Pro238Asp Mutation

Figure 19 shows the time profile of plasma hsIL-6R concentration, and Figure 20 shows the time profile of plasma antibody concentration after injection of Fv4-IgG1, Fv4-F11, and Fv4-F652. Although Fv4-F11 showed a decrease in plasma hsIL-6R concentration, Fv4-F652 showed a greater decrease in plasma hsIL-6R concentration. Fv4-F11 and Fv4-F652 showed comparable time profiles of plasma antibody concentration.

Thus, this study demonstrates that the Pro238Asp mutation can improve plasma antigen elimination while maintaining antibody pharmacokinetics comparable to Fv4-IgG1. Therefore, the Pro238Asp mutation is extremely useful for improving antigen elimination by pH-dependent antigen-binding antibodies.

[Example 4] Elimination of binding of rheumatoid factor to FcRn by site-directed mutagenesis to an improved Fc variant

The clinical utility and efficacy of therapeutic antibodies can be limited by the production of anti-drug antibodies (ADA), as ADA can affect their efficacy and pharmacokinetics and sometimes lead to serious side effects. Many factors affect the immunogenicity of therapeutic antibodies, and the presence of effector T cell epitopes is one factor. In addition, from the perspective of ADA, the presence of pre-existing antibodies to therapeutic antibodies may also be problematic. In particular, in the case of therapeutic antibodies for patients with autoimmune diseases (e.g., rheumatoid arthritis), rheumatoid factor, an autoantibody to human IgG, may be a problem with pre-existing antibodies. Recently, a humanized anti-CD4 IgG1 antibody with an Asn434His mutation was reported to induce significant rheumatoid factor binding (Clin Pharmacol Ther. 2011 Feb; 89(2): 283-90). Detailed studies confirmed that the Asn434His mutation in human IgG1 increased the binding of rheumatoid factor to the Fc region of the antibody compared to the parent human IgG1.

Rheumatoid factor is a polyclonal autoantibody directed against human IgG. Its epitope in human IgG varies between clones, but its epitope appears to be located in the CH2/CH3 interface and CH3 domain, potentially overlapping with the FcRn-binding epitope. Therefore, mutations that increase binding affinity for FcRn may also increase binding affinity for a specific rheumatoid factor clone.

Previous studies have shown that Fc engineering to increase binding affinity for FcRn at acidic pH improves endosomal recycling efficiency and prolongs the pharmacokinetics of antibodies. For example, the M252Y/S254T/T256E (YTE) variant (J Biol Chem 2006 281: 23514-23524), the M428L/N434S (LS) variant (Nat Biotechnol, 2010 28: 157-159), and the N434H variant (Clinical Pharmacology & Therapeutics (2011) 89 (2): 283-290) showed an improvement in half-life relative to native IgG1.

In order to achieve antigen elimination from plasma, the Fc region of antigen-binding molecules (antibodies) that interact with FcRn (Nat Rev Immunol. 2007 Sep; 7(9): 715-25) is modified to improve its binding affinity to FcRn at neutral pH. Such modified Fc variants include F11 variant, F68 variant, F890 variant, and F947 variant. The mechanism of antigen elimination from plasma by pH-dependent antigen-binding antibodies with improved binding affinity to FcRn at neutral pH compared to conventional antibodies is shown in Figure 1.

Such Fc variants with improved FcRn binding (at pH 6.0 and/or at neutral pH) may show increased binding to rheumatoid factor, as in the case of the previously reported Asn434His mutation. Therefore, we tested whether these Fc variants with improved FcRn binding would show increased binding to rheumatoid factor. The variant antibodies used in the following studies were Fv4-hIgG1, Fv4-YTE, Fv4-LS, Fv4-N434H, Fv4-F11, Fv4-F68, Fv4-890, and Fv4-F947.

4-1. Rheumatoid Factor Binding Studies of Fc Variants with Improved FcRn Binding

The binding assay for rheumatoid factor is performed by electrochemiluminescence (ECL) at pH 7.4. The assay is performed with serum (Proteogenex) from 15 or 30 RA patients. 50-fold diluted serum samples, biotin-labeled test antibodies (1 microgram/mL) and SULFO-TAG NHS Ester (Meso Scale Discovery)-labeled test antibodies (1 microgram/mL) are mixed and incubated at room temperature for 3 hours. The mixture is then added to a streptavidin-coated MULTI-ARRAY 96-well plate (Meso Scale Discovery), the plate is incubated at room temperature for 2 hours, and washed. After Read Buffer T (x4) (Meso Scale Discovery) is added to each well, the plate is immediately placed in a SECTOR imager 2400 reader (Meso Scale Discovery) to measure chemiluminescence.

The results of this study are shown in Figures 21 and 22. Figures 21 and 22 show ECL reactions of sera from 15 or 30 RA patients. Fv4-hIgG1 (Figures 21-1 and 22-1) with native human IgG1 showed only weak rheumatoid factor binding, while all Fc variants with improved FcRn binding (Fv4-YTE (Figure 21-2), Fv4-LS (Figure 21-3), Fv4-N434H (Figure 22-2), Fv4-F11 (Figure 22-3), Fv4-F68 (Figure 22-4), Fv4-890 (Figure 22-5), and Fv4-F947 (Figure 22-6)) significantly improved rheumatoid factor binding in more than two donors. This study clearly demonstrates that immunogenicity associated with pre-existing rheumatoid factors may be a problem when considering clinical development of therapeutic antibodies with improved binding affinity to FcRn for autoimmune diseases such as rheumatoid arthritis. Figure 23 shows the mean, geometric mean and median values of the serum ECL reactivity of the above antibody variants with blood from 15 RA patients.

Therefore, in the following studies, we generated panels of variants that could potentially reduce polyclonal rheumatoid factor binding while maintaining FcRn binding capacity.

4-2. Binding of Fc variants with improved FcRn binding by introducing mutations into the Fc region to rheumatoid factor

To generate variants with reduced polyclonal rheumatoid factor binding while retaining FcRn binding ability, mutations were rationally introduced into surface residues near the CH2/CH3 interface that were assumed not to interfere with the human FcRn/human IgG interaction.

Fv4-F890 was selected as the parent Fc variant, and single mutations and combination mutations were introduced into Fv4-F890. This resulted in the novel Fc variants F1058-F1073, F1107-F1114, F1104-F1106, and F1230-F1232 described in Table 21. Additionally, Fv4-F947 was selected as the parent Fc variant, and the same single mutations and combination mutations were introduced into Fv4-F947. This resulted in the novel Fc variants F1119-F1124 described in Table 21. The variants were first evaluated for their binding affinity to human FcRn at pH 7.0. The results are also described in Table 21. These variants did not show a significant decrease in binding affinity to human FcRn compared to either parent Fv4-F890 or Fv4-F947, indicating that these mutations did not affect human FcRn binding.

[Table 21]

We then performed rheumatoid factor binding studies at pH 7 for the variants in Table 21. The results of this study are shown in Figures 24 to 29. These figures show the ECL reactivity of sera from 15 RA patients to the following antibody variants: Fv4-IgG1, Fv4-F890, Fv4-F1058-Fv4-1073 (Figure 24), Fv4-F1104-Fv4-F1106 (Figure 26), Fv4-F1107-Fv4-F1114 (Figure 27), Fv4-F1230-Fv4-F1232 (Figure 28), Fv4-947, and Fv4-F1119-Fv4-F1124 (Figure 29). Figures 25-1, 25-2, and 25-3 show the average, geometric mean, and median values of the ECL reactivity of sera from 15 RA patients to the variants Fv4-IgG1, Fv4-F890, and Fv4-F1058-Fv4-1073.

Unexpectedly, compared to F890, which shows strong rheumatoid factor binding, new Fc variants with single mutations to F890, such as F1062, F1064-F1072, and F1107-F1114, showed a significant reduction in rheumatoid factor binding. In particular, F1062, F1064, F1068, F1070, F1072, F1107-F1109, and F1111-F1113 showed rheumatoid factor binding comparable to native IgG1, indicating that the increased immunogenicity risk of F890 variants was completely eliminated by introducing additional single mutations that reduced rheumatoid factor binding without affecting human FcRn binding. Because rheumatoid factor in patients is a polyclonal antibody that binds to multiple epitopes in the Fc region, it was unexpected that a single mutation significantly eliminated the binding of rheumatoid factor to the Fc region.

Furthermore, the double-mutated FcF1106 (Q438R/S440E) showed a significant reduction in rheumatoid factor binding compared to the single mutations Fc F1070 (Q438R) or F1072 (S440E). Similarly, the double-mutated FcF1230 (Q438R/S440D), F1231 (Q438K/S440E), and F1232 (Q438K/S440D) also showed additional reduction in rheumatoid factor binding through the combination of mutations. Meanwhile, F1104 (V422E/S424R) or F1105 (V422S/S424R) did not show any combination effect.

In addition, Fv4-F939 was selected as the parent Fc variant, and additional mutations were evaluated for improving FcRn binding (S254T or T256E) and reducing rheumatoid factor binding (H433D). The novel Fc variants described in Table 22 (F1291, F1268, F1269, F1243, F1245, F1321, F1340, and F1323) were generated. Initially, the variants were evaluated for their binding affinity to human FcRn at pH 7.0. The results are also shown in Table 22.

[Table 22]

Then, as mentioned above, we have carried out rheumatoid factor binding research for these variants.The results of this research are shown in Figure 30.Unexpectedly, compared with F939, F1291 (single H433D mutation of F939) is presented at the remarkable reduction that rheumatoid factor is combined in some donors.Similarly, compared with F1321, F1323 (single H433D mutation of F1321) is presented at the remarkable reduction that rheumatoid factor is combined in some donors.In addition, Q438R/S440E, Q438K/S440D and Q438K/S440E mutation display have for improving the rheumatoid factor of the variant of other mutation (S254T or T256E) that FcRn is combined and significantly reduce.

4-3. Reduced FcRn binding by introducing additional N-glycosylation in the Fc region Improved Fc variants and rheumatoid arthritis The combination of

Due to the steric hindrance of the bulky N-glycosylation, the introduction of additional N-glycosylation near the rheumatoid factor binding epitope can also eliminate rheumatoid factor binding. Mutations can be selected from sites that allow the introduction of mutations into the N-glycosylation sequence (Asn-Xxx-Ser/Thr) while maintaining FcRn binding. To introduce additional N-glycosylation sequences into the Fc region, single or double mutations were introduced into Fv4-F11. The new Fc variants described in Table 23 (F1077-F1083, F1094-F1097) were generated. The variants were evaluated by SDS-Page for their binding affinity to human FcRn at pH 7.0 and the presence of additional glycosylation. The results are shown in Table 23. F1077 (K248N), F1080 (S424N), F1081 (Y436N/Q438T), and F1082 (Q438N) were found to have additional glycosylation, and especially F1080 (S424N) retained binding affinity to human FcRn.

[Table 23]

Therefore, in the following studies, the S424N mutation was introduced into Fv4-F890 to generate Fv4-F1115 as described in Table 24, and its binding affinity to human FcRn was evaluated at pH 7.0. The results are also shown in Table 24.

[Table 24]

Then, as described above, we performed rheumatoid factor binding studies on these variants. The results of this study are shown in Figure 31. Unexpectedly, the single S424N mutant Fv4-F1115 showed a significant reduction in rheumatoid factor binding. This result suggests that introducing additional N-glycosylation is an effective method to eliminate rheumatoid factor binding.

4-4. Reduced rheumatoid factor binding of YTE, N434H, and LS variants

To reduce rheumatoid factor binding in the Fv4-YTE, Fv4-N434H, and Fv4-LS variants (which improve FcRn binding at acidic pH and prolong antibody pharmacokinetics), the Q438R/S440E mutation or the S424N mutation was introduced into these variants. These new Fc variants (F1166, F1167, F1172, F1173, F1170, and F1171) were generated as described in Table 25. The variants were first evaluated for their binding affinity to human FcRn at pH 6.0. The results are also shown in Table 25.

[Table 25]

We then performed rheumatoid factor binding studies on these variants (Fv4-F1166, F1167, F1172, F1173, F1170, and F1171) as described above. The results of this study are shown in Figure 32. Compared to YTE, which showed strong rheumatoid factor binding in two donors (90216S and 90214S), F1166 (Q438R/S440E) and F1167 (S424N) showed a significant reduction in rheumatoid factor binding. In addition, F1173 and F1171 showed that the S424N mutation could also eliminate rheumatoid factor binding of the N434H and LS variants. However, the Q438R/S440E mutation was unable to completely eliminate rheumatoid factor binding of the N434H and LS variants, with rheumatoid factor binding observed in one or two donors.

4-5. Alternative mutations that reduce rheumatoid factor binding of LS variants

Novel single mutations were introduced into Fv4-LS to generate the Fc variants described in Table 26 (Fv4-F1380-Fv4-F1392).

[Table 26]

We then performed rheumatoid factor binding studies on variants that maintain FcRn binding at pH 6.0 (Fv4-F1380, F1384-F1386, F1388, and F1389). The results of this study are shown in Figure 33. These variants showed a significant reduction in rheumatoid factor binding in some donors. In particular, Fv4-F1389 showed rheumatoid factor binding comparable to native IgG1.

Therefore, for reducing the immunogenicity of antigen-binding molecules containing an Fc region with improved FcRn binding (e.g., F1-F1434), such as pH-dependent antigen-binding antibodies with improved binding affinity to FcRn at neutral pH that can eliminate antigens from plasma and antibodies with improved binding affinity to FcRn at acidic pH that can improve antibody pharmacokinetics, mutations such as Pro387Arg, Val422Glu, Val422Arg, Val422Ser, Val422Asp, Val422Lys, Val422Th Ser426Asp, Ser426Ala, Ser426Gln, Ser426Tyr, His433Asp, Tyr436Thr, Gln438Glu, Gln438Arg, Gln438Ser, Gln438Lys, Ser440Glu, Ser440Asp, Ser440Gln (positions provided in EU numbering) are extremely useful.

Mutation sites other than EU387, EU422, EU424, EU426, EU433, EU436, EU438, and EU440 for reducing rheumatoid factor binding without affecting human FcRn binding can be selected from 248-257, 305-314, 342-352, 380-386, 388, 414-421, 423, 425-437, 439, and 441-444 by EU numbering.

[Example 5] Reduction of rheumatoid factor binding of new Fc variants with improved binding to human FcRn at neutral pH

The novel Fc variants described in Table 27 (F939, F1378, F1379, F1262, F1138, F1344, F1349, F1350, F1351, F1261, F1263, F1305, F1306, F1268, F1269, F1413, F1416, F1419, F1420, F1370, F1381, F1390, F1401, F1413, F1416, F1419, F1420, F1391, F1392, F1403, F1414, F1415, F1426, F1427, F1428, F1429, F1430, F1431, F1432, F1433, F1434, F1435, F1436, F1437, F1438, F1439, F1440, F1441, F1442 Variants were first evaluated for their binding affinity to human FcRn at pH 7.0. The results are also shown in Table 27.

[Table 27]

We then performed rheumatoid factor binding studies at pH 7.4 for the variants in Table 27. The results of this study are shown in Figures 34-94.

As for other mutations for improving FcRn binding at neutral pH, double mutations for reducing rheumatoid factor binding (Q438R/S440E, Q438R/S440D, Q438K/S440E, and Q438K/S440D) showed a significant decrease in rheumatoid factor binding.

5-1. Reduction of rheumatoid factor binding by novel Fc variants with improved human FcRn binding at acidic pH

The novel Fc variants described in Table 28 (F1718-F1721, F1671, F1670, F1711-F1713, F1722-F1725, F1675, F1714-F1717, F1683, F1756-F1759, F1681, F1749-F1751, F1760-F1763, F1752-F1755, F1685) were generated. The variants were first evaluated for their binding affinity to human FcRn at pH 6.0. The results are also shown in Table 28.

[Table 28]

We then performed rheumatoid factor binding studies at pH 7.4 for the variants in Table 28. The results of this study are shown in Figures 95-130.

As for other mutations for improving FcRn binding at acidic pH, double mutations for reducing rheumatoid factor binding (Q438R/S440E, Q438R/S440D, Q438K/S440E, and Q438K/S440D) showed a significant decrease in rheumatoid factor binding.

[Example 6] In vivo antigen elimination study of novel Fc variants using human FcRn transgenic mice in a human IL-6 receptor steady-state infusion model

6-1. Antibody Preparation for In Vivo Studies

pH-dependent anti-human IL6 receptor IgG1 antibodies: Fv4-IgG1 comprising VH3-IgG1 (SEQ ID NO: 1) and VL3-CK (SEQ ID NO: 3), novel Fc variants - Fv4-F1243 comprising VH3-F1243 (SEQ ID NO: 8) and VL3-CK (SEQ ID NO: 3) and Fv4-F1245 comprising VH3-F1245 (SEQ ID NO: 9) and VL3-CK (SEQ ID NO: 3) were expressed and purified by methods known to those skilled in the art described in Example 2 of WO2011/122011.

As described in Example 4 , Fv4-F1243 and Fv4-F1245 have novel Fc regions with improved binding affinity for human FcRn at neutral pH but significantly reduced binding to rheumatoid factor. To evaluate the antigen elimination effects of these variants, in vivo studies of Fv4-IgG1, Fv4-F1243, and Fv4-F1245 were conducted in a human IL-6 receptor steady-state infusion model using human FcRn transgenic mice.

6-2. In vivo antibody studies using a steady-state infusion model using human FcRn transgenic mouse strain 32

In vivo experiments were performed using the human FcRn transgenic mouse strain 32 by the same method described in Example 13 of WO2011/122011 using a steady-state infusion model.

6-3. Research Results: In vivo Antigen Elimination of Novel Fc Variants

Figure 131 shows the plasma hsIL-6R concentration time profile, and Figure 132 shows the plasma antibody concentration time profile after injection of Fv4-IgG1, Fv4-F1243, and Fv4-F1245. Compared to Fv4-IgG1 and the control (no antibody injection), Fv4-F1243 and Fv4-F1245, novel Fc variants with improved binding to FcRn at neutral pH, showed significantly reduced plasma hsIL-6R concentrations, demonstrating in vivo antigen elimination of pH-dependent antigen-binding antibodies with improved binding to FcRn at neutral pH. On day 21 or day 7, Fv4-F1243 and Fv4-F1245 each showed 10-fold greater antigen elimination than Fv4-IgG1, while the plasma antibody concentration time profiles of Fv4-F1243 and Fv4-F1245 were comparable to those of Fv4-IgG1.

[Example 7] In vivo PK study of new Fc variants using human FcRn transgenic mice

7-1. Preparation of antibodies for in vivo studies

pH-dependent anti-human IL6 receptor IgG1 antibodies: Fv4-IgG1 comprising VH3-IgG1 (SEQ ID NO: 1) and VL3-CK (SEQ ID NO: 3), and a new Fc variant - Fv4-F1389 comprising VH3-F1389 (SEQ ID NO: 10) and VL3-CK (SEQ ID NO: 3) were expressed and purified by methods known to those skilled in the art and described in Reference Example 2 of WO2011/122011.

As described in Examples 4 and 5 , Fv4-F1389 has a novel Fc region with improved binding affinity to human FcRn at acidic pH but significantly reduced binding to rheumatoid factor. To evaluate the pharmacokinetics of this variant, in vivo studies of Fv4-IgG1 and Fv4-F1389 were performed using human FcRn transgenic mice.

7-2. In vivo studies of 32 pairs of antibodies using human FcRn transgenic mouse strains

In vivo experiments were performed using the human FcRn transgenic mouse strain 32 by the same method described in Example 13 of WO2011/122011.

7-3. In vivo PK study results of new Fc variants

Figure 133 shows the plasma antibody concentration time profiles after injection of Fv4-IgG1 and Fv4-F1389. Compared to Fv4-IgG1, Fv4-F1389, a novel Fc variant with improved binding to FcRn at acidic pH and reduced binding to rheumatoid factor, showed improved pharmacokinetics. The novel Fc variants described in Table 28 had improved binding affinity to FcRn at pH 6.0 to the same level as F1389. Therefore, it is also expected that these variants will show improved pharmacokinetics when using human FcRn transgenic mouse strain 32, while having reduced binding to rheumatoid factor.

[Example 8] Preparation of antibodies that bind to human IgA in a calcium-dependent manner

8-1. Preparation of human IgA (hIgA)

Human IgA (hereinafter abbreviated as "hIgA") was prepared as an antigen using the following recombinant technology. hIgA (the variable region of which was derived from an anti-human IL-6 receptor antibody) was expressed by culturing host cells harboring a recombinant vector containing H(WT)-IgA1 (SEQ ID NO: 12) and L(WT) (SEQ ID NO: 13), and then purified using ion exchange chromatography and gel filtration chromatography using methods known to those skilled in the art.

8-2. Expression and purification of antibodies binding to hIgA

GA2-IgG1 (heavy chain SEQ ID NO: 14; light chain SEQ ID NO: 15) is an antibody that binds to hIgA. DNA sequences encoding the heavy chain (SEQ ID NO: 14) and light chain (SEQ ID NO: 15) of GA2-IgG1 were inserted into an animal cell expression plasmid using methods known to those skilled in the art. The antibody was expressed using the following method: FreeStyle293-F (Invitrogen), a human fetal kidney cell-derived cell line, was suspended in FreeStyle293 expression medium (Invitrogen). The cell suspension was seeded into a 6-well plate (3 mL/well) at a cell density of 1.33 x ¹⁰ cells/ml. The constructed plasmid was then introduced into the cells using lipofection. The cells were cultured in a _CO2 incubator (37°C, 8% _CO2 , 90 rpm) for 4 days. Antibodies were purified from the isolated culture supernatant using rProtein A Sepharose ^™ Fast Flow (Amersham Biosciences) by methods known to those skilled in the art. The absorbance of the purified antibody solution was measured spectrophotometrically (wavelength: 280 nm). The antibody concentration was determined from the measured value using the absorption coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

8-3. Evaluation of the Obtained Antibodies for Calcium-Dependent hIgA Binding Activity

The antibodies isolated as described in 8-2 were evaluated for their hIgA binding activity (dissociation constant _KD (M)) using Biacore T200 (GE Healthcare). The running buffer used for the measurement was 0.05% tween 20/20 mmol/L ACES/150 mmol/L NaCl (pH 7.4 or 5.8) containing 3 μM or 1.2 mM _CaCl2 .

The antibody was bound to a Sensor chip CM5 (GE Healthcare) immobilized with an appropriate amount of recombinant protein A/G (Thermo Scientific) by an amino coupling method. An appropriate concentration of hIgA (described in 8-1) was then injected as the analyte to allow it to interact with the antibody on the sensor chip. The measurement was performed at 37°C. After the measurement, 10 mmol/L glycine-HCl (pH 1.5) was injected to regenerate the sensor chip. Biacore T200 evaluation software (GE Healthcare) was used to calculate the dissociation constant _K (M) from the measurement results through curve fitting analysis and equilibrium parameter analysis. The results and the obtained sensorgrams are shown in Table 29 and Figure 134, respectively. The results showed that GA2-IgG1 strongly bound to hIgA at a Ca ²⁺ concentration of 1.2 mM, while the antibody weakly bound to hIgA at a Ca ²⁺ concentration of 3 μM. Furthermore, at a Ca ²⁺ concentration of 1.2 mM, GA2-IgG1 showed strong binding to human IgA at pH 7.4 but weak binding at pH 5.8. More specifically, GA2-IgG1 was shown to bind to human IgA in a pH-dependent and calcium-dependent manner.

[Table 29]

[Example 9] Preparation of antibodies having modified Fc regions that bind to hIgA in a calcium-dependent manner

Next, to evaluate the effect of FcRn binding on antigen (hIgA) elimination from plasma, GA2-F760 (heavy chain SEQ ID NO: 16; light chain SEQ ID NO: 15) was constructed by introducing amino acid substitutions L235R and S239K into GA2-IgG1 to eliminate FcγR binding. Furthermore, GA2-F1331 (heavy chain SEQ ID NO: 17; light chain SEQ ID NO: 15) was constructed by introducing amino acid substitutions G236R, M252Y, S254T, T256E, N434Y, Y436V, Q438R, and S440E into GA2-F760. This construct exhibits stronger FcRn binding than GA2-F760 at pH 7.4. The modified antibodies were expressed using animal expression plasmids containing DNA sequences encoding GA2-F1331 (heavy chain, SEQ ID NO: 17; light chain, SEQ ID NO: 15) and GA2-F760 (heavy chain, SEQ ID NO: 16; light chain, SEQ ID NO: 15) by methods known to those skilled in the art. After purification, antibody concentrations were measured. GA2-F760 was evaluated for binding to various mouse FcγRs (mFcγRI, mFcγRII, mFcγRIII, and mFcγRIV). The results showed that GA2-F760 did not bind to any receptors.

[Example 10] Evaluation of the Effect of Ca-dependent hIgA-binding Antibodies on Antigen Plasma Retention Using Human FcRn Transgenic Mice

10-1. In vivo studies using human FcRn transgenic mice

hIgA (human IgA; prepared as described in Example 8) was administered alone or in combination with an anti-hIgA antibody to human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line32+/+ mice, Jackson Laboratories; Methods Mol Biol. (2010) 602:93-104). The pharmacokinetics of hIgA and the anti-hIgA antibody were evaluated. The mixture of hIgA and the anti-hIgA antibody was administered once via the tail vein at a dose of 10 mL/kg. The anti-hIgA antibodies used were GA2-F760 and GA2-F1331.

In each mixture, the hIgA concentration was 80 μg/mL and the anti-hIgA antibody concentration was 2.69 mg/mL. Under these conditions, it is expected that the majority of the hIgA will bind to the antibody, as the anti-hIgA antibody is present in a substantial excess over the hIgA. Blood was collected from the mice 15 minutes, 1 hour, 2 hours, 7 hours, 1 day, 3 days, 7 days, and 14 days after administration. The collected blood was immediately centrifuged at 12,000 rpm and 4°C for 15 minutes to obtain plasma. The separated plasma was stored in a freezer at -20°C or below until assayed.

10-2. Measurement of plasma anti-hIgA antibody concentration by ELISA in human FcRn transgenic mice

The concentration of anti-hIgA antibodies in mouse plasma was determined by ELISA. First, an anti-human IgG (γ chain specific) F(ab')2 fragment antibody (SIGMA) was aliquoted into each well of a Nunc-ImmunoPlate, MaxiSorp (Nalge nunc International) and the plate was allowed to stand overnight at 4°C to prepare an anti-human IgG-immobilized plate. Anti-hIgA antibody standard curve samples prepared as standard solutions with plasma concentrations of 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.01563, and 0.007813 μg/mL and assay samples prepared by diluting mouse plasma samples 100-fold or more were aliquoted into the anti-human IgG-immobilized plate, and the plate was incubated at 25°C for 1 hour. Next, goat anti-human IgG (γ chain specific) biotin (BIOT) conjugate (Southern Biotechnology Associates Inc.) was aliquoted into each well of the plate, and the plate was incubated at 25°C for 1 hour. Then, streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was added to each well of the plate, and the plate was incubated at 25°C for 1 hour. The color development reaction using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a substrate was terminated with 1N sulfuric acid (Showa Chemical), and the reaction mixture in each well was measured using a microplate reader to measure the absorbance at 450nm. The analysis software SOFTmax PRO (Molecular Devices) was used to calculate the anti-hIgA antibody concentration in mouse plasma from the absorbance of the standard curve. The time course of the plasma antibody concentration of GA2-F1331 and GA2-F760 in human FcRn transgenic mice determined by the above method is shown in Figure 135.

10-3. Determination of plasma hIgA concentration by ELISA

Mouse plasma hIgA concentration was determined by ELISA. First, goat anti-human IgA antibody (BETHYL) was aliquoted into each well of a Nunc-ImmunoPlate, MaxiSorp (Nalge nunc International), and the plate was allowed to stand overnight at 4°C to prepare an anti-human IgA-immobilized plate. hIgA standard curve samples were prepared as standard solutions with plasma concentrations of 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, and 0.00625 μg/mL, and assay samples were prepared by diluting the mouse plasma sample 100 times or more. Each sample (100 μL) was mixed with 200 μL of 500 ng/mL hsIL-6R at room temperature for 1 hour, and then aliquoted into an anti-human IgA-immobilized plate at 100 μL/well. The resulting plate was allowed to stand at room temperature for 1 hour. Next, after adding biotinylated anti-human IL-6R antibody (R&D) to each well of the plate, the plate was incubated at room temperature for 1 hour. Then, after aliquoting streptavidin-PolyHRP80 (Stereospecific Detection Technologies) into each well of the plate, the plate was incubated at room temperature for 1 hour. The color reaction using the substrate TMB One Component HRP Microwell Substrate (BioFX Laboratories) was terminated with 1N sulfuric acid (Showa Chemical), and the reaction mixture in each well was measured using a microplate reader to measure the absorbance at 450 nm. The analysis software SOFTmax PRO (Molecular Devices) was used to calculate the concentration in mouse plasma from the absorbance of the standard curve. After intravenous administration, the time course of plasma hIgA concentration in human FcRn transgenic mice determined by the above method is shown in Figure 136.

The results showed that hIgA elimination was significantly accelerated when hIgA was administered in combination with GA2-F1331, an antibody showing strong human FcRn binding, compared to when hIgA was administered in combination with GA2-F760, which has very weak affinity to human FcRn.

[Example 11] Preparation of pH-dependent anti-IgE antibodies

11-1. Preparation of anti-human IgE antibodies

To prepare pH-dependent anti-human IgE antibodies, human IgE (heavy chain SEQ ID NO: 18; light chain SEQ ID NO: 19) (variable region derived from anti-human glypican 3 antibody) was expressed using FreeStyle293 (Life Technologies) as an antigen. Human IgE was prepared by purifying the expressed human IgE using conventional chromatography methods known to those skilled in the art.

From the various antibodies obtained, an antibody that binds to human IgE in a pH-dependent manner was selected. The selected anti-human IgE antibody was expressed using the human IgG1 heavy chain constant region and the human light chain constant region, and then purified. The resulting antibody was designated clone 278 (heavy chain SEQ ID NO: 20; light chain SEQ ID NO: 21).

11-2. Evaluation of Anti-Human IgE Antibodies for Their Binding Activity and pH-Dependent Binding Activity

Antibodies capable of dissociating from antigens in endosomes can be generated by designing them to bind to antigens not only in a pH-dependent manner but also in a Ca-dependent manner. Therefore, clone 278 and a control, Xolair (omalizumab; Novartis), whose IgE binding activity is independent of pH/Ca, were evaluated for pH and pH/Ca dependence of their human IgE (hIgE) binding activity.

More specifically, the hIgE-binding activity (dissociation constant K _D (M)) of clone 278 and Xolair was evaluated using Biacore T200 (GE Healthcare). The running buffer used in the assay was:

1.2mmol/l CaCl ₂ /0.05% tween20, 20mmol/lACES, 150mmol/l NaCl, pH7.4;

1.2mmol/l CaCl2 _/ 0.05% tween 20, 20mmol/l ACES, 150mmol/l NaCl, pH 5.8; and

3μM/lCaCl2 _/ 0.05%tween20, 20mmol/lACES, 150mmol/lNaCl, pH5.8.

Based on the affinity between biotin and streptavidin, an appropriate amount of a chemically synthesized peptide (hereinafter referred to as "biotinylated GPC3 peptide") having a biotinylated C-terminal Lys sequence derived from human glypican 3 protein (SEQ ID NO: 22) was added and immobilized on a Sensor chip SA (GE Healthcare). Human IgE was immobilized on the chip by injecting it at an appropriate concentration so that it was captured by the biotinylated GPC3 peptide. As the analyte, clone 278 was injected at an appropriate concentration to interact with the human IgE on the sensor chip. 10 mmol/L glycine-HCl (pH 1.5) was then injected to regenerate the sensor chip. The interaction was always measured at 37°C. The measurement results were analyzed by curve fitting using Biacore T200 evaluation software (GE Healthcare) to calculate the association rate constant ka (1/Ms) and the dissociation rate constant kd (1/s). The dissociation constant K _D (M) was calculated from the above constants. In addition, the KD ratio of each antibody at [pH 5.8, 1.2 mM Ca] was calculated compared to the KD ratio at [pH 7.4, 1.2 mM Ca] to evaluate pH-dependent binding. The KD ratio of each antibody at [pH 5.8, 3 μM Ca] was calculated compared to the KD ratio at [pH 7.4, 1.2 mM Ca] to evaluate pH/Ca-dependent binding. The results are shown in Table 30.

[Table 30]

[Example 12] Preparation of antibodies with modified Fc regions that bind to human IgE for in vivo testing

Next, to evaluate the effect of FcRn binding on antigen (human IgE) elimination from plasma, 278-F760 (heavy chain SEQ ID NO: 23; light chain SEQ ID NO: 21) was constructed to eliminate FcγR binding. Furthermore, 278-F1331 (heavy chain SEQ ID NO: 24; light chain SEQ ID NO: 21) was constructed by introducing amino acid substitutions G236R, M252Y, S254T, T256E, N434Y, Y436V, Q438R, and S440E into 278-F760. This construct showed stronger FcRn binding than 278-F760 at pH 7.4. The modified antibodies were expressed using an animal expression plasmid into which DNA sequences encoding 278-F1331 (heavy chain SEQ ID NO: 24; light chain SEQ ID NO: 21) and 278-F760 (heavy chain SEQ ID NO: 23; light chain SEQ ID NO: 21) were inserted by methods known to those skilled in the art. Antibody concentrations were measured after purification.

[Example 13] In vivo evaluation of clone 278

13-1. Preparation of human IgE (hIgE(Asp6)) for in vivo evaluation

hIgE(Asp6) (variable region derived from anti-human glypican 3 antibody), a human IgE composed of a heavy chain (SEQ ID NO: 25) and a light chain (SEQ ID NO: 19), was generated for in vivo evaluation by the same method as described in Example 11. hIgE(Asp6) is a modified molecule generated by changing asparagine to aspartic acid at six N-linked glycosylation sites in human IgE, thereby unaffecting the heterogeneity of the N-linked sugar chains of human IgE by time-dependent changes in the plasma concentration of human IgE as an antigen.

13-2. Evaluation of Clone 278 for its Effect on Accelerating Human IgE Elimination Using Human FcRn Transgenic Mice

The pharmacokinetics of hIgE(Asp6) and the anti-human IgE antibody were evaluated after administration of hIgE(Asp6) in combination with anti-hIgE antibodies (278-F760 and 278-F1331) and Sanglopor (human normal immunoglobulin, CSL Behring) to human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line32+/+ mice, Jackson Laboratories; Methods Mol Biol. (2010) 602: 93-104). A mixture of hIgE(Asp6), anti-human IgE antibody, and Sanglopor (concentrations shown in Table 31) was administered once at a dose of 10 mL/kg via the tail vein. Under these conditions, hIgE(Asp6) is expected to bind almost completely to the antibody because the amount of each antibody present is sufficiently greater than that of hIgE(Asp6). Blood was collected from mice 5 minutes, 2 hours, 7 hours, 1 day, 2 days, 4 or 5 days, 7 days, 14 days, 21 days, and 28 days after administration. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 5 minutes to obtain plasma. The separated plasma was stored in a refrigerator at -20°C or lower before measurement.

[Table 31]

13-3. Measurement of Plasma Anti-Human IgE Antibody Concentration in Human FcRn Transgenic Mice

Anti-hIgE antibody concentrations in mouse plasma were determined by electrochemiluminescence (ECL) assay. Standard curve samples were prepared at plasma concentrations of 32, 16, 8, 4, 2, 1, 0.5, and 0.25 micrograms/mL. Standard curve samples and mouse plasma assay samples were aliquoted into ECL plates fixed with hIgE (Asp6). The plates were allowed to stand overnight at 4°C. SULFO-TAG-labeled anti-rabbit antibodies (goat) (Meso Scale Discovery) were then reacted at room temperature for 1 hour. Immediately after dispensing Read Buffer T (x4) (Meso Scale Discovery), measurements were taken using a Sector Imager 2400 reader (Meso Scale Discovery). Analysis software SOFTmax PRO (Molecular Devices) was used to calculate the concentration of mouse plasma from the reaction of the standard curve. The time course of plasma antibody concentration after intravenous administration determined by the above method is shown in Figure 137.

13-4. Measurement of Plasma hIgE (Asp6) Concentration in Human FcRn Transgenic Mice

The hIgE (Asp6) concentration in mouse plasma was determined by ELISA. Standard curve samples were prepared at plasma concentrations of 192, 96, 48, 24, 12, 6, and 3 ng/mL. 10 micrograms/mL of Xolair (Novartis) was added to the standard curve samples and mouse plasma assay samples to balance the immune complex of hIgE (Asp6) and anti-hIgE antibodies. After incubation at room temperature for 30 minutes, the standard curve samples and mouse plasma assay samples were aliquoted onto an immunoplate (MABTECH) fixed with anti-human IgE antibodies or an immunoplate (Nunc F96 Micro Well Plate (Nalgenunc International)) fixed with anti-human IgE antibodies (clone 107; MABTECH). The plates were allowed to stand at room temperature for 2 hours or overnight at 4°C. Then, human GPC3 core protein (SEQ ID NO: 26), an anti-GPC3 antibody biotinylated with NHS-PEG4-Biotin (Thermo Fisher Scientific) (manufactured by Chugai Pharmaceutical Co., Ltd.), and streptavidin-PolyHRP80 (Stereospecific Detection Technologies) were sequentially reacted for 1 hour each. The color development reaction using the substrate TMB One Component HRP Microwell Substrate (BioFX Laboratories) was terminated with 1N sulfuric acid (Showa Chemical), and the mouse plasma concentration was determined using a method in which color development was evaluated by measuring absorbance at 450 nm using a microplate reader, or a method in which a luminescent reaction was performed using SuperSignal(r) ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific) as a substrate and luminescence intensity was measured using a microplate reader. Mouse plasma concentrations were calculated from the absorbance or luminescence intensity of the standard curve using the analysis software SOFTmax PRO (Molecular Devices). The time course of plasma hIgE(Asp6) concentration after intravenous administration, measured by the above method, is shown in FIG138 .

The results showed that when human IgE is administered in combination with 278-F1331 (which binds to human FcRn much more strongly than 278-F760), the elimination of human IgE is significantly accelerated. Specifically, the results indicate that pH-dependent antigen-binding antibodies with enhanced FcRn-binding activity not only for IL6R and IgA, but also for IgE, can accelerate antigen clearance from plasma and reduce plasma antigen concentrations.

[Reference Example A1] Preparation of soluble human IL-6 receptor (hsIL-6R)

Recombinant human IL-6 receptor was prepared as an antigen as follows. A cell line constitutively expressing a soluble human IL-6 receptor (hereinafter also referred to as hsIL-6R) having an amino acid sequence from positions 1 to 357 from the N-terminus was established by methods known to those skilled in the art (as reported in J. Immunol. 152: 4958-4968 (1994)). The cells were cultured to express hsIL-6R. hsIL-6R was purified from the culture supernatant 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 A2] Preparation of human FcRn

FcRn is a heterodimer of the FcRn α chain and β2-microglobulin. Oligomeric DNA primers were prepared based on the published human FcRn gene sequence (J Exp Med. 1994 Dec 1; 180(6): 2377-81). Using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template and the prepared primers, a DNA fragment encoding the entire gene was prepared by PCR. 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, oligomeric DNA primers were prepared based on the published human β2-microglobulin gene sequence (Proc. Natl. Acad. Sci. U.S.A. 99(26): 16899-16903 (2002)). A DNA fragment encoding the entire gene was prepared by PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template and the prepared primers. The resulting DNA fragment was used as a template to amplify a DNA fragment encoding the entire protein containing the signal region (Met1-Met119) by PCR and inserted into a mammalian cell expression vector.

Soluble human FcRn was expressed by the following method. The constructed plasmids for expressing human FcRn α chain (SEQ ID NO: 27) and β2-microglobulin (SEQ ID NO: 28) were introduced into HEK293H (Invitrogen), a human embryonic renal carcinoma-derived cell line, using PEI (Polyscience) by lipofection. The resulting culture supernatant was collected and FcRn was purified using IgG Sepharose 6 Fast Flow (Amersham Biosciences), followed by further purification using HiTrap Q HP (GE Healthcare) (J Immunol. 2002 Nov 1; 169(9): 5171-80). [Reference Example A3] Preparation of human IgA (hIgA)

hlgA comprising H(WT)-IgA1 (SEQ ID NO: 12) and L(WT) (SEQ ID NO: 13) was expressed and purified using rProtein L-agarose (ACTIgen) followed by gel filtration chromatography by methods known to those skilled in the art.

[Reference Example A4] Preparation of soluble human plexin A1 (hsPlexinA1)

Recombinant soluble human plexin A1 (hereinafter also referred to as hsPlexin A1) was prepared as an antigen as follows. hsPlexin A1 was constructed with reference to the NCBI reference sequence (NP_115618). Specifically, hsPlexin A1 consists of amino acid sequences 27-1243 from the NCBI reference, with a FLAG tag (DYKDDDDK, SEQ ID NO: 29) attached to its C-terminus. hsPlexin A1 was transiently expressed using FreeStyle293 (Invitrogen) and purified from the culture supernatant via two steps: anti-FLAG column chromatography and gel filtration chromatography. The fraction eluting as the main peak in the final step was used as the final purified product.

Industrial Applicability

When the conventional antibodies of targeting soluble antigens are given to the experimenter, antigen and antibody are combined, and stably persist in blood plasma.Due to the antigen ratio with antibody combination only antigen has significantly longer half-life, therefore after conventional antibodies are injected, blood plasma total antigen concentration increases to about 10-1000 times of baseline.This increase of blood plasma total antigen concentration is not preferred for therapeutic antibodies, because when compared with not taking place blood plasma total antigen concentration significantly increase, antibody concentration (dosage) must be 10-1000 times of required concentration.Therefore, compared with conventional antibodies, it is extremely useful to eliminate antigen from blood plasma and also reduce the antibody of blood plasma total antigen concentration, because required dosage will be 1/10-1/1000 of conventional antibody required dosage.

The present inventors have conducted intensive research into modified FcRn-binding domains with improved affinity for FcRn at neutral pH, and antigen-binding molecules comprising such FcRn-binding domains that have low immunogenicity, high stability, and minimal aggregate formation. They have discovered that substitutions at specific positions in the FcRn-binding domain improve affinity for FcRn at neutral pH without significantly increasing immunogenicity or the ratio of high molecular weight species, and without significantly reducing the stability of the antigen-binding molecules comprising the FcRn-binding domain. Antigen-binding molecules comprising the FcRn-binding domain of the present invention exhibit excellent pharmacokinetics, promoting a reduction in plasma antigen concentration, and meet the developability criteria of low immunogenicity, high stability, and minimal aggregate formation.

In addition, Fc engineering to improve binding affinity for FcRn at neutral or acidic pH can improve the endosomal recycling efficiency and pharmacokinetics of antibodies. However, modifications of the antibody amino acid sequence (such as amino acid substitutions and insertions) may also increase the immunogenicity of therapeutic antibodies, which in turn can lead to the generation of cytokine storms and/or anti-drug antibodies (ADAs).

The present inventors have conducted intensive research on antigen-binding molecules comprising modified FcRn-binding domains. Due to substitutions in the FcRn-binding domain that increase affinity for FcRn at neutral or acidic pH, the modified FcRn-binding domains exhibit increased binding activity for pre-existing anti-drug antibodies (ADA) at neutral pH. As a result, it was discovered that additional substitutions at specific positions in the FcRn-binding domain reduced binding activity for pre-existing anti-drug antibodies (ADA) at neutral pH, while maintaining the increased FcRn binding activity within the corresponding pH range to a high degree. The antigen-binding molecules of the present invention are extremely superior in terms of pharmacokinetics, promoting a decrease in plasma antigen concentration without increasing antibody clearance.

Claims (25)

1. An antigen-binding molecule comprising a modified human IgG Fc region, wherein the modified human IgG Fc region comprises an amino acid substituted with glutamic acid, aspartic acid, or glutamine at the EU440 position, wherein the antigen-binding molecule does not significantly increase the binding affinity for pre-existing anti-drug antibodies (ADAs) at neutral pH compared to the binding affinity of an antigen-binding molecule comprising a complete human IgG Fc region. 2. The antigen-binding molecule of claim 1, wherein the pre-existing anti-drug antibody is rheumatoid factor. 3. The antigen-binding molecule of claim 1, wherein the modified human IgG Fc region comprises an amino acid substitution, wherein the amino acid substitution is one of the following combinations: . 4. The antigen-binding molecule of claim 1, wherein the antigen-binding molecule further enhances its binding affinity for FcRn in a neutral or acidic pH range. 5. The antigen-binding molecule of claim 4, wherein the modified human IgG Fc region comprises the following amino acid substitutions: i)a)EU438R/EU440E and ii) a) N434H; b) M252Y/S254T/T256E; c) M428L/N434S; or d) T250Q and M428L (EU designations). 6. The antigen-binding molecule of claim 4, wherein the modified human IgG Fc region comprises three or more amino acid substitutions, wherein the three or more substitutions are one of the following combinations . 7. The antigen-binding molecule of claim 4, wherein the modified human IgG Fc region comprises three or more amino acid substitutions, wherein the three or more amino acid substitutions are one of the following combinations: . 8. The antigen-binding molecule of claim 4, wherein the modified human IgG Fc region comprises three or more amino acid substitutions, wherein the three or more substitutions are one of the following combinations . 9. The antigen-binding molecule of claim 4, wherein the modified human IgG Fc region comprises three or more amino acid substitutions, wherein the three or more amino acid substitutions are one of the following combinations . 10. A method for reducing the binding activity of an antigen-binding molecule containing a human IgG Fc region to pre-existing ADA, wherein the IgG Fc region exhibits increased binding activity to FcRn at neutral or acidic pH and increased binding activity to pre-existing ADA at neutral pH, the method comprising the following steps: a) Provide an antigen-binding molecule with an IgG Fc region exhibiting enhanced binding activity to human FcRn at neutral or acidic pH and enhanced binding activity to pre-existing ADA at neutral pH; and b) Replace the amino acid in the human IgG Fc region at the EU440 position with glutamic acid, aspartic acid, or glutamine to obtain an antigen-binding molecule with a modified human IgG Fc region. 11. The method of claim 10, wherein step b) comprises introducing an amino acid substitution into the Fc region of human IgG, said amino acid substitution being one of the following combinations . 12. A method for increasing the total number of antigens that a single antigen-binding molecule can bind without significantly improving binding activity to pre-existing ADA at neutral pH compared to parental antibodies, the method comprising the following steps: a) Provide an antigen-binding molecule containing the Fc region of the parent's IgG. b) Modify the parental IgG Fc region of step a) by introducing one of the following substitutions or combinations: ; and c) Modify the human IgG Fc region modified in step b) by introducing one of the following substitutions or combinations: . 13. A method for promoting the extracellular release of an antigen-free antigen-binding molecule taken up in cells in an antigen-binding form without significantly increasing the binding activity of said antigen-binding molecule to pre-stored ADA at neutral pH compared to a parental antibody, the method comprising the steps of: a) Provide an antigen-binding molecule containing the Fc region of the parent's IgG. b) Modify the parental IgG Fc region of step a) by introducing one of the following substitutions or combinations: ; and c) Modify the human IgG Fc region modified in step b) by introducing one of the following substitutions or combinations: . 14. A method for enhancing the ability of antigen-binding molecules to eliminate plasma antigens without significantly increasing binding activity to pre-existing ADA at neutral pH compared to parental antibodies, the method comprising the following steps: a) Provide an antigen-binding molecule containing the Fc region of the parent's IgG. b) Modify the parental IgG Fc region of step a) by introducing one of the following substitutions or combinations: ; and c) Modify the human IgG Fc region modified in step b) by introducing one of the following substitutions or combinations: . 15. A method for improving the pharmacokinetics of an antigen-binding molecule without significantly increasing its binding activity to pre-existing ADA at neutral pH compared to a parental antibody, the method comprising the following steps: a) Provide an antigen-binding molecule containing the Fc region of the parent's IgG. b) Modify the parental IgG Fc region of step a) by introducing one of the following substitutions or combinations: 1 EU252/EU434/EU307/EU311/EU436/EU286 2 EU252/EU434/EU307/EU311/EU436/EU308 3 EU252/EU434/EU307/EU311/EU436/EU286/EU308 4 EU252/EU434/EU307/EU311/EU436/EU428 5 EU252/EU434/EU307/EU311/EU436/EU308/EU428 6 EU252/EU434/EU307/EU311/EU436/EU250/EU428 7 EU252/EU434/EU307/EU311/EU436/EU250/EU308 8 EU252/EU434/EU307/EU311/EU436/EU250/EU286/EU308 9 EU252/EU434/EU307/EU311/EU436/EU250/EU286/EU308/EU428 10 EU252/EU434/EU307/EU311/EU286 11 EU252/EU434/EU307/EU311/EU286/EU254 12 EU252/EU434/EU307/EU311/EU436 13 EU252/EU434/EU307/EU311/EU436/EU254 14 EU252/EU434/EU307/EU311/EU436/EU250 15 EU252/EU434/EU308/EU250 16 EU252/EU434/EU308/EU250/EU436 17 EU252/EU434/EU308/EU250/EU307/EU311 18 EU252/EU315/EU434 19 EU252/EU434/EU436 20 EU252/EU254/EU434/EU436 twenty one EU307/EU311/EU434 twenty two EU307/EU309/EU311/EU434 twenty three EU307/EU309/EU311/EU434 twenty four EU250/EU252/EU434/EU436 and c) Modify the human IgG Fc region modified in step b) by introducing one of the following substitutions or combinations: . 16. A method for reducing the concentration of total plasma antigen or free plasma antigen without significantly increasing the binding activity to pre-existing ADA at neutral pH compared to parental antibodies, the method comprising the following steps: a) Provides an antigen-binding molecule comprising the parent's IgG Fc region, wherein the antigen-binding molecule comprises an antigen-binding domain capable of binding the antigen. b) Modify the parental IgG Fc region of step a) by introducing one of the following substitutions or combinations: 1 EU252/EU434/EU307/EU311/EU436/EU286 2 EU252/EU434/EU307/EU311/EU436/EU308 3 EU252/EU434/EU307/EU311/EU436/EU286/EU308 4 EU252/EU434/EU307/EU311/EU436/EU428 5 EU252/EU434/EU307/EU311/EU436/EU308/EU428 6 EU252/EU434/EU307/EU311/EU436/EU250/EU428 7 EU252/EU434/EU307/EU311/EU436/EU250/EU308 8 EU252/EU434/EU307/EU311/EU436/EU250/EU286/EU308 9 EU252/EU434/EU307/EU311/EU436/EU250/EU286/EU308/EU428 10 EU252/EU434/EU307/EU311/EU286 11 EU252/EU434/EU307/EU311/EU286/EU254 12 EU252/EU434/EU307/EU311/EU436 13 EU252/EU434/EU307/EU311/EU436/EU254 14 EU252/EU434/EU307/EU311/EU436/EU250 15 EU252/EU434/EU308/EU250 16 EU252/EU434/EU308/EU250/EU436 17 EU252/EU434/EU308/EU250/EU307/EU311 18 EU252/EU315/EU434 19 EU252/EU434/EU436 20 EU252/EU254/EU434/EU436 twenty one EU307/EU311/EU434 twenty two EU307/EU309/EU311/EU434 twenty three EU307/EU309/EU311/EU434 twenty four EU250/EU252/EU434/EU436 and c) Modify the human IgG Fc region modified in step b) by introducing one of the following substitutions or combinations: . 17. An antigen-binding molecule comprising a modified human IgG Fc region, wherein the modified human IgG Fc region comprises glutamic acid, aspartic acid, or glutamine at the EU440 position, wherein the antigen-binding molecule does not significantly increase its binding affinity for pre-existing antidrug antibodies (ADAs) at neutral pH compared to the binding affinity of an antigen-binding molecule comprising an intact human IgG Fc region. 18. The antigen-binding molecule of claim 17, wherein the modified human IgG Fc region comprises any combination of the following amino acids. . 19. The antigen-binding molecule of claim 17, wherein the antigen-binding molecule further enhances its binding affinity for FcRn in a neutral or acidic pH range. 20. The antigen-binding molecule of claim 19, wherein the modified human IgG Fc region comprises the following amino acids: i)a)EU438R/EU440E and ii) a) EU434H; b) EU252Y/EU254T/EU256E; c) EU428L/EU434S; or d) EU250Q and EU428L. 21. The antigen-binding molecule of claim 19, wherein the modified human IgG Fc region comprises any one of the following combinations of amino acids: . 22. The antigen-binding molecule of claim 19, wherein the modified human IgG Fc region comprises any one of the following combinations of amino acids: . 23. The antigen-binding molecule of claim 19, wherein the modified human IgG Fc region comprises any one of the following combinations of amino acids: . 24. The antigen-binding molecule of claim 19, wherein the modified human IgG Fc region comprises any one of the following combinations of amino acids: . 25. The antigen-binding molecule of claim 19, wherein the antigen-binding molecule comprises a pH-dependent antigen-binding domain or a calcium ion-dependent antigen-binding domain.