Antigen-binding molecules that bind to PDGF-B and PDGF-D and methods of using the same

Abstract

The present invention provides a multispecific antigen-binding molecule that binds to PDGF-D and PDGF-B, an antigen-binding molecule that binds to PDGF-D, and an antigen-binding molecule that binds to PDGF-B. The present invention also relates to a composition and a treatment method for using these antigen-binding molecules for the treatment and / or prevention of PDGF-mediated diseases, disorders, or pathological conditions.

Description

Technology Field

[0001] The present invention relates to an antigen-binding molecule that binds to PDGF-B and / or PDGF-D, a pharmaceutical composition comprising the same, and a method for using the same. Background Technology

[0002] Many chronic diseases are characterized by persistent and tenacious inflammation, tissue damage, and fibrosis. For example, progressive kidney diseases such as diabetic nephropathy, IgA nephropathy, and proliferative lupus nephritis exhibit histological features such as interstitial cell expansion and glomerular and tubular interstitial fibrosis.

[0003] Platelet-derived growth factor (PDGF) signaling pathways play a significant role in fibrosis. Interstitial mesenchymal cells express both PDGF receptor (PDGFR) alpha and beta, and their activation induces cell proliferation and migration, as well as the production of extracellular matrix—that is, the major processes of fibrosis (Non-patent Literature 1). PDGF is also involved in many human diseases, including but not limited to atherosclerosis, restenosis, retinal vascular disease, organ fibrosis, rheumatoid arthritis, osteoarthritis, tumorigenesis, and systemic sclerosis (SSc; scleroderma) (Non-patent Literatures 2-5).

[0004] The PDGF family includes five isoforms: PDGF-AA, -AB, -BB, -CC, and -DD. These isoforms bind to the tyrosine kinase PDGF receptor (PDGFR) dimers alpha-alpha, alpha-beta, or beta-beta. Specifically, PDGF-A and -C bind primarily to the PDGFR-alpha chain, PDGF-B binds to both the -alpha and -beta chains, whereas PDGF-D binds only to the -beta chain (Non-patent Literature 6). Upon ligand binding, PDGFR is phosphorylated and activates various downstream signaling pathways and transcription factors, e.g., phospholipase C, ras GTPase-activated protein, phosphatidylinositol 3-kinase (PI3K), Janus kinase, mitogen-activated protein kinase, p38, or calcium release, thereby inducing gene expression and cellular effects of PDGF (Non-patent Literature 7).

[0005] Fibrosis is characterized by pathological remodeling in many tissues and contributes to many clinical diseases. In fact, all chronic progressive diseases are associated with fibrosis and represent a huge number of patients worldwide. However, there are few effective treatment options (Non-Patent Literature 8). Therefore, there is a long-term need for new potential therapeutic agents that can effectively treat or improve these diseases / disorders, and the present invention meets this need. Prior art literature

[0006] B. M. Klinkhammer et al. Molecular Aspects of Medicine 62 (2018) 44-62Trojanowska, 2008, Rheumatology 47:v2-v4Andrae et al. 2008 Genes Dev. 22: 1276-1312.Ying et al. Mol Med Rep. Dec 2017; 16(6): 7879-7889P. Boor et al. Nephrology Dialysis Transplantation, Volume 29, issue suppl_1, February 2014 i45-i54Chen et al. Biochim Biophys Acta. October 2013; 1834(10): 2176-2186.Heldin Cell Communication and Signaling 2013 11:97Don C. Rockey et al. N Engl J Med 2015; 372:1138-1149 The problem to be solved

[0007] The object of the present invention is to provide an antigen-binding molecule that effectively inhibits PDGF-mediated diseases or disorders such as fibrotic diseases or fibrosis. means of solving the problem

[0008] The inventors have discovered that dual blockade of PDGF-B and PDGF-D can effectively inhibit or treat PDGF-mediated diseases or disorders such as fibrosis. Accordingly, the inventors have successfully prepared novel antigen-binding molecules that specifically bind to platelet-derived growth factor B (PDGF-B), e.g., antibodies and antibody fragments thereof, and antigen-binding molecules that specifically bind to platelet-derived growth factor D (PDGF-D). The inventors have also prepared novel multispecific antigen-binding molecules that specifically bind to both PDGF-B and PDGF-D. These antigen-binding molecules are effective in blocking the activity of both PDGF-B and PDGF-D, and the combined inhibitory effect is effective in inhibiting, preventing, or treating PDGF-mediated diseases or disorders, including but not limited to fibrotic diseases or fibrosis, e.g., chronic kidney disease. Another aspect of the present invention relates to a composition comprising the aforementioned antigen-binding molecule or a multispecific antigen-binding molecule, and a method of using the same as a pharmaceutical agent.

[0009] Challenges in the development of therapeutic multispecific antibodies include immunogenicity issues, poor plasma pharmacokinetics (PK), and coupling errors between antibody heavy and light chains of different specificities upon co-expression, which reduce the purity and yield of the desired multispecific antibody when properly assembled. In some embodiments, through careful antibody engineering and optimization (including charge mutations in the constant region and Fc region and Fc region selection), the present disclosure provides an antigen-binding molecule having favorable plasma PK, manufacturability / producibility, and structural uniformity, as well as low immunogenicity.

[0010] [Embodiments of the present invention]

[0011] The present invention is based on these findings and specifically includes the exemplary aspects and embodiments described below. More specifically, the present invention relates to the following:

[0012] (A-1)

[0013] As a multispecific antigen-binding molecule,

[0014] It includes a first antigen-binding domain and a second antigen-binding domain, and

[0015] Herein, the first antigen-binding domain specifically binds to PDGF-D and comprises an antibody heavy chain variable region comprising a heavy chain region (complementarity determining region, CDR) 1 having the amino acid sequence presented in SEQ ID NO. 292, a heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 293, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 294, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 300, a light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 301, and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 302;

[0016] Herein, the second antigen-binding domain comprises any one of the following (a1) to (a5), a multispecific antigen-binding molecule:

[0017] (a1) an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 258, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 259, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 260;

[0018] (a2) an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 236, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 266, a light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 267, and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 268;

[0019] (a3) an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 274, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 275, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 276;

[0020] (a4) an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 242, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 243, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 244, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 274, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 275, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 276; and

[0021] (a5) Antibody heavy chain variable region comprising heavy chain CDR 1 having the amino acid sequence presented in SEQ No. 234, heavy chain CDR 2 having the amino acid sequence presented in SEQ No. 235, and heavy chain CDR 3 having the amino acid sequence presented in SEQ No. 236, and antibody light chain variable region comprising light chain CDR 1 having the amino acid sequence presented in SEQ No. 282, light chain CDR 2 having the amino acid sequence presented in SEQ No. 283, and light chain CDR 3 having the amino acid sequence presented in SEQ No. 284.

[0022] (A-2)

[0023] As a multispecific antigen-binding molecule,

[0024] It includes a first antigen-binding domain and a second antigen-binding domain, and

[0025] Herein, the first antigen binding domain comprises an antibody heavy chain variable region comprising a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence presented in SEQ ID NO. 292, a heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 293, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 294, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 300, a light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 301, and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 302;

[0026] Here, the second antigen-binding domain comprises an antibody heavy chain variable region including the following (a1) or (a2):

[0027] (a1) heavy chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 234, heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 235, and heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 236; and

[0028] (a2) Heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 242, heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 243, heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 244,

[0029] Herein, the second antigen-binding domain comprises a variable antibody light chain region comprising any one of (b1) to (b4), a multispecific antigen-binding molecule:

[0030] (b1) light chain CDR 1 having the amino acid sequence presented in SEQ No. 258, light chain CDR 2 having the amino acid sequence presented in SEQ No. 259, and light chain CDR 3 having the amino acid sequence presented in SEQ No. 260;

[0031] (b2) light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 266, light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 267, and light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 268;

[0032] (b3) light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 274, light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 275, and light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 276; and

[0033] (b4) Light chain CDR 1 having the amino acid sequence presented in SEQ No. 282, light chain CDR 2 having the amino acid sequence presented in SEQ No. 283, and light chain CDR 3 having the amino acid sequence presented in SEQ No. 284.

[0034] (A-3)

[0035] In (A-2),

[0036] A multispecific antigen-binding molecule comprising any one of the following (a1) to (a5), wherein the second antigen-binding domain is a multispecific antigen-binding molecule:

[0037] (a1) an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 258, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 259, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 260;

[0038] (a2) an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 236, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 266, a light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 267, and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 268;

[0039] (a3) an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 274, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 275, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 276;

[0040] (a4) an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 242, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 243, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 244, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 274, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 275, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 276; and

[0041] (a5) Antibody heavy chain variable region comprising heavy chain CDR 1 having the amino acid sequence presented in SEQ No. 234, heavy chain CDR 2 having the amino acid sequence presented in SEQ No. 235, and heavy chain CDR 3 having the amino acid sequence presented in SEQ No. 236, and antibody light chain variable region comprising light chain CDR 1 having the amino acid sequence presented in SEQ No. 282, light chain CDR 2 having the amino acid sequence presented in SEQ No. 283, and light chain CDR 3 having the amino acid sequence presented in SEQ No. 284.

[0042] (A-4)

[0043] In (A-2) or (A-3),

[0044] A multispecific antigen-binding molecule in which a first antigen-binding domain specifically binds to PDGF-D (preferably human PDGF-D) and a second antigen-binding domain specifically binds to PDGF-B (preferably human PDGF-B).

[0045] (A-4a)

[0046] In any one of (A-1) to (A-4),

[0047] A multispecific antigen-binding molecule in which at least one of the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 comprises one or more conservative amino acid substitutions.

[0048] (A-5)

[0049] In any one of (A-1) to (A-4a),

[0050] The first antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 291, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 299;

[0051] Herein, the second antigen-binding domain comprises an antibody heavy chain variable region comprising any one of the following (a1) to (a2):

[0052] (a1) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 233, and

[0053] (a2) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 241;

[0054] Herein, the second antigen-binding domain comprises a variable antibody light chain region comprising any one of (b1) to (b4), a multispecific antigen-binding molecule:

[0055] (b1) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 257,

[0056] (b2) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence described in SEQ ID NO. 265,

[0057] (b3) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 273, and

[0058] (b4) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence described in SEQ ID NO. 281.

[0059] (A-6)

[0060] In any one of (A-1) to (A-5),

[0061] The first antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 291, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 299;

[0062] A multispecific antigen-binding molecule comprising any one of the following (a1) to (a5), wherein the second antigen-binding domain is a multispecific antigen-binding molecule:

[0063] (a1) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 257;

[0064] (a2) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 265;

[0065] (a3) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 273;

[0066] (a4) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 241, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 273; and

[0067] (a5) A heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 281.

[0068] (A-7)

[0069] In any one of (A-1) to (A-6),

[0070] The first antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence presented in SEQ ID NO. 291 and a light chain variable region comprising the amino acid sequence presented in SEQ ID NO. 299;

[0071] A multispecific antigen-binding molecule comprising any one of the following (a1) to (a5), wherein the second antigen-binding domain is a multispecific antigen-binding molecule:

[0072] (a1) a heavy chain variable region comprising the amino acid sequence presented in SEQ ID NO. 233 and a light chain variable region comprising the amino acid sequence presented in SEQ ID NO. 257;

[0073] (a2) a heavy chain variable region comprising the amino acid sequence presented in SEQ ID NO. 233 and a light chain variable region comprising the amino acid sequence presented in SEQ ID NO. 265;

[0074] (a3) a heavy chain variable region comprising the amino acid sequence presented in SEQ ID NO. 233 and a light chain variable region comprising the amino acid sequence presented in SEQ ID NO. 273;

[0075] (a4) a heavy chain variable region comprising the amino acid sequence presented in SEQ ID NO. 241 and a light chain variable region comprising the amino acid sequence presented in SEQ ID NO. 273; and

[0076] (a5) A heavy chain variable region comprising the amino acid sequence presented in sequence number 233, and a light chain variable region comprising the amino acid sequence presented in sequence number 281.

[0077] (A-7a)

[0078] A multispecific antigen-binding molecule that binds to the same epitope of PDGF-B and / or PDGF-D as that of any one of (A-1) to (A-7).

[0079] (A-7b)

[0080] A multispecific antigen-binding molecule competing with any one of (A-1) to (A-7) for binding to PDGF-B and / or PDGF-D proteins.

[0081] (A-8)

[0082] In any one of (A-1) to (A-7),

[0083] A multispecific antigen-binding molecule comprising a protein-L non-binding motif in which the second antigen-binding domain selectively includes human variable kappa subgroup 2 or any subgroup of human or mouse variable lambda.

[0084] (A-9)

[0085] In any one of (A-1) to (A-8),

[0086] A multispecific antigen-binding molecule in which the second antigen-binding domain comprises framework region 1 of human variable kappa subgroup 2.

[0087] (A-10)

[0088] In any one of (A-1) to (A-9),

[0089] A multispecific antigen-binding molecule having at least one of the features selected from the group consisting of (a) to (k):

[0090] (a) Neutralizing PDGF-DD-induced PDGFR beta phosphorylation in vivo or in vitro,

[0091] (b) Neutralizing the combination of PDGF-BB and PDGF-DD-induced PDGFR beta phosphorylation in vivo or in vitro,

[0092] (c) In vivo or in vitro, selectively inhibits PDGF-DD-induced cell proliferation in NIH3T3 cells and / or IMR90 cells,

[0093] (d) selectively inhibiting the combination of PDGF-BB / DD-induced cell proliferation in NIH3T3 cells and / or IMR90 cells in vivo or in vitro,

[0094] (e) Selectively inhibiting PDGF-AB-induced PDGFR alpha phosphorylation in IMR90 cells in vivo or in vitro,

[0095] (f) Selectively inhibits PDGF-AB-induced cell proliferation in IMR90 cells in vivo or in vitro,

[0096] (g) Selectively inhibits the combination of PDGF-BB, PDGF-DD, and PDGF-AB-induced cell proliferation in IMR90 cells in vivo or in vitro,

[0097] (h) Human, mouse, synomolgus, and rat may cross-reactively bind to PDGF-BB and / or PDGF-DD,

[0098] (i) 10 hPDGF-BB and hPDGF-DD -7 M or less, even more preferably 10 -8 M or less, even more preferably 10 -9 M or less, and most preferably 10 -8 M to 10 -10 Combined with KD of M or less,

[0099] (j) Can block the function of human PDGF-AB, PDGF-BB and / or PDGF-DD, or

[0100] (k) Fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma and retinitis pigmentosa (RP), preferably chronic It can inhibit, suppress, treat, or prevent kidney disease (CKD), particularly glomerulonephritis accompanied by vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis in vivo.

[0101] (A-11)

[0102] In any one of (A-1) to (A-10),

[0103] A multispecific antigen-binding molecule, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises a heavy chain constant region and a light chain constant region, optionally wherein the heavy chain constant regions of the first antigen-binding domain and the second antigen-binding domain associate to form an antibody Fc domain.

[0104] (A-11a)

[0105] In any one of (A-1) to (A-11),

[0106] A multispecific antigen-binding molecule in which the antibody Fc domain is a human Fc domain derived from IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

[0107] (A-12)

[0108] In (A-11) or (A-11a),

[0109] A multispecific antigen-binding molecule comprising one or more of the following amino acid residues (i) to (xviii):

[0110] (i) Glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region;

[0111] (ii) Glutamic acid at position 147 (EU numbering) in the heavy chain constant region;

[0112] (iii) Glutamic acid or lysine at position 131 (EU numbering) in the light chain constant region;

[0113] (iv) Asparagine at position 145 (EU numbering) in the light chain invariant region;

[0114] (v) Lee Sin at position 152 (EU numbering) within the light chain invariant zone;

[0115] (vi) Glutamic acid or lysine at position 160 (EU numbering) in the light chain constant region;

[0116] (vii) Lee Sin at position 196 (EU numbering) within the heavy chain invariant region;

[0117] (viii) Arginine at position 235 (EU numbering) in the heavy chain constant region;

[0118] (ix) Arginine at position 236 (EU numbering) in the heavy chain constant region;

[0119] (x) Lee Sin at position 356 (EU numbering) within the heavy chain invariant region;

[0120] (xi) Lee Sin at position 399 (EU numbering) within the heavy chain invariant region;

[0121] (xii) Glutamic acid at position 409 (EU numbering) in the heavy chain constant region;

[0122] (xiii) Glutamic acid at position 419 (EU numbering) in the heavy chain constant region;

[0123] (xiv) Leucine at position 428 (EU numbering) in the heavy chain invariant region;

[0124] (xv) Alanine at position 434 (EU numbering) in the heavy chain invariant region;

[0125] (xvi) Arginine at position 438 (EU numbering) in the heavy chain constant region;

[0126] (xvii) Glutamic acid at position 439 (EU numbering) in the heavy chain constant region;

[0127] (xviii) Glutamic acid at position 440 (EU numbering) in the heavy chain constant region.

[0128] (A-13)

[0129] In any one of (A-11) to (A-12),

[0130] The heavy chain invariant region and light chain invariant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (f):

[0131] (a) A first amino acid residue in the heavy chain constant region (CH1) at position 147 according to EU numbering, and a second amino acid residue in the light chain constant region (CL) at position 131 according to EU numbering,

[0132] (b) a first amino acid residue in CH1 at position 147 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0133] (c) A first amino acid residue in CH1 at position 175 according to EU numbering, and a second amino acid residue in CL at position 131 according to EU numbering,

[0134] (d) a first amino acid residue in CH1 at position 175 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0135] (e) a first amino acid residue in CH1 at position 196 according to EU numbering, and a second amino acid residue in CL at position 131 according to EU numbering, and

[0136] (f) A first amino acid residue in CH1 at position 196 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0137] A multispecific antigen-binding molecule in which the first amino acid residue and the second amino acid residue are oppositely charged amino acid residues.

[0138] (A-14)

[0139] In any one of (A-11) to (A-13),

[0140] A multispecific antigen-binding molecule wherein the heavy chain invariant region and the light chain invariant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (d):

[0141] (a) The heavy chain constant region of the first antigen-binding domain comprises at least one of Lys at position 175 and Lys at position 196 (EU numbering),

[0142] (b) The light chain constant region of the first antigen-binding domain comprises at least one of Glu at position 131, Glu at position 160, and Lys at position 152 (EU numbering),

[0143] (c) The heavy chain constant region of the second antigen-binding domain comprises at least one of Glu at position 147 and Glu at position 175 (EU numbering), and

[0144] (d) The light chain constant region of the second antigen-binding domain comprises at least one of Lys at position 131 and Lys at position 160 (EU numbering).

[0145] (A-15)

[0146] In any one of (A-11) to (A-14),

[0147] A multispecific antigen-binding molecule wherein the first heavy chain constant region and / or the second heavy chain constant region comprises an amino acid substitution at at least one of the amino acid positions (according to EU numbering) of the group consisting of the following:

[0148] (a) Location 356 of the first heavy chain invariant region and location 439 of the second heavy chain invariant region,

[0149] (b) Location 439 of the first heavy chain invariant region and location 356 of the second heavy chain invariant region,

[0150] (c) Location 357 of the first heavy chain invariant region and location 370 of the second heavy chain invariant region,

[0151] (d) Location 370 of the first heavy chain invariant region and location 357 of the second heavy chain invariant region,

[0152] (e) the location 399 of the first heavy chain invariant region and the location 409 of the second heavy chain invariant region, and

[0153] (f) Location 409 of the first heavy chain invariant region and location 399 of the second heavy chain invariant region.

[0154] (A-16)

[0155] In any one of (A-11) to (A-15),

[0156] The first heavy chain invariant region comprises at least one of Lys at position 399 and Glu (EU numbering) at position 439, and

[0157] A multispecific antigen-binding molecule in which the second heavy chain constant region comprises at least one of Lys at position 356, Glu at position 409, and Glu at position 419 (EU numbering).

[0158] (A-17)

[0159] In any one of (A-11) to (A-16),

[0160] A multispecific antigen-binding molecule in which at least one of the first heavy chain constant region or the second heavy chain constant region comprises Arg at position 235 and Arg at position 236 (according to EU numbering).

[0161] (A-18)

[0162] In any one of (A-11) to (A-17),

[0163] A multispecific antigen-binding molecule in which at least one of the first heavy chain constant region or the second heavy chain constant region comprises Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (according to EU numbering).

[0164] (A-19)

[0165] A multispecific antigen-binding molecule comprising or composed of any one of the following (a) to (e):

[0166] (a) a first heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 372, a first light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 373, a second heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 370, and a second light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 371;

[0167] (b) a first heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 376, a first light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 377, a second heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 374, and a second light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 375;

[0168] (c) a first heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 380, a first light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 381, a second heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 378, and a second light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 379;

[0169] (d) a first heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 384, a first light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 385, a second heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 382, ​​and a second light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 383; and

[0170] (e) a first heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 388, a first light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 389, a second heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 386, and a second light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 387.

[0171] (A-20)

[0172] In any one of (A-1) to (A-19),

[0173] A multispecific antigen-binding molecule comprising a Fab, a single-stranded Fv(scFv), a single-stranded antibody, Fv, a single-stranded Fv2(scFv2), or a F(ab')2 structure.

[0174] (A-21)

[0175] A pharmaceutical composition comprising any one of (A-1) to (A20) a multispecific antigen-binding molecule.

[0176] (A-22)

[0177] Optionally, fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma, and retinitis pigmentosa (RP), preferably chronic kidney A pharmaceutical composition of any one of (A-1) to (A-20) or (A-21) for use in a therapy for the treatment of a disease (CKD), particularly glomerulonephritis accompanied by vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis.

[0178] (A-23)

[0179] A method for treating an individual having a pathological condition, comprising the step of administering to the individual an effective amount of a multispecific antigen-binding molecule of any one of (A-1) to (A-20) or a pharmaceutical composition of (A-21).

[0180] The pathological conditions include fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, ocular disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma, and retinitis pigmentosa (RP), preferably chronic A method of renal disease (CKD), particularly glomerulonephritis with vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis.

[0181] (A-24)

[0182] In the manufacture of a drug for treatment or prevention, a multispecific antigen-binding molecule of any one of (A-1) to (A-20) or a pharmaceutical composition of (A-21) is used,

[0183] Fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma, and retinitis pigmentosa (RP), preferably chronic kidney Use of a multispecific antigen-binding molecule or pharmaceutical composition in the manufacture of a drug for the treatment or prevention of disease (CKD), particularly glomerulonephritis accompanied by vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis.

[0184] (A-25)

[0185] An isolated polynucleotide or a plurality of polynucleotides encoding any one of (A-1) to (A-20) a multispecific antigen-binding molecule.

[0186] (A-26)

[0187] A host cell comprising a polynucleotide of (A-25) or a plurality of polynucleotides.

[0188] (A-27)

[0189] A method for generating any one of (A-1) to (A-20) a multispecific antigen-binding molecule, comprising the steps of: (a) culturing a host cell of (A-26) under conditions suitable for the expression of the multispecific antigen-binding molecule; and (b) recovering the multispecific antigen-binding molecule.

[0190] (B-1)

[0191] As a multispecific antigen-binding molecule,

[0192] It includes a first antigen-binding domain and a second antigen-binding domain, and

[0193] Herein, the first antigen binding domain comprises an antibody heavy chain variable region comprising a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence presented in SEQ ID NO. 292, a heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 293, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 294, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 300, a light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 301, and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 302;

[0194] A multispecific antigen-binding molecule comprising, wherein the second antigen-binding domain comprises an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 258, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 259, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 260.

[0195] (B-2)

[0196] In (B-1),

[0197] A multispecific antigen-binding molecule in which at least one of the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 comprises one or more conservative amino acid substitutions.

[0198] (B-3)

[0199] In (B-1) or (B-2),

[0200] The first antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 291, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 299;

[0201] A multispecific antigen-binding molecule comprising a second antigen-binding domain comprising a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 257.

[0202] (B-4)

[0203] In any one of (B-1) to (B-3),

[0204] A multispecific antigen-binding molecule, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises a heavy chain constant region and a light chain constant region, optionally wherein the heavy chain constant regions of the first antigen-binding domain and the second antigen-binding domain associate to form an antibody Fc domain.

[0205] (B-5)

[0206] (B-4) A multispecific antigen-binding molecule in which the antibody Fc domain is a human Fc domain derived from IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

[0207] (B-6)

[0208] In (B-4) or (B-5),

[0209] A multispecific antigen-binding molecule comprising one or more of the following amino acid residues (i) to (xviii):

[0210] (i) Glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region;

[0211] (ii) Glutamic acid at position 147 (EU numbering) in the heavy chain constant region;

[0212] (iii) Glutamic acid or lysine at position 131 (EU numbering) in the light chain constant region;

[0213] (iv) Asparagine at position 145 (EU numbering) in the light chain invariant region;

[0214] (v) Lee Sin at position 152 (EU numbering) within the light chain invariant zone;

[0215] (vi) Glutamic acid or lysine at position 160 (EU numbering) in the light chain constant region;

[0216] (vii) Lee Sin at position 196 (EU numbering) within the heavy chain invariant region;

[0217] (viii) Arginine at position 235 (EU numbering) in the heavy chain constant region;

[0218] (ix) Arginine at position 236 (EU numbering) in the heavy chain constant region;

[0219] (x) Lee Sin at position 356 (EU numbering) within the heavy chain invariant region;

[0220] (xi) Lee Sin at position 399 (EU numbering) within the heavy chain invariant region;

[0221] (xii) Glutamic acid at position 409 (EU numbering) in the heavy chain constant region;

[0222] (xiii) Glutamic acid at position 419 (EU numbering) in the heavy chain constant region;

[0223] (xiv) Leucine at position 428 (EU numbering) in the heavy chain invariant region;

[0224] (xv) Alanine at position 434 (EU numbering) in the heavy chain invariant region;

[0225] (xvi) Arginine at position 438 (EU numbering) in the heavy chain constant region;

[0226] (xvii) Glutamic acid at position 439 (EU numbering) in the heavy chain constant region;

[0227] (xviii) Glutamic acid at position 440 (EU numbering) in the heavy chain constant region.

[0228] (B-7)

[0229] In any one of (B-4) to (B-6),

[0230] The heavy chain invariant region and light chain invariant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (f):

[0231] (a) A first amino acid residue in the heavy chain constant region (CH1) at position 147 according to EU numbering, and a second amino acid residue in the light chain constant region (CL) at position 131 according to EU numbering,

[0232] (b) a first amino acid residue in CH1 at position 147 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0233] (c) A first amino acid residue in CH1 at position 175 according to EU numbering, and a second amino acid residue in CL at position 131 according to EU numbering,

[0234] (d) a first amino acid residue in CH1 at position 175 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0235] (e) a first amino acid residue in CH1 at position 196 according to EU numbering, and a second amino acid residue in CL at position 131 according to EU numbering, and

[0236] (f) A first amino acid residue in CH1 at position 196 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0237] A multispecific antigen-binding molecule in which the first amino acid residue and the second amino acid residue are oppositely charged amino acid residues.

[0238] (B-8)

[0239] In any one of (B-4) to (B-7),

[0240] A multispecific antigen-binding molecule wherein the heavy chain invariant region and the light chain invariant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (d):

[0241] (a) The heavy chain constant region of the first antigen-binding domain comprises at least one of Lys at position 175 and Lys at position 196 (EU numbering),

[0242] (b) The light chain constant region of the first antigen-binding domain comprises at least one of Glu at position 131, Glu at position 160, and Lys at position 152 (EU numbering),

[0243] (c) The heavy chain constant region of the second antigen-binding domain comprises at least one of Glu at position 147 and Glu at position 175 (EU numbering), and

[0244] (d) The light chain constant region of the second antigen-binding domain comprises at least one of Lys at position 131 and Lys at position 160 (EU numbering).

[0245] (B-9)

[0246] In any one of (B-4) to (B-8),

[0247] A multispecific antigen-binding molecule wherein the first heavy chain constant region and / or the second heavy chain constant region comprises at least one amino acid substitution at amino acid positions (according to EU numbering) of the group consisting of the following:

[0248] (a) Location 356 of the first heavy chain invariant region and location 439 of the second heavy chain invariant region,

[0249] (b) Location 439 of the first heavy chain invariant region and location 356 of the second heavy chain invariant region,

[0250] (c) Location 357 of the first heavy chain invariant region and location 370 of the second heavy chain invariant region,

[0251] (d) Location 370 of the first heavy chain invariant region and location 357 of the second heavy chain invariant region,

[0252] (e) the location 399 of the first heavy chain invariant region and the location 409 of the second heavy chain invariant region, and

[0253] (f) Location 409 of the first heavy chain invariant region and location 399 of the second heavy chain invariant region.

[0254] (B-10)

[0255] In any one of (B-4) to (B-9),

[0256] The first heavy chain invariant region comprises at least one of Lys at position 399 and Glu (EU numbering) at position 439, and

[0257] A multispecific antigen-binding molecule in which the second heavy chain constant region comprises at least one of Lys at position 356, Glu at position 409, and Glu at position 419 (EU numbering).

[0258] (B-11)

[0259] In any one of (B-4) to (B-10),

[0260] A multispecific antigen-binding molecule in which the antibody Fc domain comprises at least one of Arg at position 235 and Arg at position 236 (according to EU numbering).

[0261] (B-12)

[0262] In any one of (B-4) to (B-11),

[0263] A multispecific antigen-binding molecule in which the antibody Fc domain comprises at least one of Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (according to EU numbering).

[0264] (B-13)

[0265] As a multispecific antigen-binding molecule,

[0266] A multispecific antigen-binding molecule comprising a first heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 372, a first light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 373, a second heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 370, and comprising or composed of the amino acid sequence presented in SEQ ID NO. 371.

[0267] (B-14)

[0268] As a multispecific antigen-binding molecule,

[0269] It includes a first antigen-binding domain and a second antigen-binding domain, and

[0270] Herein, the first antigen binding domain comprises an antibody heavy chain variable region comprising a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence presented in SEQ ID NO. 292, a heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 293, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 294, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 300, a light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 301, and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 302;

[0271] A multispecific antigen-binding molecule comprising, wherein the second antigen-binding domain comprises an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 266, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 267, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 268.

[0272] (B-15)

[0273] In (B-14),

[0274] A multispecific antigen-binding molecule in which at least one of the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 comprises one or more conservative amino acid substitutions.

[0275] (B-16)

[0276] In (B-14) or (B-15),

[0277] The first antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 291, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 299;

[0278] A multispecific antigen-binding molecule comprising a second antigen-binding domain comprising a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 265.

[0279] (B-17)

[0280] In any one of (B-15) to (B-16),

[0281] A multispecific antigen-binding molecule, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises a heavy chain constant region and a light chain constant region, optionally wherein the heavy chain constant regions of the first antigen-binding domain and the second antigen-binding domain associate to form an antibody Fc domain.

[0282] (B-18)

[0283] In (B-17),

[0284] A multispecific antigen-binding molecule in which the antibody Fc domain is a human Fc domain derived from IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

[0285] (B-19)

[0286] In (B-17) or (B-18),

[0287] A multispecific antigen-binding molecule comprising one or more of the following amino acid residues (i) to (xviii):

[0288] (i) Glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region;

[0289] (ii) Glutamic acid at position 147 (EU numbering) in the heavy chain constant region;

[0290] (iii) Glutamic acid or lysine at position 131 (EU numbering) in the light chain constant region;

[0291] (iv) Asparagine at position 145 (EU numbering) in the light chain invariant region;

[0292] (v) Lee Sin at position 152 (EU numbering) within the light chain invariant zone;

[0293] (vi) Glutamic acid or lysine at position 160 (EU numbering) in the light chain constant region;

[0294] (vii) Lee Sin at position 196 (EU numbering) within the heavy chain invariant region;

[0295] (viii) Arginine at position 235 (EU numbering) in the heavy chain constant region;

[0296] (ix) Arginine at position 236 (EU numbering) in the heavy chain constant region;

[0297] (x) Lee Sin at position 356 (EU numbering) within the heavy chain invariant region;

[0298] (xi) Lee Sin at position 399 (EU numbering) within the heavy chain invariant region;

[0299] (xii) Glutamic acid at position 409 (EU numbering) in the heavy chain constant region;

[0300] (xiii) Glutamic acid at position 419 (EU numbering) in the heavy chain constant region;

[0301] (xiv) Leucine at position 428 (EU numbering) in the heavy chain invariant region;

[0302] (xv) Alanine at position 434 (EU numbering) in the heavy chain invariant region;

[0303] (xvi) Arginine at position 438 (EU numbering) in the heavy chain constant region;

[0304] (xvii) Glutamic acid at position 439 (EU numbering) in the heavy chain constant region;

[0305] (xviii) Glutamic acid at position 440 (EU numbering) in the heavy chain constant region.

[0306] (B-20)

[0307] In any one of (B-17) to (B-19),

[0308] The heavy chain invariant region and light chain invariant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (f):

[0309] (a) A first amino acid residue in the heavy chain constant region (CH1) at position 147 according to EU numbering, and a second amino acid residue in the light chain constant region (CL) at position 131 according to EU numbering,

[0310] (b) a first amino acid residue in CH1 at position 147 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0311] (c) A first amino acid residue in CH1 at position 175 according to EU numbering, and a second amino acid residue in CL at position 131 according to EU numbering,

[0312] (d) a first amino acid residue in CH1 at position 175 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0313] (e) a first amino acid residue in CH1 at position 196 according to EU numbering, and a second amino acid residue in CL at position 131 according to EU numbering, and

[0314] (f) A first amino acid residue in CH1 at position 196 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0315] A multispecific antigen-binding molecule in which the first amino acid residue and the second amino acid residue are oppositely charged amino acid residues.

[0316] (B-21)

[0317] In any one of (B-17) to (B-20),

[0318] A multispecific antigen-binding molecule wherein the heavy chain invariant region and the light chain invariant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (d):

[0319] (a) The heavy chain constant region of the first antigen-binding domain comprises at least one of Lys at position 175 and Lys at position 196 (EU numbering),

[0320] (b) The light chain constant region of the first antigen-binding domain comprises at least one of Glu at position 131, Glu at position 160, and Lys at position 152 (EU numbering),

[0321] (c) The heavy chain constant region of the second antigen-binding domain comprises at least one of Glu at position 147 and Glu at position 175 (EU numbering), and

[0322] (d) The light chain constant region of the second antigen-binding domain comprises at least one of Lys at position 131 and Lys at position 160 (EU numbering).

[0323] (B-22)

[0324] In any one of (B-17) to (B-21),

[0325] A multispecific antigen-binding molecule wherein the first heavy chain constant region and / or the second heavy chain constant region comprises at least one amino acid substitution at amino acid positions (according to EU numbering) of the group consisting of the following:

[0326] (a) Location 356 of the first heavy chain invariant region and location 439 of the second heavy chain invariant region,

[0327] (b) Location 439 of the first heavy chain invariant region and location 356 of the second heavy chain invariant region,

[0328] (c) Location 357 of the first heavy chain invariant region and location 370 of the second heavy chain invariant region,

[0329] (d) Location 370 of the first heavy chain invariant region and location 357 of the second heavy chain invariant region,

[0330] (e) the location 399 of the first heavy chain invariant region and the location 409 of the second heavy chain invariant region, and

[0331] (f) Location 409 of the first heavy chain invariant region and location 399 of the second heavy chain invariant region.

[0332] (B-23)

[0333] In any one of (B-17) to (B-22),

[0334] The first heavy chain invariant region comprises at least one of Lys at position 399 and Glu (EU numbering) at position 439, and

[0335] A multispecific antigen-binding molecule in which the second heavy chain constant region comprises at least one of Lys at position 356, Glu at position 409, and Glu at position 419 (EU numbering).

[0336] (B-24)

[0337] In any one of (B-17) to (B-23),

[0338] A multispecific antigen-binding molecule in which the antibody Fc domain comprises at least one of Arg at position 235 and Arg at position 236 (according to EU numbering).

[0339] (B-25)

[0340] In any one of (B-17) to (B-24),

[0341] A multispecific antigen-binding molecule in which the antibody Fc domain comprises at least one of Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (according to EU numbering).

[0342] (B-26)

[0343] As a multispecific antigen-binding molecule,

[0344] A multispecific antigen-binding molecule comprising a first heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 376, a first light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 377, a second heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 374, and a multispecific antigen-binding molecule comprising or composed of the amino acid sequence presented in SEQ ID NO. 375.

[0345] (B-27)

[0346] As a multispecific antigen-binding molecule,

[0347] It includes a first antigen-binding domain and a second antigen-binding domain, and

[0348] Herein, the first antigen binding domain comprises an antibody heavy chain variable region comprising a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence presented in SEQ ID NO. 292, a heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 293, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 294, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 300, a light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 301, and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 302;

[0349] A multispecific antigen-binding molecule comprising, wherein the second antigen-binding domain comprises an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 274, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 275, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 276.

[0350] (B-28)

[0351] In (B-27),

[0352] A multispecific antigen-binding molecule in which at least one of the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 comprises one or more conservative amino acid substitutions.

[0353] (B-29)

[0354] In (B-27) or (B-28),

[0355] The first antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 291, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 299;

[0356] A multispecific antigen-binding molecule comprising a second antigen-binding domain comprising a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 273.

[0357] (B-30)

[0358] In any one of (B-27) to (B-29),

[0359] A multispecific antigen-binding molecule, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises a heavy chain constant region and a light chain constant region, optionally wherein the heavy chain constant regions of the first antigen-binding domain and the second antigen-binding domain associate to form an antibody Fc domain.

[0360] (B-31)

[0361] In (B-30),

[0362] A multispecific antigen-binding molecule in which the antibody Fc domain is a human Fc domain derived from IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

[0363] (B-32)

[0364] In (B-30) or (B-31),

[0365] A multispecific antigen-binding molecule comprising one or more of the following amino acid residues (i) to (xviii):

[0366] (i) Glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region;

[0367] (ii) Glutamic acid at position 147 (EU numbering) in the heavy chain constant region;

[0368] (iii) Glutamic acid or lysine at position 131 (EU numbering) in the light chain constant region;

[0369] (iv) Asparagine at position 145 (EU numbering) in the light chain invariant region;

[0370] (v) Lee Sin at position 152 (EU numbering) within the light chain invariant zone;

[0371] (vi) Glutamic acid or lysine at position 160 (EU numbering) in the light chain constant region;

[0372] (vii) Lee Sin at position 196 (EU numbering) within the heavy chain invariant region;

[0373] (viii) Arginine at position 235 (EU numbering) in the heavy chain constant region;

[0374] (ix) Arginine at position 236 (EU numbering) in the heavy chain constant region;

[0375] (x) Lee Sin at position 356 (EU numbering) within the heavy chain invariant region;

[0376] (xi) Lee Sin at position 399 (EU numbering) within the heavy chain invariant region;

[0377] (xii) Glutamic acid at position 409 (EU numbering) in the heavy chain constant region;

[0378] (xiii) Glutamic acid at position 419 (EU numbering) in the heavy chain constant region;

[0379] (xiv) Leucine at position 428 (EU numbering) in the heavy chain invariant region;

[0380] (xv) Alanine at position 434 (EU numbering) in the heavy chain invariant region;

[0381] (xvi) Arginine at position 438 (EU numbering) in the heavy chain constant region;

[0382] (xvii) Glutamic acid at position 439 (EU numbering) in the heavy chain constant region;

[0383] (xviii) Glutamic acid at position 440 (EU numbering) in the heavy chain constant region.

[0384] (B-33)

[0385] In any one of (B-30) to (B-32),

[0386] The heavy chain invariant region and light chain invariant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (f):

[0387] (a) A first amino acid residue in the heavy chain constant region (CH1) at position 147 according to EU numbering, and a second amino acid residue in the light chain constant region (CL) at position 131 according to EU numbering,

[0388] (b) a first amino acid residue in CH1 at position 147 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0389] (c) A first amino acid residue in CH1 at position 175 according to EU numbering, and a second amino acid residue in CL at position 131 according to EU numbering,

[0390] (d) a first amino acid residue in CH1 at position 175 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0391] (e) a first amino acid residue in CH1 at position 196 according to EU numbering, and a second amino acid residue in CL at position 131 according to EU numbering, and

[0392] (f) A first amino acid residue in CH1 at position 196 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0393] A multispecific antigen-binding molecule in which the first amino acid residue and the second amino acid residue are oppositely charged amino acid residues.

[0394] (B-34)

[0395] In any one of (B-30) to (B-33),

[0396] A multispecific antigen-binding molecule wherein the heavy chain invariant region and the light chain invariant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (d):

[0397] (a) The heavy chain constant region of the first antigen-binding domain includes Lys(EU numbering) at position 175,

[0398] (b) The light chain constant region of the first antigen-binding domain comprises at least one of Glu at position 131, Glu at position 160, and Asn at position 145 (EU numbering),

[0399] (c) The heavy chain constant region of the second antigen-binding domain comprises at least one of Glu at position 147 and Glu at position 175 (EU numbering), and

[0400] (d) The light chain constant region of the second antigen-binding domain comprises at least one of Lys at position 131 and Lys at position 160 (EU numbering).

[0401] (B-35)

[0402] In any one of (B-30) to (B-34),

[0403] A multispecific antigen-binding molecule wherein the first heavy chain constant region and / or the second heavy chain constant region comprises an amino acid substitution at at least one of the amino acid positions (according to EU numbering) of the group consisting of the following:

[0404] (a) Location 356 of the first heavy chain invariant region and location 439 of the second heavy chain invariant region,

[0405] (b) Location 439 of the first heavy chain invariant region and location 356 of the second heavy chain invariant region,

[0406] (c) Location 357 of the first heavy chain invariant region and location 370 of the second heavy chain invariant region,

[0407] (d) Location 370 of the first heavy chain invariant region and location 357 of the second heavy chain invariant region,

[0408] (e) the location 399 of the first heavy chain invariant region and the location 409 of the second heavy chain invariant region, and

[0409] (f) Location 409 of the first heavy chain invariant region and location 399 of the second heavy chain invariant region.

[0410] (B-36)

[0411] In any one of (B-30) to (B-35),

[0412] The first heavy chain invariant region contains Glu at position 439 (EU numbering), and

[0413] A multispecific antigen-binding molecule in which the second heavy chain constant region comprises at least one of Lys at position 356 and Glu (EU numbering) at position 419.

[0414] (B-37)

[0415] In any one of (B-30) to (B-36),

[0416] A multispecific antigen-binding molecule in which the antibody Fc domain comprises at least one of Arg at position 235 and Arg at position 236 (according to EU numbering).

[0417] (B-38)

[0418] In any one of (B-30) to (B-37),

[0419] A multispecific antigen-binding molecule in which the antibody Fc domain comprises at least one of Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (according to EU numbering).

[0420] (B-39)

[0421] As a multispecific antigen-binding molecule,

[0422] A multispecific antigen-binding molecule comprising a first heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 380, a first light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 381, a second heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 378, and a second light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 379.

[0423] (B-40)

[0424] As a multispecific antigen-binding molecule,

[0425] It includes a first antigen-binding domain and a second antigen-binding domain, and

[0426] Herein, the first antigen binding domain comprises an antibody heavy chain variable region comprising a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence presented in SEQ ID NO. 292, a heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 293, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 294, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 300, a light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 301, and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 302;

[0427] A multispecific antigen-binding molecule comprising, wherein the second antigen-binding domain comprises an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 242, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 243, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 244, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 274, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 275, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 276.

[0428] (B-41)

[0429] In (B-40),

[0430] A multispecific antigen-binding molecule in which at least one of the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 comprises one or more conservative amino acid substitutions.

[0431] (B-42)

[0432] In (B-40) or (B-41),

[0433] The first antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 291, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 299;

[0434] A multispecific antigen-binding molecule comprising a second antigen-binding domain comprising a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 241, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 273.

[0435] (B-43)

[0436] In any one of (B-40) to (B-42),

[0437] A multispecific antigen-binding molecule, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises a heavy chain constant region and a light chain constant region, optionally wherein the heavy chain constant regions of the first antigen-binding domain and the second antigen-binding domain associate to form an antibody Fc domain.

[0438] (B-44)

[0439] In (B-43),

[0440] A multispecific antigen-binding molecule in which the antibody Fc domain is a human Fc domain derived from IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

[0441] (B-45)

[0442] In (B-43) or (B-44),

[0443] A multispecific antigen-binding molecule comprising one or more of the following amino acid residues (i) to (xviii):

[0444] (i) Glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region;

[0445] (ii) Glutamic acid at position 147 (EU numbering) in the heavy chain constant region;

[0446] (iii) Glutamic acid or lysine at position 131 (EU numbering) in the light chain constant region;

[0447] (iv) Asparagine at position 145 (EU numbering) in the light chain invariant region;

[0448] (v) Lee Sin at position 152 (EU numbering) within the light chain invariant zone;

[0449] (vi) Glutamic acid or lysine at position 160 (EU numbering) in the light chain constant region;

[0450] (vii) Lee Sin at position 196 (EU numbering) within the heavy chain invariant region;

[0451] (viii) Arginine at position 235 (EU numbering) in the heavy chain constant region;

[0452] (ix) Arginine at position 236 (EU numbering) in the heavy chain constant region;

[0453] (x) Lee Sin at position 356 (EU numbering) within the heavy chain invariant region;

[0454] (xi) Lee Sin at position 399 (EU numbering) within the heavy chain invariant region;

[0455] (xii) Glutamic acid at position 409 (EU numbering) in the heavy chain constant region;

[0456] (xiii) Glutamic acid at position 419 (EU numbering) in the heavy chain constant region;

[0457] (xiv) Leucine at position 428 (EU numbering) in the heavy chain invariant region;

[0458] (xv) Alanine at position 434 (EU numbering) in the heavy chain invariant region;

[0459] (xvi) Arginine at position 438 (EU numbering) in the heavy chain constant region;

[0460] (xvii) Glutamic acid at position 439 (EU numbering) in the heavy chain constant region;

[0461] (xviii) Glutamic acid at position 440 (EU numbering) in the heavy chain constant region.

[0462] (B-46)

[0463] In any one of (B-43) to (B-45),

[0464] The heavy chain invariant region and light chain invariant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (f):

[0465] (a) A first amino acid residue in the heavy chain constant region (CH1) at position 147 according to EU numbering, and a second amino acid residue in the light chain constant region (CL) at position 131 according to EU numbering,

[0466] (b) a first amino acid residue in CH1 at position 147 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0467] (c) A first amino acid residue in CH1 at position 175 according to EU numbering, and a second amino acid residue in CL at position 131 according to EU numbering,

[0468] (d) a first amino acid residue in CH1 at position 175 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0469] (e) a first amino acid residue in CH1 at position 196 according to EU numbering, and a second amino acid residue in CL at position 131 according to EU numbering, and

[0470] (f) A first amino acid residue in CH1 at position 196 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0471] A multispecific antigen-binding molecule in which the first amino acid residue and the second amino acid residue are oppositely charged amino acid residues.

[0472] (B-47)

[0473] In any one of (B-43) to (B-46),

[0474] A multispecific antigen-binding molecule wherein the heavy chain invariant region and the light chain invariant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (d):

[0475] (a) The heavy chain constant region of the first antigen-binding domain includes Lys(EU numbering) at position 175,

[0476] (b) The light chain constant region of the first antigen-binding domain comprises at least one of Glu at position 131, Glu at position 160, and Asn at position 145 (EU numbering),

[0477] (c) The heavy chain constant region of the second antigen-binding domain comprises at least one of Glu at position 147 and Glu at position 175 (EU numbering), and

[0478] (d) The light chain constant region of the second antigen-binding domain comprises at least one of Lys at position 131 and Lys at position 160 (EU numbering).

[0479] (B-48)

[0480] In any one of (B-43) to (B-47),

[0481] A multispecific antigen-binding molecule wherein the first heavy chain constant region and / or the second heavy chain constant region comprises an amino acid substitution at at least one of the amino acid positions (according to EU numbering) of the group consisting of the following:

[0482] (a) Location 356 of the first heavy chain invariant region and location 439 of the second heavy chain invariant region,

[0483] (b) Location 439 of the first heavy chain invariant region and location 356 of the second heavy chain invariant region,

[0484] (c) Location 357 of the first heavy chain invariant region and location 370 of the second heavy chain invariant region,

[0485] (d) Location 370 of the first heavy chain invariant region and location 357 of the second heavy chain invariant region,

[0486] (e) the location 399 of the first heavy chain invariant region and the location 409 of the second heavy chain invariant region, and

[0487] (f) Location 409 of the first heavy chain invariant region and location 399 of the second heavy chain invariant region.

[0488] (B-49)

[0489] In any one of (B-43) to (B-48),

[0490] The first heavy chain invariant region contains Glu at position 439 (EU numbering), and

[0491] A multispecific antigen-binding molecule in which the second heavy chain constant region comprises at least one of Lys at position 356 and Glu (EU numbering) at position 419.

[0492] (B-50)

[0493] In any one of (B-43) to (B-49),

[0494] A multispecific antigen-binding molecule in which the antibody Fc domain comprises at least one of Arg at position 235 and Arg at position 236 (according to EU numbering).

[0495] (B-51)

[0496] In any one of (B-43) to (B-50),

[0497] A multispecific antigen-binding molecule in which the antibody Fc domain comprises at least one of Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (according to EU numbering).

[0498] (B-52)

[0499] As a multispecific antigen-binding molecule,

[0500] A multispecific antigen-binding molecule comprising a first heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 384, a first light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 385, a second heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 382, ​​and a second light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 383.

[0501] (B-53)

[0502] As a multispecific antigen-binding molecule,

[0503] It includes a first antigen-binding domain and a second antigen-binding domain, and

[0504] Herein, the first antigen binding domain comprises an antibody heavy chain variable region comprising a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence presented in SEQ ID NO. 292, a heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 293, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 294, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 300, a light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 301, and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 302;

[0505] A multispecific antigen-binding molecule comprising, wherein the second antigen-binding domain comprises an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 282, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 283, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 284.

[0506] (B-54)

[0507] In (B-53),

[0508] A multispecific antigen-binding molecule in which at least one of the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 comprises one or more conservative amino acid substitutions.

[0509] (B-55)

[0510] In (B-53) or (B-54),

[0511] The first antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 291, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 299;

[0512] A multispecific antigen-binding molecule comprising a second antigen-binding domain comprising a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 281.

[0513] (B-56)

[0514] In any one of (B-53) to (B-55),

[0515] A multispecific antigen-binding molecule, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises a heavy chain constant region and a light chain constant region, optionally wherein the heavy chain constant regions of the first antigen-binding domain and the second antigen-binding domain associate to form an antibody Fc domain.

[0516] (B-57)

[0517] In (B-56),

[0518] A multispecific antigen-binding molecule in which the antibody Fc domain is a human Fc domain derived from IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

[0519] (B-58)

[0520] In (B-56) or (B-57),

[0521] A multispecific antigen-binding molecule comprising one or more of the following amino acid residues (i) to (xviii):

[0522] (i) Glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region;

[0523] (ii) Glutamic acid at position 147 (EU numbering) in the heavy chain constant region;

[0524] (iii) Glutamic acid or lysine at position 131 (EU numbering) in the light chain constant region;

[0525] (iv) Asparagine at position 145 (EU numbering) in the light chain invariant region;

[0526] (v) Lee Sin at position 152 (EU numbering) within the light chain invariant zone;

[0527] (vi) Glutamic acid or lysine at position 160 (EU numbering) in the light chain constant region;

[0528] (vii) Lee Sin at position 196 (EU numbering) within the heavy chain invariant region;

[0529] (viii) Arginine at position 235 (EU numbering) in the heavy chain constant region;

[0530] (ix) Arginine at position 236 (EU numbering) in the heavy chain constant region;

[0531] (x) Lee Sin at position 356 (EU numbering) within the heavy chain invariant region;

[0532] (xi) Lee Sin at position 399 (EU numbering) within the heavy chain invariant region;

[0533] (xii) Glutamic acid at position 409 (EU numbering) in the heavy chain constant region;

[0534] (xiii) Glutamic acid at position 419 (EU numbering) in the heavy chain constant region;

[0535] (xiv) Leucine at position 428 (EU numbering) in the heavy chain invariant region;

[0536] (xv) Alanine at position 434 (EU numbering) in the heavy chain invariant region;

[0537] (xvi) Arginine at position 438 (EU numbering) in the heavy chain constant region;

[0538] (xvii) Glutamic acid at position 439 (EU numbering) in the heavy chain constant region;

[0539] (xviii) Glutamic acid at position 440 (EU numbering) in the heavy chain constant region.

[0540] (B-59)

[0541] In any one of (B-56) to (B-58),

[0542] The heavy chain invariant region and light chain invariant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (f):

[0543] (a) A first amino acid residue in the heavy chain constant region (CH1) at position 147 according to EU numbering, and a second amino acid residue in the light chain constant region (CL) at position 131 according to EU numbering,

[0544] (b) a first amino acid residue in CH1 at position 147 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0545] (c) A first amino acid residue in CH1 at position 175 according to EU numbering, and a second amino acid residue in CL at position 131 according to EU numbering,

[0546] (d) a first amino acid residue in CH1 at position 175 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0547] (e) a first amino acid residue in CH1 at position 196 according to EU numbering, and a second amino acid residue in CL at position 131 according to EU numbering, and

[0548] (f) A first amino acid residue in CH1 at position 196 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0549] A multispecific antigen-binding molecule in which the first amino acid residue and the second amino acid residue are oppositely charged amino acid residues.

[0550] (B-60)

[0551] In any one of (B-56) to (B-59),

[0552] A multispecific antigen-binding molecule wherein the heavy chain invariant region and the light chain invariant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (d):

[0553] (a) The heavy chain constant region of the first antigen-binding domain comprises at least one of Lys at position 175 and Lys at position 196 (EU numbering),

[0554] (b) The light chain constant region of the first antigen-binding domain comprises at least one of Glu at position 131, Glu at position 160, and Lys at position 152 (EU numbering),

[0555] (c) The heavy chain constant region of the second antigen-binding domain comprises at least one of Glu at position 147 and Glu at position 175 (EU numbering), and

[0556] (d) The light chain constant region of the second antigen-binding domain comprises at least one of Lys at position 131 and Lys at position 160 (EU numbering).

[0557] (B-61)

[0558] In any one of (B-56) to (B-60),

[0559] A multispecific antigen-binding molecule wherein the first heavy chain constant region and / or the second heavy chain constant region comprises an amino acid substitution at at least one of the amino acid positions (according to EU numbering) of the group consisting of the following:

[0560] (a) Location 356 of the first heavy chain invariant region and location 439 of the second heavy chain invariant region,

[0561] (b) Location 439 of the first heavy chain invariant region and location 356 of the second heavy chain invariant region,

[0562] (c) Location 357 of the first heavy chain invariant region and location 370 of the second heavy chain invariant region,

[0563] (d) Location 370 of the first heavy chain invariant region and location 357 of the second heavy chain invariant region,

[0564] (e) the location 399 of the first heavy chain invariant region and the location 409 of the second heavy chain invariant region, and

[0565] (f) Location 409 of the first heavy chain invariant region and location 399 of the second heavy chain invariant region.

[0566] (B-62)

[0567] In any one of (B-56) to (B-61),

[0568] The first heavy chain invariant region comprises at least one of Lys at position 399 and Glu (EU numbering) at position 439, and

[0569] A multispecific antigen-binding molecule in which the second heavy chain constant region comprises at least one of Lys at position 356, Glu at position 409, and Glu at position 419 (EU numbering).

[0570] (B-63)

[0571] In any one of (B-56) to (B-62),

[0572] A multispecific antigen-binding molecule in which the antibody Fc domain comprises at least one of Arg at position 235 and Arg at position 236 (according to EU numbering).

[0573] (B-64)

[0574] In any one of (B-56) to (B-63),

[0575] A multispecific antigen-binding molecule in which the antibody Fc domain comprises at least one of Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (according to EU numbering).

[0576] (B-65)

[0577] As a multispecific antigen-binding molecule,

[0578] A multispecific antigen-binding molecule comprising a first heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 388, a first light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 389, a second heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 386, and a second light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 387.

[0579] (B-66)

[0580] In any one of (B-1) to (B-65),

[0581] A multispecific antigen-binding molecule in which a first antigen-binding domain specifically binds to PDGF-D (preferably human PDGF-D) and a second antigen-binding domain specifically binds to PDGF-B (preferably human PDGF-B).

[0582] (B-66a)

[0583] A multispecific antigen-binding molecule that binds to the same epitope of PDGF-B and / or PDGF-D as that of any one of (B-1) to (B-66).

[0584] (B-66b)

[0585] A multispecific antigen-binding molecule competing with any one of (B-1) to (B-66) to bind to PDGF-B and / or PDGF-D proteins.

[0586] (B-66c)

[0587] 10 to PDGF-D (preferably human PDGF-D) -8 M or less, more preferably 10 -9 M or less, and most preferably 10 -8 M to 10 -10 Binds to M's KD and 10 to PDGF-B (preferably human PDGF-B). -8 M or less, more preferably 10 -9 M or less, and most preferably 10 -8 M to 10 -10 A multispecific antigen-binding molecule that binds to the KD of M.

[0588] (B-67)

[0589] In any one of (B-1) to (B-66b),

[0590] A multispecific antigen-binding molecule comprising a protein-L non-binding motif in which the second antigen-binding domain selectively includes human variable kappa subgroup 2 or any subgroup of human or mouse variable lambda.

[0591] (B-68)

[0592] In any one of (B-1) to (B-67),

[0593] A multispecific antigen-binding molecule in which the second antigen-binding domain comprises framework region 1 of human variable kappa subgroup 2.

[0594] (B-69)

[0595] In any one of (B-1) to (B-68),

[0596] A multispecific antibody is a multispecific antigen-binding molecule having at least one of the features selected from the group consisting of (a) to (j):

[0597] (a) Neutralizing PDGF-DD-induced PDGFR beta phosphorylation in vivo or in vitro,

[0598] (b) Neutralizing the combination of PDGF-BB and PDGF-DD-induced PDGFR beta phosphorylation in vivo or in vitro,

[0599] (c) In vivo or in vitro, selectively inhibits PDGF-DD-induced cell proliferation in NIH3T3 cells and / or IMR90 cells,

[0600] (d) selectively inhibiting the combination of PDGF-BB / DD-induced cell proliferation in NIH3T3 cells and / or IMR90 cells in vivo or in vitro,

[0601] (e) Selectively inhibiting PDGF-AB-induced PDGFR alpha phosphorylation in IMR90 cells in vivo or in vitro,

[0602] (f) Selectively inhibits PDGF-AB-induced cell proliferation in IMR90 cells in vivo or in vitro,

[0603] (g) Selectively inhibits the combination of PDGF-BB, PDGF-DD, and PDGF-AB-induced cell proliferation in IMR90 cells in vivo or in vitro,

[0604] (h) 10 hPDGF-BB and hPDGF-DD -7 M or less, even more preferably 10 -8 M or less, even more preferably 10 -9 M or less, and most preferably 10 -8 M to 10 -10 Combined with KD of M or less,

[0605] (i) Human, mouse, synomolgus, and rat may cross-reactively bind to PDGF-BB and / or PDGF-DD, or

[0606] (j) Fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma, and retinitis pigmentosa (RP), preferably chronic It can inhibit, suppress, treat, or prevent kidney disease (CKD), particularly glomerulonephritis accompanied by vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis in vivo.

[0607] (B-70)

[0608] In any one of (B-1) to (B-69),

[0609] A multispecific antigen-binding molecule comprising a Fab, a single-stranded Fv(scFv), a single-stranded antibody, Fv, a single-stranded Fv2(scFv2), or a F(ab')2 structure.

[0610] (B-71)

[0611] A pharmaceutical composition comprising any one of (B-1) to (B-70) a multispecific antigen-binding molecule.

[0612] (B-72)

[0613] Optionally, fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma, and retinitis pigmentosa (RP), preferably chronic kidney A pharmaceutical composition of any one of (B-1) to (B-70) or (B-71) for use in a therapy for the treatment of a disease (CKD), particularly glomerulonephritis accompanied by vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis.

[0614] (B-73)

[0615] A method for treating an individual having a pathological condition comprising the step of administering to the individual an effective amount of a pharmaceutical composition of any one of (B-1) to (B-70) multispecific antigen-binding molecules or (B-71), wherein the pathological condition is fibrosis (e.g., cardiomyofibrosis, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related disease, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, A method for stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-associated age-related macular degeneration (NVAMD), glaucoma and retinitis pigmentosa (RP), preferably chronic kidney disease (CKD), particularly glomerulonephritis with vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis.

[0616] (B-74)

[0617] In the manufacture of a drug for treatment or prevention, a multispecific antigen-binding molecule of any one of (B-1) to (B-70) or a pharmaceutical composition of (B-71) is used,

[0618] Fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma, and retinitis pigmentosa (RP), preferably chronic kidney Use of a multispecific antigen-binding molecule or pharmaceutical composition in the manufacture of a drug for the treatment or prevention of disease (CKD), particularly glomerulonephritis accompanied by vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis.

[0619] (B-75)

[0620] An isolated polynucleotide or a plurality of polynucleotides encoding any one of (B-1) to (B-70) a multispecific antigen-binding molecule.

[0621] (B-76)

[0622] A host cell comprising a polynucleotide of (B-75) or a plurality of polynucleotides.

[0623] (B-77)

[0624] A method for generating any one of (B-1) to (B-70) multiple specific antigen-binding molecules,

[0625] A method comprising (a) culturing host cells of (B-76) under conditions suitable for the expression of a multispecific antigen-binding molecule and (b) recovering the multispecific antigen-binding molecule.

[0626] (C-1)

[0627] As a multispecific antigen-binding molecule,

[0628] It includes a first antigen-binding domain and a second antigen-binding domain, and

[0629] Herein, the first antigen binding domain comprises an antibody heavy chain variable region comprising a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence presented in SEQ ID NO. 292, a heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 293, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 294, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 300, a light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 301, and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 302;

[0630] A multispecific antigen-binding molecule comprising, wherein the second antigen-binding domain comprises an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 226, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 227, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 228, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 250, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 251, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 252.

[0631] (C-2)

[0632] In (C-1),

[0633] The first antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 291, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 299;

[0634] A multispecific antigen-binding molecule comprising a second antigen-binding domain comprising a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 225, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 249.

[0635] (C-3)

[0636] In any one of (C-1) to (C-2),

[0637] A multispecific antigen-binding molecule, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises a heavy chain constant region and a light chain constant region, optionally wherein the heavy chain constant regions of the first antigen-binding domain and the second antigen-binding domain associate to form an antibody Fc domain.

[0638] (C-4)

[0639] In (C-3),

[0640] A multispecific antigen-binding molecule in which the antibody Fc domain is a human Fc domain derived from IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

[0641] (C-5)

[0642] In (C-3) or (C-4),

[0643] A multispecific antigen-binding molecule comprising one or more of the following amino acid residues (i) to (xviii):

[0644] (i) Glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region;

[0645] (ii) Glutamic acid at position 147 (EU numbering) in the heavy chain constant region;

[0646] (iii) Glutamic acid or lysine at position 131 (EU numbering) in the light chain constant region;

[0647] (iv) Asparagine at position 145 (EU numbering) in the light chain invariant region;

[0648] (v) Lee Sin at position 152 (EU numbering) within the light chain invariant zone;

[0649] (vi) Glutamic acid or lysine at position 160 (EU numbering) in the light chain constant region;

[0650] (vii) Lee Sin at position 196 (EU numbering) within the heavy chain invariant region;

[0651] (viii) Arginine at position 235 (EU numbering) in the heavy chain constant region;

[0652] (ix) Arginine at position 236 (EU numbering) in the heavy chain constant region;

[0653] (x) Lee Sin at position 356 (EU numbering) within the heavy chain invariant region;

[0654] (xi) Lee Sin at position 399 (EU numbering) within the heavy chain invariant region;

[0655] (xii) Glutamic acid at position 409 (EU numbering) in the heavy chain constant region;

[0656] (xiii) Glutamic acid at position 419 (EU numbering) in the heavy chain constant region;

[0657] (xiv) Leucine at position 428 (EU numbering) in the heavy chain invariant region;

[0658] (xv) Alanine at position 434 (EU numbering) in the heavy chain invariant region;

[0659] (xvi) Arginine at position 438 (EU numbering) in the heavy chain constant region;

[0660] (xvii) Glutamic acid at position 439 (EU numbering) in the heavy chain constant region;

[0661] (xviii) Glutamic acid at position 440 (EU numbering) in the heavy chain constant region.

[0662] (C-6)

[0663] In any one of (C-3) to (C-5),

[0664] The heavy chain invariant region and light chain invariant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (f):

[0665] (a) A first amino acid residue in the heavy chain constant region (CH1) at position 147 according to EU numbering, and a second amino acid residue in the light chain constant region (CL) at position 131 according to EU numbering,

[0666] (b) a first amino acid residue in CH1 at position 147 according to EU numbering, and a second amino acid residue in CL at position 160 according to Kabat numbering,

[0667] (c) A first amino acid residue in CH1 at position 175 according to EU numbering, and a second amino acid residue in CL at position 131 according to EU numbering,

[0668] (d) a first amino acid residue in CH1 at position 175 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0669] (e) a first amino acid residue in CH1 at position 196 according to EU numbering, and a second amino acid residue in CL at position 131 according to EU numbering, and

[0670] (f) A first amino acid residue in CH1 at position 196 according to EU numbering, and a second amino acid residue in CL at position 160 according to EU numbering,

[0671] A multispecific antigen-binding molecule in which the first amino acid residue and the second amino acid residue are oppositely charged amino acid residues.

[0672] (C-7)

[0673] In any one of (C-3) to (C-6),

[0674] A multispecific antigen-binding molecule wherein the heavy chain invariant region and the light chain invariant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (d):

[0675] (a) The heavy chain constant region of the first antigen-binding domain comprises at least one of Glu at position 147, Glu at position 175, and Lys at position 196 (EU numbering),

[0676] (b) The light chain constant region of the first antigen-binding domain comprises at least one of Lys at position 131 and Lys at position 160 (EU numbering),

[0677] (c) The heavy chain constant region of the second antigen-binding domain comprises at least one of Lys (EU numbering) at position 175, and

[0678] (d) The light chain constant region of the second antigen-binding domain comprises at least one of Glu at position 131, Lys at position 152, and Glu at position 160 (EU numbering),

[0679] (C-8)

[0680] In any one of (C-3) to (C-7),

[0681] A multispecific antigen-binding molecule wherein the first heavy chain constant region and / or the second heavy chain constant region comprises an amino acid substitution at at least one of the amino acid positions (according to EU numbering) of the group consisting of the following:

[0682] (a) Location 356 of the first heavy chain invariant region and location 439 of the second heavy chain invariant region,

[0683] (b) Location 439 of the first heavy chain invariant region and location 356 of the second heavy chain invariant region,

[0684] (c) Location 357 of the first heavy chain invariant region and location 370 of the second heavy chain invariant region,

[0685] (d) Location 370 of the first heavy chain invariant region and location 357 of the second heavy chain invariant region,

[0686] (e) the location 399 of the first heavy chain invariant region and the location 409 of the second heavy chain invariant region, and

[0687] (f) Location 409 of the first heavy chain invariant region and location 399 of the second heavy chain invariant region.

[0688] (C-9)

[0689] In any one of (C-3) to (C-8),

[0690] The first heavy chain invariant region contains Lys at position 356 (EU numbering), and

[0691] A multispecific antigen-binding molecule in which the second heavy chain constant region comprises at least one of Glu at position 419 and Glu at position 439 (EU numbering).

[0692] (C-10)

[0693] In any one of (C-3) to (C-9),

[0694] A multispecific antigen-binding molecule in which the antibody Fc domain comprises at least one of Arg at position 235 and Arg at position 236 (according to EU numbering).

[0695] (C-11)

[0696] In any one of (C-3) to (C-10),

[0697] A multispecific antigen-binding molecule in which the antibody Fc domain comprises at least one of Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (according to EU numbering).

[0698] (C-12)

[0699] As a multispecific antigen-binding molecule,

[0700] A multispecific antigen-binding molecule comprising a first heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 368, a first light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 369, a second heavy chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 366, and a second light chain comprising or composed of the amino acid sequence presented in SEQ ID NO. 367.

[0701] (C-13)

[0702] As a multispecific antigen-binding molecule,

[0703] A first heavy chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 368; a first light chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 369; and a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 366. A multispecific antigen-binding molecule comprising a second heavy chain comprising or composed of an amino acid sequence having identity, and a second light chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 367.

[0704] (C-14)

[0705] As a multispecific antigen-binding molecule,

[0706] A first heavy chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 372; a first light chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 373; and a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 370. A multispecific antigen-binding molecule comprising a second heavy chain comprising or composed of an amino acid sequence having identity, and a second light chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 371.

[0707] (C-15)

[0708] As a multispecific antigen-binding molecule,

[0709] A first heavy chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 376; a first light chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 377; and a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 374. A multispecific antigen-binding molecule comprising a second heavy chain comprising or composed of an amino acid sequence having identity, and a second light chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 375.

[0710] (C-16)

[0711] As a multispecific antigen-binding molecule,

[0712] A first heavy chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 380; a first light chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 381; and a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 378. A multispecific antigen-binding molecule comprising a second heavy chain comprising or composed of an amino acid sequence having identity, and a second light chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 379.

[0713] (C-17)

[0714] As a multispecific antigen-binding molecule,

[0715] A first heavy chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 384; a first light chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 385; and a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 382. A multispecific antigen-binding molecule comprising a second heavy chain comprising or composed of an amino acid sequence having identity, and a second light chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 383.

[0716] (C-18)

[0717] As a multispecific antigen-binding molecule,

[0718] A first heavy chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 388; a first light chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 389; and a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 386. A multispecific antigen-binding molecule comprising a second heavy chain comprising or composed of an amino acid sequence having identity, and a second light chain comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 387.

[0719] (C-19)

[0720] In any one of (C-12) to (C-18),

[0721] A multispecific antigen-binding molecule in which a first light chain and a first heavy chain are contained within a first antigen-binding domain that specifically binds to PDGF-D (preferably human PDGF-D), and a second light chain and a second heavy chain are contained within a second antigen-binding domain that specifically binds to PDGF-B (preferably human PDGF-B).

[0722] (C-20)

[0723] In any one of (C-1) to (C-11),

[0724] A multispecific antigen-binding molecule in which a first antigen-binding domain specifically binds to PDGF-D (preferably human PDGF-D) and a second antigen-binding domain specifically binds to PDGF-B (preferably human PDGF-B).

[0725] (C-20a)

[0726] A multispecific antigen-binding molecule that binds to the same epitope of PDGF-B and / or PDGF-D as that of any one of (C-1) to (C-20).

[0727] (C-20b)

[0728] A multispecific antigen-binding molecule competing with any one of (C-1) to (C-20) to bind to PDGF-B and / or PDGF-D proteins.

[0729] (C-21)

[0730] In any one of (C-1) to (C-20),

[0731] A multispecific antigen-binding molecule comprising a protein-L non-binding motif in which the second antigen-binding domain selectively includes human variable kappa subgroup 2 or any subgroup of human or mouse variable lambda.

[0732] (C-22)

[0733] In either (C-1) or (C-21),

[0734] A multispecific antigen-binding molecule in which the second antigen-binding domain comprises framework region 1 of human variable kappa subgroup 2.

[0735] (C-23)

[0736] In any one of (C-1) to (C-22),

[0737] A multispecific antibody is a multispecific antigen-binding molecule having at least one of the features selected from the group consisting of (a) to (j):

[0738] (a) Neutralizing PDGF-DD-induced PDGFR beta phosphorylation in vivo or in vitro,

[0739] (b) Neutralizing the combination of PDGF-BB and PDGF-DD-induced PDGFR beta phosphorylation in vivo or in vitro,

[0740] (c) In vivo or in vitro, selectively inhibits PDGF-DD-induced cell proliferation in NIH3T3 cells and / or IMR90 cells,

[0741] (d) selectively inhibiting the combination of PDGF-BB / DD-induced cell proliferation in NIH3T3 cells and / or IMR90 cells in vivo or in vitro,

[0742] (e) Selectively inhibiting PDGF-AB-induced PDGFR alpha phosphorylation in IMR90 cells in vivo or in vitro,

[0743] (f) Selectively inhibits PDGF-AB-induced cell proliferation in IMR90 cells in vivo or in vitro,

[0744] (g) Selectively inhibits the combination of PDGF-BB, PDGF-DD, and PDGF-AB-induced cell proliferation in IMR90 cells in vivo or in vitro,

[0745] (h) 10 hPDGF-BB and hPDGF-DD -7 M or less, even more preferably 10 -8 M or less, even more preferably 10 -9 M or less, and most preferably 10 -8 M to 10 -10 Combined with KD of M or less,

[0746] (i) Human, mouse, synomolgus, and rat may cross-reactively bind to PDGF-BB and / or PDGF-DD, or

[0747] (j) Fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma, and retinitis pigmentosa (RP), preferably chronic It can inhibit, suppress, treat, or prevent kidney disease (CKD), particularly glomerulonephritis accompanied by vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis in vivo.

[0748] (C-24)

[0749] In any one of (C-1) to (C-23),

[0750] A multispecific antigen-binding molecule comprising a Fab, a single-stranded Fv(scFv), a single-stranded antibody, Fv, a single-stranded Fv2(scFv2), or a F(ab')2 structure.

[0751] (C-25)

[0752] A pharmaceutical composition comprising any one of (C-1) to (C-24) a multispecific antigen-binding molecule.

[0753] (C-26)

[0754] As a pharmaceutical composition of any one of (C-1) to (C-24) multispecific antigen-binding molecules or (C-25) for use in therapy,

[0755] Optionally, fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma, and retinitis pigmentosa (RP), preferably chronic kidney A multispecific antigen-binding molecule or pharmaceutical composition for use in the treatment of diseases (CKD), particularly glomerulonephritis accompanied by vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis.

[0756] (C-27)

[0757] A method for treating an individual having a pathological condition, comprising the step of administering to the individual an effective amount of a pharmaceutical composition of any one of (C-1) to (C-24) multispecific antigen-binding molecules or (C-25), wherein the pathological condition is fibrosis (e.g., cardiomyofibrosis, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related disease, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, A method for stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-associated age-related macular degeneration (NVAMD), glaucoma and retinitis pigmentosa (RP), preferably chronic kidney disease (CKD), particularly glomerulonephritis with vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis.

[0758] (C-28)

[0759] In the manufacture of a drug for treatment or prevention, a multispecific antigen-binding molecule of any one of (C-1) to (C-24) or a pharmaceutical composition of (C-25) is used,

[0760] Fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma, and retinitis pigmentosa (RP), preferably chronic kidney Use of a multispecific antigen-binding molecule or pharmaceutical composition in the manufacture of a drug for the treatment or prevention of disease (CKD), particularly glomerulonephritis accompanied by vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis.

[0761] (C-29)

[0762] An isolated polynucleotide or a plurality of polynucleotides encoding any one of (C-1) to (C-24) a multispecific antigen-binding molecule.

[0763] (C-30)

[0764] A host cell comprising a polynucleotide of (C-29) or a plurality of polynucleotides.

[0765] (C-31)

[0766] A method for generating any one of (C-1) to (C-24) a multispecific antigen-binding molecule,

[0767] A method comprising (a) culturing host cells of (C-30) under conditions suitable for the expression of a multispecific antigen-binding molecule and (b) recovering the multispecific antigen-binding molecule.

[0768] (D-1)

[0769] As an antigen-binding molecule,

[0770] An antigen-binding molecule comprising an antibody heavy chain variable region comprising a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence presented in SEQ No. 292, a heavy chain CDR 2 having the amino acid sequence presented in SEQ No. 293, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ No. 294, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ No. 300, a light chain CDR 2 having the amino acid sequence presented in SEQ No. 301, and a light chain CDR 3 having the amino acid sequence presented in SEQ No. 302.

[0771] (D-2)

[0772] As an antigen-binding molecule,

[0773] An antigen-binding molecule comprising a heavy chain variable region comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences presented in Table 1, and a light chain variable region comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences presented in Table 1.

[0774] (D-3)

[0775] As an antigen-binding molecule,

[0776] It includes a heavy chain variable region comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences presented in Table 1, and a light chain variable region comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences presented in Table 1, wherein

[0777] An antigen-binding molecule in which at least one of the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 further comprises one or more conservative amino acid substitutions.

[0778] (D-4)

[0779] In any one of (D-1) to (D-3),

[0780] An antigen-binding molecule, wherein the heavy chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 291, and the light chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 299.

[0781] (D-5)

[0782] In any one of (D-1) to (D-4),

[0783] An antigen-binding molecule, wherein the heavy chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the VH amino acid sequence presented in Table 1, and the light chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the VL amino acid sequence presented in Table 1.

[0784] (D-6)

[0785] As an antigen-binding molecule,

[0786] An antigen-binding molecule comprising a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 291, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 299.

[0787] (D-7)

[0788] As an antigen-binding molecule,

[0789] An antigen-binding molecule comprising a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in Table 1, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in Table 1.

[0790] (D-8)

[0791] In any one of (D-1) to (D-7),

[0792] The antigen-binding molecule is an antigen-binding molecule that specifically binds to PDGF-D (preferably human PDGF-D).

[0793] (D-8a)

[0794] An antigen-binding molecule that binds to the same epitope of PDGF-D as that of any one of (D-1) to (D-8).

[0795] (D-8b)

[0796] An antigen-binding molecule that competes with any one of (D-1) to (D-8) to bind to the PDGF-D protein.

[0797] (D-9)

[0798] In any one of (D-1) to (D-8),

[0799] The antigen-binding molecule is an antigen-binding molecule that exhibits at least one of the features selected from the group consisting of (a) to (f):

[0800] (a) Neutralizing PDGF-DD-induced PDGFR beta phosphorylation in vivo or in vitro,

[0801] (b) selectively inhibiting PDGF-DD-induced cell proliferation in NIH3T3 cells and / or IMR90 cells in vivo or in vitro,

[0802] (c) Can block the function of human PDGF-DD,

[0803] (d) 10 hPDGF-DD -7 M or less, even more preferably 10 -8 M or less, even more preferably 10 -9 M or less, and most preferably 10 -8 M to 10 -10 Combined with KD of M or less,

[0804] (e) Can cross-reactively bind to human, mouse, synomolgus PDGF-DD, or

[0805] (f) Fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma and retinitis pigmentosa (RP), preferably chronic It can inhibit, suppress, treat, or prevent kidney disease (CKD), particularly glomerulonephritis accompanied by vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis in vivo.

[0806] (D-10)

[0807] In any one of (D-1) to (D-9),

[0808] An antigen-binding molecule further comprising at least one heavy chain invariant region and at least one light chain invariant region.

[0809] (D-11)

[0810] In (D-10),

[0811] The antigen-binding molecule comprises one or more of the following amino acid residues (i) to (xviii):

[0812] (i) Glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region;

[0813] (ii) Glutamic acid at position 147 (EU numbering) in the heavy chain constant region;

[0814] (iii) Glutamic acid or lysine at position 131 (EU numbering) in the light chain constant region;

[0815] (iv) Asparagine at position 145 (EU numbering) in the light chain invariant region;

[0816] (v) Lee Sin at position 152 (EU numbering) within the light chain invariant zone;

[0817] (vi) Glutamic acid or lysine at position 160 (EU numbering) in the light chain constant region;

[0818] (vii) Lee Sin at position 196 (EU numbering) within the heavy chain invariant region;

[0819] (viii) Arginine at position 235 (EU numbering) in the heavy chain constant region;

[0820] (ix) Arginine at position 236 (EU numbering) in the heavy chain constant region;

[0821] (x) Lee Sin at position 356 (EU numbering) within the heavy chain invariant region;

[0822] (xi) Lee Sin at position 399 (EU numbering) within the heavy chain invariant region;

[0823] (xii) Glutamic acid at position 409 (EU numbering) in the heavy chain constant region;

[0824] (xiii) Glutamic acid at position 419 (EU numbering) in the heavy chain constant region;

[0825] (xiv) Leucine at position 428 (EU numbering) in the heavy chain invariant region;

[0826] (xv) Alanine at position 434 (EU numbering) in the heavy chain invariant region;

[0827] (xvi) Arginine at position 438 (EU numbering) in the heavy chain constant region;

[0828] (xvii) Glutamic acid at position 439 (EU numbering) in the heavy chain constant region;

[0829] (xviii) Glutamic acid at position 440 (EU numbering) in the heavy chain constant region.

[0830] (D-12)

[0831] In (D-10) or (D-11),

[0832] An antigen-binding molecule in which at least one heavy chain constant region comprises Arg at position 235 and Arg at position 236 (according to EU numbering).

[0833] (D-13)

[0834] In any one of (D-10) to (D-12),

[0835] An antigen-binding molecule, wherein at least one heavy chain constant region comprises Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (according to EU numbering).

[0836] (E-1)

[0837] As an antigen-binding molecule,

[0838] It includes an antibody heavy chain variable region and an antibody light chain variable region, wherein

[0839] The antibody heavy chain variable region comprises any one of the following (a1) to (a2):

[0840] (a1) a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence presented in SEQ NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236; or

[0841] (a2) heavy chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 242, heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 243, and heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 244;

[0842] The antibody light chain variable region comprises any one of the following (b1) to (b4), an antigen-binding molecule:

[0843] (b1) Light chain CDR 1 having the amino acid sequence presented in SEQ NO. 258, light chain CDR 2 having the amino acid sequence presented in SEQ NO. 259, and light chain CDR 3 having the amino acid sequence presented in SEQ NO. 260,

[0844] (b2) Light chain CDR 1 having the amino acid sequence presented in SEQ NO. 266, light chain CDR 2 having the amino acid sequence presented in SEQ NO. 267, and light chain CDR 3 having the amino acid sequence presented in SEQ NO. 268,

[0845] (b3) light chain CDR 1 having the amino acid sequence presented in SEQ NO. 274, light chain CDR 2 having the amino acid sequence presented in SEQ NO. 275, and light chain CDR 3 having the amino acid sequence presented in SEQ NO. 276, or

[0846] (b4) Light chain CDR 1 having the amino acid sequence presented in SEQ No. 282, light chain CDR 2 having the amino acid sequence presented in SEQ No. 283, and light chain CDR 3 having the amino acid sequence presented in SEQ No. 284.

[0847] (E-2)

[0848] In (E-1),

[0849] Antigen-binding molecule comprising any one of the following (a) to (e):

[0850] (a) a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236, a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 258, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 259, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 260,

[0851] (b) heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 234, heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236, light chain CDR 1 having the amino acid sequence presented in SEQ NO. 266, light chain CDR 2 having the amino acid sequence presented in SEQ NO. 267, and light chain CDR 3 having the amino acid sequence presented in SEQ NO. 268,

[0852] (c) heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 234, heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236, light chain CDR 1 having the amino acid sequence presented in SEQ NO. 274, light chain CDR 2 having the amino acid sequence presented in SEQ NO. 275, and light chain CDR 3 having the amino acid sequence presented in SEQ NO. 276,

[0853] (d) a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 242, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 243, a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 244, a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 274, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 275, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 276, or

[0854] (e) heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 234, heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236, light chain CDR 1 having the amino acid sequence presented in SEQ NO. 282, light chain CDR 2 having the amino acid sequence presented in SEQ NO. 283, and light chain CDR 3 having the amino acid sequence presented in SEQ NO. 284.

[0855] (E-3)

[0856] In (E-1) or (E-2),

[0857] An antigen-binding molecule in which at least one of the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 further comprises one or more conservative amino acid substitutions.

[0858] (E-4)

[0859] In any one of (E-1) to (E-3),

[0860] The heavy chain variable region comprises any one of the following (a1) to (a2):

[0861] (a1) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 233, and

[0862] (a2) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 241;

[0863] Herein, the antibody light chain variable region comprises any one of the following (b1) to (b4), an antigen-binding molecule:

[0864] (b1) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 257,

[0865] (b2) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 265,

[0866] (b3) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 273, and

[0867] (b4) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence described in SEQ ID NO. 281.

[0868] (E-5)

[0869] In any one of (E-1) to (E-4),

[0870] Antigen-binding molecule comprising any one of the following (a1) to (a5):

[0871] (a1) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 257;

[0872] (a2) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 265;

[0873] (a3) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 273;

[0874] (a4) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 241, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 273; and

[0875] (a5) A heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 281.

[0876] (E-6)

[0877] Antigen-binding molecule comprising any one of the following (a1) to (a5):

[0878] (a1) a heavy chain variable region comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 257;

[0879] (a2) a heavy chain variable region comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 265;

[0880] (a3) a heavy chain variable region comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 273;

[0881] (a4) a heavy chain variable region comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 241, and a light chain variable region comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 273; and

[0882] (a5) a heavy chain variable region comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region comprising or composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 281.

[0883] (E-7)

[0884] In any one of (E-1) to (E-6),

[0885] The antigen-binding molecule is an antigen-binding molecule that specifically binds to PDGF-B (preferably human PDGF-B).

[0886] (E-7a)

[0887] An antigen-binding molecule that binds to the same epitope of PDGF-B as that of any one of (E-1) to (E-7).

[0888] (E-7b)

[0889] An antigen-binding molecule that competes with any one of (E-1) to (E-7) antigen-binding molecules to bind to PDGF-B protein.

[0890] (E-8)

[0891] In any one of (E-1) to (E-7),

[0892] The antigen-binding molecule exhibits at least one of the features selected from the group consisting of (a) to (d):

[0893] (a) Can cross-reactively bind to PDGF-BB in humans, mice, synomolgus, and rats,

[0894] (b) 10 hPDGF-BB -7 M or less, even more preferably 10 -8 M or less, even more preferably 10 -9 M or less, and most preferably 10 -8 M to 10 -10 Combined with KD of M or less,

[0895] (c) May block the function of human PDGF-AB and / or PDGF-BB, or

[0896] (d) Fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma, and retinitis pigmentosa (RP), preferably chronic It can inhibit, suppress, treat, or prevent kidney disease (CKD), particularly glomerulonephritis accompanied by vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis in vivo.

[0897] (E-9)

[0898] In any one of (E-1) to (E-8),

[0899] An antigen-binding molecule further comprising at least one heavy chain invariant region and at least one light chain invariant region.

[0900] (E-10)

[0901] In (E-9),

[0902] The antigen-binding molecule comprises one or more of the following amino acid residues (i) to (xviii):

[0903] (i) Glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region;

[0904] (ii) Glutamic acid at position 147 (EU numbering) in the heavy chain constant region;

[0905] (iii) Glutamic acid or lysine at position 131 (EU numbering) in the light chain constant region;

[0906] (iv) Asparagine at position 145 (EU numbering) in the light chain invariant region;

[0907] (v) Lee Sin at position 152 (EU numbering) within the light chain invariant zone;

[0908] (vi) Glutamic acid or lysine at position 160 (EU numbering) in the light chain constant region;

[0909] (vii) Lee Sin at position 196 (EU numbering) within the heavy chain invariant region;

[0910] (viii) Arginine at position 235 (EU numbering) in the heavy chain constant region;

[0911] (ix) Arginine at position 236 (EU numbering) in the heavy chain constant region;

[0912] (x) Lee Sin at position 356 (EU numbering) within the heavy chain invariant region;

[0913] (xi) Lee Sin at position 399 (EU numbering) within the heavy chain invariant region;

[0914] (xii) Glutamic acid at position 409 (EU numbering) in the heavy chain constant region;

[0915] (xiii) Glutamic acid at position 419 (EU numbering) in the heavy chain constant region;

[0916] (xiv) Leucine at position 428 (EU numbering) in the heavy chain invariant region;

[0917] (xv) Alanine at position 434 (EU numbering) in the heavy chain invariant region;

[0918] (xvi) Arginine at position 438 (EU numbering) in the heavy chain constant region;

[0919] (xvii) Glutamic acid at position 439 (EU numbering) in the heavy chain constant region;

[0920] (xviii) Glutamic acid at position 440 (EU numbering) in the heavy chain constant region.

[0921] (E-11)

[0922] In any one of (D-1) to (E-10),

[0923] The antigen-binding molecule is an antigen-binding molecule comprising Fab, single-chain Fv(scFv), single-chain antibody, Fv, single-chain Fv2(scFv2), or F(ab')2 structure.

[0924] (E-12)

[0925] A pharmaceutical composition comprising any one of the antigen-binding molecules (D-1) to (E-11).

[0926] (E-13)

[0927] As a pharmaceutical composition of any one of (D-1) to (E-11) antigen-binding molecules or (E-12) for use in therapy,

[0928] Optionally, fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma, and retinitis pigmentosa (RP), preferably chronic kidney An antigen-binding molecule or pharmaceutical composition for use in vivo in the treatment of diseases (CKD), particularly glomerulonephritis accompanied by vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis.

[0929] (E-14)

[0930] A method for treating an individual having a pathological condition, comprising the step of administering to the individual an effective amount of an antigen-binding molecule of any one of (D-1) to (E-11) or a pharmaceutical composition of (E-12), wherein the pathological condition is fibrosis (e.g., cardiomyofibrosis, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related disease, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, ocular A method comprising a disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-associated age-related macular degeneration (NVAMD), glaucoma and retinitis pigmentosa (RP), preferably chronic kidney disease (CKD), particularly glomerulonephritis with vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis.

[0931] (E-15)

[0932] As a use of any one of the antigen-binding molecules of (D-1) to (E-11) or a pharmaceutical composition of (E-12) in the manufacture of a drug for treatment or prevention,

[0933] Fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma, and retinitis pigmentosa (RP), preferably chronic kidney Use of a multispecific antigen-binding molecule or pharmaceutical composition in the manufacture of a drug for the treatment or prevention of disease (CKD), particularly glomerulonephritis accompanied by vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis.

[0934] (E-16)

[0935] An isolated polynucleotide or a plurality of polynucleotides encoding any one of (D-1) to (E-11) antigen-binding molecules.

[0936] (E-17)

[0937] A host cell comprising a polynucleotide of (E-16) or a plurality of polynucleotides.

[0938] (E-18)

[0939] A method for generating any one of the antigen-binding molecules (D-1) to (E-11),

[0940] A method comprising (a) culturing host cells of (E-17) under conditions suitable for the expression of antigen-binding molecules and (b) recovering antigen-binding molecules. Brief explanation of the drawing

[0941] Fig. 1 Figure 1 relates to the evaluation of the neutralizing activity of monoclonal antibodies against both human and mouse PDGF-DD in a cell-based receptor tyrosine kinase (PDGFR beta) activity assay. Fig. 2 Figure 2 relates to the evaluation of the neutralizing activity of monoclonal antibodies against both human and mouse PDGF-DD in the BrdU cell proliferation assay in human and mouse fibroblasts. Fig. 3 Figure 3 shows the results of the PDGFR beta-kinase activity assay. PNC0004, PNC0019, PNC0024, PNC0089, PNC0139, PNC0154, and CR002 are anti-PDGF-D antibodies. IC17 is an anti-KLH antibody used as a negative control. NT (untreated) are cells not treated with ligands and antibodies. Fig. 4 Figure 4 shows the results of the BrdU cell proliferation assay in the mouse fibroblast cell line NIH3T3. PNC0004, PNC0019, PNC0024, PNC0089, PNC0139, PNC0154, and CR002 are anti-PDGF-D antibodies. IC17 is an anti-KLH antibody used as a negative control. NT (untreated) are cells not treated with ligands and antibodies. Fig. 5 Figure 5 shows the results of the BrdU cell proliferation assay in the human fibroblast cell line IMR90. PNC0004, PNC0019, PNC0024, PNC0089, PNC0139, PNC0154, and CR002 are anti-PDGF-D antibodies. IC17 is an anti-KLH antibody used as a negative control. NT (untreated) are cells not treated with ligands and antibodies. Fig. 6a Figure 6a illustrates the physicochemical properties of the bispecific antibodies Ro006vk2 / / hPNC139-SG191FAST, Ro006vk2AM1 / / hPNC139-SG191FAST, Ro006vk2AM3 / / hPNC139-SG191FAST, Ro006vk2AM5 / / hPNC139-SG191FAST, Ro006vk2AM7 / / hPNC139-SG191FAST and Ro006vk2AM8 / / hPNC139-SG191FAST. Fig. 6b Figure 6b illustrates the physicochemical properties of the bispecific antibodies Ro006vk2 / / hPNC139-SG191FAST, Ro006vk2AM1 / / hPNC139-SG191FAST, Ro006vk2AM3 / / hPNC139-SG191FAST, Ro006vk2AM5 / / hPNC139-SG191FAST, Ro006vk2AM7 / / hPNC139-SG191FAST and Ro006vk2AM8 / / hPNC139-SG191FAST. Fig. 6c Figure 6c illustrates the physicochemical properties of the bispecific antibodies Ro006vk2 / / hPNC139-SG191FAST, Ro006vk2AM1 / / hPNC139-SG191FAST, Ro006vk2AM3 / / hPNC139-SG191FAST, Ro006vk2AM5 / / hPNC139-SG191FAST, Ro006vk2AM7 / / hPNC139-SG191FAST and Ro006vk2AM8 / / hPNC139-SG191FAST. Fig. 7 Figure 7 shows the results of Biacore binding analysis of Ro006vk2AM1 / / hPNC139-SG191FAST for PDGF-BB and PDGF-DD from different species. Fig. 8 Figure 8 shows the results of immunogenicity data (FIL). Fig. 9 Figure 9 shows the results of the PDGFR beta-kinase activity assay. CPR001 / / CR002, Ro006vk2 / / hPNC139-SG191FAST, Ro006vk2AM1 / / hPNC139-SG191FAST, Ro006vk2AM3 / / hPNC139-SG191FAST, Ro006vk2AM5 / / hPNC139-SG191FAST, Ro006vk2AM7 / / hPNC139-SG191FAST, and Ro006vk2AM8 / / hPNC139-SG191FAST are anti-PDGF-B / D bispecific antibodies. IC17 is an anti-KLH antibody used as a negative control. Fig. 10 Figure 10 shows the results of the PDGFR beta-kinase activity assay. CPR001 / / CR002, Ro006vk2 / / hPNC139-SG191FAST, Ro006vk2AM1 / / hPNC139-SG191FAST, Ro006vk2AM3 / / hPNC139-SG191FAST, Ro006vk2AM5 / / hPNC139-SG191FAST, Ro006vk2AM7 / / hPNC139-SG191FAST, and Ro006vk2AM8 / / hPNC139-SG191FAST are anti-PDGF-B / D bispecific antibodies. IC17 is an anti-KLH antibody used as a negative control. Fig. 11 Figure 11 shows the results of the BrdU cell proliferation assay in mouse fibroblast NIH3T3. CPR001 / / CR002, Ro006vk2 / / hPNC139-SG191FAST, Ro006vk2AM1 / / hPNC139-SG191FAST, Ro006vk2AM3 / / hPNC139-SG191FAST, Ro006vk2AM5 / / hPNC139-SG191FAST, Ro006vk2AM7 / / hPNC139-SG191FAST, and Ro006vk2AM8 / / hPNC139-SG191FAST are anti-PDGF-B / D bispecific antibodies. IC17 is an anti-KLH antibody used as a negative control. Fig. 12 Figure 12 shows the results of the BrdU cell proliferation assay in human fibroblast IMR90. CPR001 / / CR002, Ro006vk2 / / hPNC139-SG191FAST, Ro006vk2AM1 / / hPNC139-SG191FAST, Ro006vk2AM3 / / hPNC139-SG191FAST, Ro006vk2AM5 / / hPNC139-SG191FAST, Ro006vk2AM7 / / hPNC139-SG191FAST, and Ro006vk2AM8 / / hPNC139-SG191FAST are anti-PDGF-B / D bispecific antibodies. IC17 is an anti-KLH antibody used as a negative control. Fig. 13 Figure 13 shows the results of the BrdU cell proliferation assay in mouse fibroblast NIH3T3. CPR001 / / CR002, Ro006vk2 / / hPNC139-SG191FAST, Ro006vk2AM1 / / hPNC139-SG191FAST, Ro006vk2AM3 / / hPNC139-SG191FAST, Ro006vk2AM5 / / hPNC139-SG191FAST, Ro006vk2AM7 / / hPNC139-SG191FAST, and Ro006vk2AM8 / / hPNC139-SG191FAST are anti-PDGF-B / D bispecific antibodies. IC17 is an anti-KLH antibody used as a negative control. Fig. 14 Figure 14 shows the results of the BrdU cell proliferation assay in human fibroblast IMR90. CPR001 / / CR002, Ro006vk2 / / hPNC139-SG191FAST, Ro006vk2AM1 / / hPNC139-SG191FAST, Ro006vk2AM3 / / hPNC139-SG191FAST, Ro006vk2AM5 / / hPNC139-SG191FAST, Ro006vk2AM7 / / hPNC139-SG191FAST, and Ro006vk2AM8 / / hPNC139-SG191FAST are anti-PDGF-B / D bispecific antibodies. IC17 is an anti-KLH antibody used as a negative control. Fig. 15 Figure 15 shows the results of the PDGFR alpha-kinase activity assay in human fibroblast IMR90. CPR001 / / CR002, Ro006vk2 / / hPNC139-SG191FAST, Ro006vk2AM1 / / hPNC139-SG191FAST, Ro006vk2AM3 / / hPNC139-SG191FAST, Ro006vk2AM5 / / hPNC139-SG191FAST, Ro006vk2AM7 / / hPNC139-SG191FAST, and Ro006vk2AM8 / / hPNC139-SG191FAST are anti-PDGF-B / D bispecific antibodies. IC17 is an anti-KLH antibody used as a negative control. Fig. 16 Figure 16 shows the results of the BrdU cell proliferation assay in human fibroblast IMR90. CPR001 / / CR002, Ro006vk2 / / hPNC139-SG191FAST, Ro006vk2AM1 / / hPNC139-SG191FAST, Ro006vk2AM3 / / hPNC139-SG191FAST, Ro006vk2AM5 / / hPNC139-SG191FAST, Ro006vk2AM7 / / hPNC139-SG191FAST, and Ro006vk2AM8 / / hPNC139-SG191FAST are anti-PDGF-B / D bispecific antibodies. IC17 is an anti-KLH antibody used as a negative control. Fig. 17 Figure 17 shows the results of the BrdU cell proliferation assay in human fibroblast IMR90. CPR001 / / CR002, Ro006vk2 / / hPNC139-SG191FAST, Ro006vk2AM1 / / hPNC139-SG191FAST, Ro006vk2AM3 / / hPNC139-SG191FAST, Ro006vk2AM5 / / hPNC139-SG191FAST, Ro006vk2AM7 / / hPNC139-SG191FAST, and Ro006vk2AM8 / / hPNC139-SG191FAST are anti-PDGF-B / D bispecific antibodies. IC17 is an anti-KLH antibody used as a negative control. Fig. 18 Figure 18 illustrates the results of evaluating collagen type 1 alpha 1 (Col1a1) mRNA levels and hydroxyproline content in the kidney. The efficacy of bispecific antibodies (Ro006vk2 / / hPNC139-SG191FAST, Ro006vk2AM1 / / hPNC139-SG191FAST, Ro006vk2AM3 / / hPNC139-SG191FAST, Ro006vk2AM5 / / hPNC139-SG191FAST, Ro006vk2AM7 / / hPNC139-SG191FAST, and Ro006vk2AM8 / / hPNC139-SG191FAST) was evaluated in a mouse renal fibrosis model induced by unilateral ureteral obstruction (UUO). The sham surgery group represents the non-disease-induced control group. Col1a1 mRNA and renal fibrosis were inhibited by treatment with the anti-PDGF B / D bispecific antibody. IC17dK is an anti-KLH antibody used as a negative control. Specific details for implementing the invention

[0942] General technology

[0943] The techniques and procedures described or referenced herein are generally understood and used by those skilled in the art in ordinary ways, such as the widely used methods described below: Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988); Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).

[0944] definition

[0945] amino acids

[0946] The following definitions and detailed descriptions are provided to facilitate understanding of the invention as illustrated in this specification.

[0947] In the present invention, amino acids are described using three-letter or one-letter codes or both, for example, alanine is described as Ala / A, leucine as Leu / L, arginine as Arg / R, lysine as Lys / K, asparagine as Asn / N, methionine as Met / M, aspartic acid as Asp / D, phenylalanine as Phe / F, cysteine ​​as Cys / C, proline as Pro / P, glutamine as Gln / Q, serine as Ser / S, glutamic acid as Glu / E, threonine as Thr / T, glycine as Gly / G, tryptophan as Trp / W, histidine as His / H, tyrosine as Tyr / Y, isoleucine as Ile / I, or valine as Val / V.

[0948] Changes in amino acids

[0949] For amino acid modification in the amino acid sequence of an antigen-binding molecule, known methods, such as site-directed mutation methods (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and nested extension PCR, may be appropriately used. Additionally, several known methods for amino acid modification for substitution with non-natural amino acids may also be used (Annu Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, it is suitable to use a cell-free translation system (Clover Direct (Protein Express)) containing tRNA with a non-natural amino acid bound to an amber repressor tRNA complementary to the UAG codon (amber codon), which is one of the stop codons.

[0950] and / or

[0951] In this specification, when describing amino acid modification sites, the meaning of the term “and / or” includes all combinations in which “and” and “or” are suitably combined. Specifically, for example, “substituted with amino acids at positions 33, 55 and / or 96” includes the following amino acid modification variations: (a) position 33, (b) position 55, (c) position 96, (d) positions 33 and 55, (e) positions 33 and 96, (f) positions 55 and 96, and (g) amino acid modification(s) at positions 33, 55 and 96.

[0952] Additionally, as an abbreviated expression indicating a specific change in amino acids in this invention, an expression combining a number (indicating the position of the changed amino acid) and a one- or three-character amino acid code (indicating the amino acids before and after the change) may be appropriately used, and such an expression may be composed of the sequence of the code for the amino acid before the change - the number indicating the position - the code for the amino acid after the change. For example, expression N100bL or Asn100bLeu, used to refer to the substitution of an amino acid included in the antibody variable region, indicates the substitution of Asn with Leu at position 100b (according to Kavat numbering). That is, the number indicates the amino acid position according to Kavat numbering, the one- or three-character amino acid code preceding the number indicates the amino acid before the substitution, and the one- or three-character amino acid code following the number indicates the amino acid after the substitution. Similarly, expression P238D or Pro238Asp, used to refer to the substitution of an amino acid in the Fc region included in the antibody constant region, indicates the substitution of Pro at position 238 (according to Kavat numbering) with Asp. That is, the number indicates the amino acid position according to EU numbering, the one- or three-letter amino acid code written before the number indicates the amino acid before substitution, and the one- or three-letter amino acid code written after the number indicates the amino acid after substitution.

[0953] Antigen-binding molecules and multispecific antigen-binding molecules

[0954] As used herein, the term “antigen-binding molecule” refers to any molecule comprising one or more antigen-binding domains or any molecule having binding activity to an antigen, and may further refer to molecules such as peptides or proteins having a length of about 5 amino acids or more. Peptides and proteins are not limited to those derived from organisms and may, for example, be polypeptides produced from artificially designed sequences. They may also be naturally occurring polypeptides, synthetic polypeptides, recombinant polypeptides, etc.

[0955] An advantageous example of an antigen-binding molecule of the present invention is an antigen-binding molecule comprising a plurality of antigen-binding domains. In certain embodiments, the antigen-binding molecule of the present invention comprises two antigen-binding domains having the same antigen-binding specificity. In certain embodiments, the antigen-binding molecule of the present invention comprises two antigen-binding domains having the same antigen-binding specificity and an FcRn binding domain contained in the antibody Fc region. In certain embodiments, the antigen-binding molecule of the present invention comprises two antigen-binding domains having different antigen-binding specificities and an FcRn binding domain contained in the antibody Fc region. As a method for extending the blood half-life of a protein administered to a living organism, a method of adding an antibody's FcRn-binding domain to a protein of interest and utilizing the function of FcRn-mediated recycling is generally known. An antigen-binding molecule having two or more antigen-binding domains having different antigen-binding specificities may be referred to as a "multispecific antigen-binding molecule."

[0956] antigen binding domain

[0957] As used herein, the term "antigen binding domain" refers to a portion of an antibody, said portion specifically binding to all or part of an antigen and including a region complementary thereto. If the molecular weight of the antigen is large, the antibody may bind only to a specific portion of the antigen. The specific portion is referred to as an "epitope." The antigen binding domain may be provided from one or more antibody variable domains. Preferably, the antigen binding domain contains an antibody variable domain comprising both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Such preferred antigen binding domains include, for example, "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv2 (scFv2)", "Fab", and "F(ab')2".

[0958] In certain embodiments, one of the antigen-binding domains of the multispecific antigen-binding molecule of the present invention specifically binds to PDGF-D, and the other specifically binds to PDGF-B. In other words, PDGF-D and PDGF-B are each preferred antigens of interest.

[0959] In certain embodiments, the antigen-binding domain of the antigen-binding molecule of the present invention specifically binds to PDGF-D. In other words, PDGF-D is the preferred antigen of interest—the antigen-binding domain has binding activity to its antigen of interest, but substantially recognizes other molecules in a sample containing a mixed population of antigenic molecules and does not bind to them.

[0960] Binding activity

[0961] As used herein, the phrase “having binding activity” refers to the activity of an antigen-binding domain, antibody, antigen-binding molecule, antibody variable fragment, or similar (hereinafter “antigen-binding domain or similar”) that binds to an antigen of interest at a specific binding level higher than a non-specific or background binding level. That is, such antigen-binding domain, etc., “have specific / significant binding activity” with respect to the antigen of interest. Specificity may be measured by any method for detecting affinity or binding activity described herein or known in the art. The aforementioned specific binding level may be sufficiently high to be recognized as significant by a person skilled in the art. For example, when a person skilled in the art can detect or observe any significant or relatively strong signal or value regarding the binding between the antigen-binding domain, etc., and the antigen of interest in a suitable binding assay, it may be said that the antigen-binding domain, etc., has “specific / significant binding activity” with respect to the antigen of interest. Alternatively, "having specific / significant binding activity" may be rephrased as "specifically / significantly binding" (to the antigen of interest). At times, the phrase "having binding activity" has substantially the same meaning as "having specific / significant binding activity" in the art. As used herein, an antigen-binding molecule or antibody "specifically binding" to an antigen refers to an antigen-binding molecule or antibody that binds with high affinity to the antigen and substantially the same antigen, which, when measured by surface plasmon resonance assay or cell binding assay, 10 -7 M or less, preferably 10 -8 M or less, more preferably 10 -9 M or less, most preferably 10 -8 M to 10 -10 It means having a KD of M or less, but does not bind to unrelated antigens with high affinity.

[0962] In some embodiments, the binding activity or binding affinity (KD) of the antigen-binding domain of the present invention for the antigen of interest (i.e., PDGF-B or PDGF-D) is evaluated at 25°C using, for example, a Biacore T200 instrument (GE Healthcare). Anti-human Fc (e.g., GE Healthcare) is immobilized on all flow cells of the CM4 sensor chip using an amine coupling kit (e.g., GE Healthcare). The antigen-binding molecule or antigen-binding domain is captured on the surface of the anti-Fc sensor, and then the antigen (e.g., PDGF-B or PDGF-D) is injected across the flow cells. All antigen-binding domains and analytes are prepared in PBS-NET (10 mM phosphate, 287 mM NaCl, 2.7 mM KCl, 3.2 mM EDTA, 0.01% P2O, 0.005% NaN3, pH 7.4). The sensor surface is regenerated with 3M MgCl2 in each cycle. Binding affinity is determined by processing data using, for example, Biacore T200 Evaluation software, version 2.0 (GE Healthcare) and fitting it to a 1:1 binding model.

[0963] PDGF-B and PDGF-D

[0964] In one embodiment, the antigen-binding molecule of the present invention is a multispecific antigen-binding molecule, preferably a multispecific antibody, comprising a first antigen-binding domain that specifically binds to PDGF-D (i.e., PDGF-D and PDGF-DD) and a second antigen-binding domain that specifically binds to PDGF-B (i.e., PDGF-B, PDGF-AB and PDGF-BB) and inhibits the interaction of PDGF-D and PDGF-B with PDGFR, thereby inhibiting the activity of PDGF-D and PDGF-B.

[0965] The term "PDGF-B" refers to any naturally occurring form of PDGF-B, which is either monomeric or dimeric, that may originate from any suitable organism. The term encompasses any dimeric form of PDGF-B, namely PDGF-AB and PDGF-BB. As used herein, "PDGF-B" refers to mammalian PDGF-B, e.g., human, rat, or mouse, as well as non-human primates, cattle, sheep, or pigs. Preferably, PDGF-B is human PDGF-B. The term "PDGF-B" also encompasses fragments, variants, isomorphs, and other conglomerates of such PDGF-B molecules. Variant PDGF-B molecules will generally be characterized by having the same type of activity as naturally occurring PDGF-B, such as the ability to bind to PDGFR, the ability to induce phosphorylation of the receptor, the ability to mediate signaling by such receptor, the ability to induce cell migration or proliferation, and the ability to induce or increase the deposition of the extracellular matrix. An exemplary amino acid sequence of human PDGF-B is presented in sequence number 172.

[0966] Similarly, the term “PDGF-D” refers to any naturally occurring form of PDGF-D, which is either monomeric or dimeric, that may originate from any suitable organism. The term encompasses PDGF-D, i.e., dimeric forms including PDGF-DD. As used herein, “PDGF-D” refers to mammalian PDGF-D, e.g., human, rat, or mouse, as well as non-human primates, cattle, sheep, or pig PDGF-D. Preferably, PDGF-B is human PDGF-D. The term “PDGF-D” also encompasses fragments, variants, isomorphs, and other conglomerates of such PDGF-B molecules. Variant PDGF-D molecules will generally be characterized by having the same type of activity as naturally occurring PDGF-D, such as the ability to bind to PDGFR, the ability to induce phosphorylation of the receptor, the ability to mediate signaling by such receptor, the ability to induce cell migration or proliferation, and the ability to induce or increase the deposition of the extracellular matrix. An exemplary amino acid sequence of human PDGF-D is presented in sequence number 171.

[0967] In one embodiment, the antigen-binding molecule of the present invention comprises an antigen-binding domain that specifically binds to PDGF-D (e.g., PDGF-D and PDGF-DD). Accordingly, the antigen-binding molecule of the present invention binds to PDGF-D and inhibits interaction with PDGFR, thereby inhibiting PDGF-D activity. The terms "PDGF-D mediated activity," "PDGF-D mediated effect," "PDGF-D activity," "PDGF-D biological activity," or "PDGF-D function," used interchangeably herein, refer to any activity mediated by the interaction of PDGF-D with a cognate receptor, said activity including, but not limited to, the binding of PDGF-D to PDGFR, phosphorylation of PDGFR, dimerization of PDGFR, increased cell migration, increased cell proliferation, increased extracellular matrix deposition, and any other PDGF-D activity known in the art or to be revealed in the future. Activities that inhibit (or reduce) PDGF-D activity (e.g., inhibiting or reducing PDGF-D binding to PDGFR, inhibiting or reducing PDGF-D-mediated phosphorylation of PDGFR, inhibiting or reducing PDGF-D-induced dimerization of PDGFR, inhibiting or reducing PDGF-D-induced cell migration, inhibiting or reducing PDGF-D-induced cell proliferation, inhibiting or reducing PDGF-D-induced extracellular matrix deposition) may be collectively referred to as "activities that neutralize (or reduce) PDGF-D", "activities that neutralize (or reduce) PDGF-D", etc. In one embodiment, the antigen-binding molecule of the present invention is an antibody that specifically binds to PDGF-D. In one embodiment, the degree of binding of the antigen-binding molecule / antibody of the present invention to an unrelated, non-PDGF-D protein is less than about 10% of the binding of said antibody to PDGF-D when measured, for example, by radioimmunoassay (RIA). In one embodiment, the non-PDGF-D is PDGF-A, PDGF-B, or PDGF-C.In certain embodiments, the antibody binding to PDGF-D is 1 micro M or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10. -8 M or less, e.g., 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 It has a dissociation constant (Kd) of M). In certain embodiments, the anti-PDGF-D antibody binds to an epitope of PDGF-D that is conserved between PDGF-D from different species.

[0968] In one embodiment, the antigen-binding molecule of the present invention that specifically binds to PDGF-D exhibits one or more of the following characteristics: increased neutralizing activity toward PDGF-D compared to a reference antibody binding to PDGF-D; increased activity that inhibits or reduces the binding of PDGF-D to a PDGF receptor compared to a reference antibody binding to PDGF-D; increased activity that inhibits or reduces PDGF-D-mediated phosphorylation of the PDGF receptor compared to a reference antibody binding to PDGF-D; increased activity that inhibits or reduces PDGF-D-induced dimerization of the PDGF receptor compared to a reference antibody binding to PDGF-D; and increased activity that inhibits or reduces PDGF-D-induced proliferation of cells expressing the PDGF receptor compared to a reference antibody binding to PDGF-D.

[0969] In the present invention, "reference antibody binding to PDGF-D" may be any known antibody that specifically binds to PDGF-D. In certain embodiments, the reference antibody is an antibody comprising a heavy chain variable region having the amino acid sequence presented in SEQ ID NO. 165 and a light chain variable region having the amino acid sequence presented in SEQ ID NO. 166, such as CR002 used in the examples.

[0970] Using known methods, it can be determined whether an antigen-binding molecule increases the activity of inhibiting or decreasing the binding of PDGF-D to the PDGF receptor, increases the activity of inhibiting or decreasing PDGF-D-mediated phosphorylation of the PDGF receptor, increases the activity of inhibiting or decreasing PDGF-D-induced dimerization of the PDGF receptor, or increases the activity of inhibiting or decreasing PDGF-D-induced proliferation of cells expressing the PDGF receptor compared to a reference antibody binding to PDGF-D. Such methods include those used in the examples described herein.

[0971] For example, the activity of an antigen-binding molecule that inhibits or reduces PDGF-D-mediated phosphorylation of the PDGF receptor can be determined using a cell-based receptor tyrosine kinase activity assay. Briefly, suitable cells are cultured without serum and treated with a test antigen-binding molecule pre-treated with a ligand (PDGF-D). PDGFR beta phosphorylation is measured using a commercially available test kit, such as the PathScan Phospho-PDGF receptor beta (Tyr751) ELISA kit (Cell Signaling Technology). The inhibitory effect of the test antigen-binding molecule on PDGF-D-mediated phosphorylation of the PDGF receptor can be determined in various known ways (e.g., inhibition percentage (%), IC50, etc.) based on the level of PDGF receptor phosphorylation when the test antigen-binding molecule is present and when the test antigen-binding molecule is absent. The inhibitory effect of a reference antibody on PDGF-D-mediated phosphorylation of the PDGF receptor can also be determined in the same way. If the inhibitory effect of the test antigen-binding molecule is stronger than that of the reference antibody, it is determined that the test antigen-binding molecule has increased activity in inhibiting or reducing PDGF-D-mediated phosphorylation of the PDGF receptor compared to the reference antibody; this also indicates that the test antigen-binding molecule has increased activity in inhibiting or reducing the binding of PDGF-D to the PDGF receptor compared to the reference antibody.

[0972] PDGF-D-induced proliferation of cells can be measured by any known cell proliferation assay, such as the BrdU cell proliferation assay, which measures the integration of BrdU into the newly synthesized DNA of proliferating cells. Briefly, suitable cells expressing the PDGF receptor, including the mouse fibroblast cell line NIH3T3 and the human lung fibroblast cell line IMR90, are cultured, serum is removed, and then treated with a test antigen-binding molecule pre-treated with the ligand (PDGF-D). The cells are further cultured in the presence of brmodeoxyuridine (BrdU). DNA synthesis is determined by measuring the integrated BrdU in the blood immunochemically. Commercially available kits (e.g., Cell Proliferation ELISA BrdU Kit (Roche Applied Science Cat. No. 11647229001)) may be suitablely used. The inhibitory effect of a test antigen-binding molecule on PDGF-D-induced proliferation of cells can be determined in various known ways based on BrdU integration, for example, in the presence or absence of the test antigen-binding molecule. The inhibitory effect of a reference antibody on PDGF-D-induced proliferation of cells can also be determined in the same way. If the inhibitory effect of the test antigen-binding molecule is stronger than that of the reference antibody, it is determined that the test antigen-binding molecule has increased activity in inhibiting or reducing PDGF-D-induced proliferation of cells expressing the PDGF receptor compared to the reference antibody, which also indicates that the test antigen-binding molecule has increased activity in inhibiting or reducing the binding of PDGF-D to the PDGF receptor compared to the reference antibody.

[0973] PDGF-D-induced dimerization of the PDGF receptor can be measured using commercially available kits. The inhibitory effect of a test antigen-binding molecule on PDGF-D-induced dimerization in cells can be determined in various known ways, for example, based on the dimerization signal detected in the presence and absence of the test antigen-binding molecule. The inhibitory effect of a reference antibody on PDGF-D-induced dimerization of the PDGF receptor can also be determined in the same way. If the inhibitory effect of the test antigen-binding molecule is stronger than that of the reference antibody, it is determined that the test antigen-binding molecule has increased activity in inhibiting or reducing PDGF-D-induced dimerization of the PDGF receptor compared to the reference antibody, which also indicates that the test antigen-binding molecule has increased activity in inhibiting or reducing the binding of PDGF-D to the PDGF receptor compared to the reference antibody.

[0974] Antigen-binding domains that specifically bind to PDGF-B (e.g., PDGF-B, PDGF-AB, and PDGF-BB) inhibit the interaction between PDGF-B and PDGFR and thus inhibit PDGF-B activity. The terms "PDGF-B mediated activity," "PDGF-B mediated effect," "PDGF-B activity," "PDGF-B biological activity," or "PDGF-B function," used interchangeably herein, refer to any activity mediated by the interaction of PDGF-B with a cognate receptor, said activity including, but not limited to, the binding of PDGF-B to PDGFR, phosphorylation of PDGFR, dimerization of PDGFR, increased cell migration, increased cell proliferation, increased extracellular matrix deposition, and any other PDGF-B activity known in the art or to be revealed in the future. Activities that inhibit (or reduce) PDGF-B activity (e.g., inhibiting or reducing PDGF-B binding to PDGFR, inhibiting or reducing PDGF-B-mediated phosphorylation of PDGFR, inhibiting or reducing PDGF-B-induced dimerization of PDGFR, inhibiting or reducing PDGF-B-induced cell migration, inhibiting or reducing PDGF-B-induced cell proliferation, inhibiting or reducing PDGF-B-induced extracellular matrix deposition) may be collectively referred to as “activities that neutralize (or reduce) PDGF-B”, “activities that neutralize (or reduce) PDGF-B”, etc. In one embodiment, the degree of binding of the antigen-binding domain that specifically binds to PDGF-B to unrelated non-PDGF-B proteins is less than about 10% of the binding of the antigen-binding domain to PDGF-B when measured, for example, by radioimmunoassay (RIA). In one embodiment, the non-PDGF-B is PDGF-A, PDGF-C, or PDGF-D.In certain embodiments, the antigen-binding domain binding to PDGF-B is 1 micro M or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10. -8 M or less, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 It has a dissociation constant (Kd) of M). In certain embodiments, the antigen-binding domain binds to an epitope of PDGF-B that is conserved between PDGF-B from different species.

[0975] The method of the present invention uses the antigen-binding molecule or antibody of the present invention to block, inhibit, or reduce (including a significant reduction) PDGF-D activity, including downstream events mediated by PDGF-D. As discussed above, the antigen-binding molecule or antibody of the present invention exhibits one or more of the following characteristics: (a) specifically binding to PDGF-D; (b) blocking the interaction between PDGF-D and cell surface receptors and downstream signaling events; (c) blocking the phosphorylation of PDGFR; (d) blocking PDGF-D-mediated induction of cell proliferation; (e) blocking PDGF-D-mediated induction of cell migration; and (f) blocking or reducing PDGF-D-mediated deposition of the extracellular matrix. In a preferred embodiment, the antigen-binding molecule or antibody of the present invention preferably reacts with PDGF-D in such a manner that it blocks the interaction between PDGF-D and a cell surface receptor, e.g., PDGFR. Additionally, the method of the present invention uses the multispecific antigen-binding molecule or antibody of the present invention to block, inhibit, or reduce (including significant reduction) PDGF-B and PDGF-D activity, including downstream events mediated by PDGF-B and / or PDGF-D. The multispecific antigen-binding molecule or antibody of the present invention exhibits one or more of the following characteristics: (a) specifically binding to PDGF-B and / or PDGF-D; (b) blocking the interaction and downstream signaling events between cell surface receptors and PDGF-B and / or PDGF-D; (c) blocking the phosphorylation of PDGFR; (d) blocking the PDGF-B and / or PDGF-D-mediated induction of cell proliferation; (e) blocking the PDGF-B and / or PDGF-D-mediated induction of cell migration; and (f) blocking or reducing PDGF-B and / or PDGF-D-mediated deposition of the extracellular matrix.In a preferred embodiment, the antigen-binding molecule or antibody of the present invention preferably reacts with PDGF-B and / or PDGF-D in a manner that blocks the interaction between a cell surface receptor, e.g., PDGFR, and PDGF-B and / or PDGF-D.

[0976] Affinity

[0977] “Affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antigen-binding molecule or antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, “binding affinity” as used herein refers to the intrinsic binding affinity reflecting the 1:1 interaction between the members of a binding pair (e.g., an antigen-binding molecule and an antigen, or an antibody and an antigen). The affinity of molecule X for partner Y can generally be expressed as a dissociation constant (KD). Affinity may be measured by general methods known in the art, including those described herein. Specific exemplary and best practices for measuring binding affinity are described below.

[0978] Methods for determining affinity

[0979] In specific embodiments, the antigen-binding domain of the antigen-binding molecule or antibody provided herein is 1 micro M or less, 120 nM or less, 100 nM or less, 80 nM or less, 70 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13It has a dissociation constant (KD) of M). In certain embodiments, the KD value of the first antigen-binding domain of the antibody / antigen-binding molecule for PDGF-B or PDGF-D is in the range of 0.1-40, 1-50, 1-70, 1-80, 30-50, 30-70, 30-80, 40-70, 40-80, or 60-80 nM.

[0980] In one embodiment, KD is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed with a Fab version of the antibody of interest and its antigen. For example, the soluble binding affinity of Fab for the antigen is measured by Fab at a minimum concentration ( 125 I) It is measured by equilibrating with a labeled antigen and then capturing the bound antigen onto an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish conditions for the assay, a MICROTITER (registered trademark) multi-well plate (Thermo Scientific) is coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w / v) bovine serum albumin in PBS for 2 to 5 hours at room temperature (approx. 23°C). On a non-absorbent plate (Nunc #269620), 100 pM or 26 pM [ 125The [I]-antigen is mixed with the serial dilution of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, incubation may be extended (e.g., about 65 hours) to ensure equilibrium is reached. Afterward, the mixture is transferred to a capture plate for incubation at room temperature (e.g., for one hour). Then, the solution is removed, and the plate is washed eight times with 0.1% polysorbate 20 in PBS (Tween-20 (registered trademark)). Once the plate is dry, 150 μl / well of scintillant (MICROSCINT-20) is added. TM Add ; Packard) and the plate for 10 minutes TOPCOUNT TM It is counted using a gamma counter (Packard). Concentrations of each Fab that provide 20% or less of the maximum binding are selected for use in competitive binding assays.

[0981] According to another embodiment, KD is measured using a BIACORE (trademark) surface plasmon resonance assay. For example, the assay using BIACORE (trademark)-2000 or BIACORE (trademark)-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C with an immobilized antigen CM5 chip in ~10 reaction units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions for use. Prior to injecting the antigen at a flow rate of 5 μl / min, the antigen was diluted to 5 μg / ml (~0.2 μM) in 10 mM sodium acetate, pH 4.8, to achieve approximately 10 reaction units (RU) of bound protein. After antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected at a flow rate of approximately 25 μl / min at 25°C in 0.05% polysorbate 20 (TWEEN-20 TM ) Inject into PBS containing a surfactant (PBST). Association rate (k on ) and dissociation rate (k off ) is calculated using a simple 1-to-1 Langmuir coupled model (BIACORE (trademark) evaluation software version 3.2) by simultaneously fitting association and dissociation sensograms. The equilibrium dissociation constant (KD) is the ratio k off / k on It is calculated as follows. For example, refer to Chen et al., J. Mol. Biol. 293:865-881 (1999). The binding rate is 10 by the above surface plasmon resonance assay. 6 M -1 s -1If it exceeds, the binding rate is measured using a spectrometer, such as a stationary-flow spectrophotometer (Aviv Instruments) or an 8000-series SLM-AMINCO equipped with a stirred cuvette. TM It can be determined using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 340 nm, 16 nm band pass; excitation = 295 nm) of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen when measured in a thermospectrophotometer.

[0982] A method for measuring the affinity of an antigen-binding domain of an antibody is described above, and a person skilled in the art can measure the affinity for other antigen-binding domains.

[0983] antibodies

[0984] In this document, the term "antibody" is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (insofar as they exhibit desired antigen-binding activity).

[0985] Classes of antibodies

[0986] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isomorphs), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the various classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0987] framework

[0988] "Framework" or "FR" refers to variable domain residues, not hypervariable region (HVR) residues. The FR of the variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0989] Common Human Framework

[0990] The "human consensus framework" is a framework representing the amino acid residues that occur most commonly in the selection of human immunoglobulin VL or VH framework sequences. Generally, human immunoglobulin VL or VH sequences are selected from a subgroup of variable domain sequences. Generally, the subgroups of sequences are the same as those in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., same as above. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., same as above.

[0991] HVR

[0992] As used herein, the terms “hypervariable region” or “HVR” refer to each region of an antibody variable domain that is hypervariable in sequence (“complementary determination region” or “CDR”) and / or forms a structurally defined loop (“hypervariable loop”) and / or includes antigen contact residues (“antigen contact region”). Generally, the antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein comprise the following:

[0993] (a) hypervariable loops occurring at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2), and 96-101(H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0994] (b) CDR occurring at amino acid residues 24-34(L1), 50-56(L2), 89-97(L3), 31-35b(H1), 50-65(H2) and 95-102(H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0995] (c) Antigen contact sites occurring at amino acid residues 27c-36(L1), 46-55(L2), 89-96(L3), 30-35b(H1), 47-58(H2) and 93-101(H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996));

[0996] (d) a combination of (a), (b) and / or (c), including HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3), and 94-102(H3).

[0997] Unless otherwise specified, HVR residues and other residues (e.g., FR residues) of the variable domain are numbered according to literature other than the above Kabat.

[0998] HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 can also be called "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2", and "LCDR3", respectively.

[0999] variable area

[1000] The term "variable region" or "variable domain" refers to the antibody heavy or light chain domains involved in the antibody's binding to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (e.g., Kindt et al. Kuby Immunology, 6 th See ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated using the VH or VL domain of the antibody binding to the antigen to screen for a library of complementary VL or VH domains. For example, see Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[1001] Identity (order identity)

[1002] The “percentage of amino acid sequence identity” for a reference polypeptide sequence is defined as the percentage of amino acid residues of candidate sequences identical to the amino acid residues of the reference polypeptide sequence, after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percentage of sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence identity may be achieved in various ways within the scope of the art in the relevant field, using, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX (registered trademark) (Genetyx Co., Ltd.). A skilled technician may determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

[1003] The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and its source code was submitted to the U.S. Copyright Office at 20559, Washington, D.C., along with user documentation, and is registered under U.S. Copyright No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from its source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are non-variable. When ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with respect to, or against, a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or containing a given % amino acid sequence identity with respect to, or against, a given amino acid sequence B) is calculated as follows:

[1004] 100 x fraction X / Y

[1005] Here, X is the number of amino acid residues scored as identical match in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A with respect to B will be recognized as not equal to the % amino acid sequence identity of B with respect to A. Unless otherwise specified, all amino acid sequence identity % values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[1006] Chimeric Antibodies

[1007] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species. Similarly, the term "chimeric antibody variable domain" refers to an antibody variable domain in which part of the heavy chain and / or light chain variable domain originates from a specific source or species, and the remainder of the heavy chain and / or light chain variable domain originates from a different source or species.

[1008] Humanized antibodies

[1009] "Humanized" antibody refers to a chimeric antibody comprising amino acid residues of non-human HVR and amino acid residues of human FR. In certain embodiments, the humanized antibody will substantially comprise at least one, and typically two, variable domains, wherein all or substantially all HVR (e.g., CDR) corresponds to that of the non-human antibody, and all or substantially all FR corresponds to that of the human antibody. The humanized antibody may optionally comprise at least a portion of an antibody constant region derived from the human antibody. The "humanized form" of the antibody, e.g., the non-human antibody, refers to the antibody that has undergone humanization. "Humanized antibody variable region" refers to the variable region of the humanized antibody.

[1010] Human antibodies

[1011] "Human antibody" refers to an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes the human antibody repertoire or other human antibody encoding sequences, possessing an amino acid sequence. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. "Human antibody variable region" refers to the variable region of a human antibody.

[1012] Method for generating antibodies with desired binding activity

[1013] Methods for generating antibodies having desired binding activity are known to those skilled in the art. The following is an example describing a method for generating antibodies that bind to PDGF-B or PDGF-D (i.e., anti-PDGF-B antibody or anti-PDGF-D antibody).

[1014] Anti-PDGF-B or PDGF-D antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. The resulting anti-PDGF-B or PDGF-D antibodies are preferably monoclonal antibodies derived from mammals. Such mammalian-derived monoclonal antibodies include antibodies produced by hybridomas or host cells transformed with expression vectors containing antibody genes by genetic engineering techniques.

[1015] A hybridoma that produces a monoclonal antibody can be generated using known techniques, for example, as described below. Specifically, a mammal is immunized by a conventional immunization method using PDGF-B or PDGF-D proteins as sensitization antigens. The generated immune cells are fused with a known parent cell by a conventional cell fusion method. Then, a hybridoma that produces an anti-PDGF-B or PDGF-D antibody can be selected by screening the monoclonal antibody-producing cells using a conventional screening method.

[1016] Specifically, monoclonal antibodies are prepared as described below. First, a polypeptide containing PDGF-B or PDGF-D to be used as a sensitizing antigen or immunogen for antibody production may be synthesized or expressed. Alternatively, a nucleic acid encoding full-length PDGF-B or PDGF-D or a truncated or other variant form of PDGF-B or PDGF-D may be expressed to produce a PDGF-B or PDGF-D-containing protein (either in monomeric or dimeric form). The desired human full-length PDGF-B or PDGF-D (monomeric or dimeric), or a truncated or other variant form of PDGF-B or PDGF-D, may be purified from host cells or their culture supernatant by known methods.

[1017] Purified full-length PDGF-B or PDGF-D, or truncated or other variant forms of PDGF-B or PDGF-D, may be used as sensitizing antigens or immunogens for the immunization of mammals. Partial peptides of full-length PDGF-B or PDGF-D may also be used as sensitizing antigens. In this case, the partial peptides may also be obtained by chemical synthesis from the human PDGF-B or PDGF-D amino acid sequence. Additionally, they may also be obtained by incorporating a portion of the PDGF-B or PDGF-D gene into an expression vector and expressing it.

[1018] Alternatively, it is possible to use a fusion protein prepared by fusing a desired partial polypeptide or peptide of full-length PDGF-B or PDGF-D or PDGF-B or PDGF-D ECD protein with a different polypeptide as a sensitizing antigen. For example, an antibody Fc fragment and a peptide tag are preferably used to generate a fusion protein used as a sensitizing antigen. A vector for the expression of such a fusion protein can be constructed by fusing genes encoding two or more desired polypeptide fragments in-frame and inserting the fusion gene into the expression vector as described above. A method for generating a fusion protein is described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47–9.58 (1989) Cold Spring Harbor Lab. Press). A method for preparing PDGF-B or PDGF-D to be used as a sensitizing antigen, and a method for immunization using PDGF-B or PDGF-D are also described in the examples in the latter part of this specification.

[1019] There are no specific restrictions on the mammals to be immunized with the sensitization antigen. However, it is desirable to select mammals considering their compatibility with the parent cells used for cell fusion. Generally, rodents such as mice, rats, and hamsters, as well as rabbits and monkeys, are preferably used.

[1020] The above animals are immunized with sensitizing antigens by known methods. Commonly practiced immunization methods include, for example, injecting the sensitizing antigen into the mammal intraperitoneal or subcutaneous. Specifically, the sensitizing antigen is appropriately diluted with PBS (phosphate-buffered saline) or physiological saline, etc. If desired, a conventional adjuvant, such as Freund's complete adjuvant, is mixed with the antigen, and the mixture is emulsified. Then, the sensitizing antigen is administered to the mammal several times at intervals of 4 to 21 days. Suitable carriers may be used for immunization with sensitizing antigens. In particular, when low molecular weight fractional peptides are used as sensitizing antigens, it is sometimes desirable to couple the sensitizing antigen peptide to a carrier protein, such as albumin or keyhole limpet hemocyanin, for immunization.

[1021] Alternatively, hybridomas that generate desired antibodies can be manufactured using DNA immunization as described below. DNA immunization is an immunization method that confers immune stimulation by expressing a sensitized antigen in an immunized animal as a result of administering vector DNA configured to allow the expression of an antigen protein-encoding gene in the animal.

[1022] To produce the monoclonal antibody of the present invention using DNA immunization, first, DNA for the expression of PDGF-B or PDGF-D proteins is administered to the animal to be immunized. PDGF-B or PDGF-D-encoding DNA can be synthesized by known methods such as PCR. The obtained DNA is inserted into a suitable expression vector, which is then administered to the animal to be immunized. Preferably used expression vectors include, for example, commercially available expression vectors such as pcDNA3.1. The vector can be administered to the organism using conventional methods. For example, DNA immunization is performed by introducing expression vector-coated gold particles into cells within the body of the animal to be immunized using a gene gun.

[1023] After immunizing the mammal as described above, an increase in the titer of PDGF-B or PDGF-D binding antibodies is confirmed in the serum. Then, immune cells are collected from the mammal, and cell fusion is performed. In particular, splenocytes are preferably used as immune cells.

[1024] Mammalian myeloma cells are used as cells to be fused with the aforementioned immune cells. Myeloma cells preferably contain suitable selection markers for selection. The selection markers confer characteristics for cell survival (or death) under specific culture conditions. Hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency) are known as selection markers. Cells with HGPRT or TK deficiency possess hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in HAT-selective medium and therefore die upon culture in HAT-selective medium; however, they are saved when fused with normal cells. Cells can continue DNA synthesis using the normal cell's recovery pathway and thus can grow in HAT-selective medium.

[1025] HGPRT-deficient cells can be selected in a medium containing 6-thioguanine or 8-azaguanine (hereinafter abbreviated as 8AG), and TK-deficient cells can be selected in a medium containing 5'-bromodeoxyuridine. Normal cells die in the selection medium because they incorporate these pyrimidine analogs into their DNA. Meanwhile, cells deficient in these enzymes can survive in the selection medium because they cannot incorporate these pyrimidine analogs. Additionally, a selection marker referred to as G418 resistance, provided by the neomycin-resistance gene, confers resistance to the 2-deoxystreptamine antibiotic (gentamicin analog). Various types of myeloma cells suitable for cell fusion are known.

[1026] For example, myeloma cells including the following cells may preferably be used:

[1027] P3(P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550);

[1028] P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978)81, 1-7);

[1029] NS-1 (C. Eur. J. Immunol. (1976)6 (7), 511-519);

[1030] MPC-11 (Cell (1976) 8 (3), 405-415);

[1031] SP2 / 0 (Nature (1978) 276 (5685), 269-270);

[1032] FO (J. Immunol. Methods (1980) 35 (1-2), 1-21);

[1033] S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323);

[1034] R210 (Nature (1979) 277 (5692), 131-133) et al.

[1035] Cell fusion between immune cells and myeloma cells is essentially carried out using known methods, for example, the method used by Kohler and Milstein et al. (Methods Enzymol. (1981) 73: 3-46).

[1036] More specifically, cell fusion can be carried out, for example, in the presence of a cell fusion promoter in a conventional culture medium. Fusion promoters include, for example, polyethylene glycol (PEG) and Sendai virus (HVJ). If necessary, an auxiliary substance such as dimethyl sulfoxide is additionally added to improve fusion efficiency.

[1037] The ratio of immune cells to myeloma cells can be appropriately determined, and a preferred example includes 1 to 10 myeloma cells per immune cell. The culture medium used for cell fusion includes, for example, media suitable for the growth of myeloma cell lines such as RPMI1640 medium and MEM medium, and other conventional culture media used for culturing these types of cells. Additionally, serum supplements such as fetal bovine serum (FCS) may preferably be added to the culture medium.

[1038] For cell fusion, a predetermined number of the immune cells and myeloma cells are thoroughly mixed in the culture medium. Then, a PEG solution (e.g., with an average molecular weight of about 1,000 to 6,000) preheated to about 37°C is added at a concentration of typically 30% to 60% (w / v). This is gently mixed to produce the desired fusion cells (hybridomas). Subsequently, the aforementioned appropriate culture medium is gradually added to the cells, and the mixture is repeatedly centrifuged to remove the supernatant. Thus, cell fusion agents and other substances unfavorable to hybridoma growth can be removed.

[1039] The hybridoma obtained in this way can be selected by culturing it in a conventional selection medium, for example, HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Cells other than the desired hybridoma (non-fusion cell) can be killed by continuing to culture them in the HAT medium for a sufficient period. Typically, the period is from several days to several weeks. Then, the hybridoma that produces the desired antibody is selected and monocloned by a conventional restriction dilution method.

[1040] The hybridoma thus obtained can be selected using a selection medium based on selection markers possessed by the myeloma cells used for cell fusion. For example, HGPRT- or TK-deficient cells can be selected by culturing them in HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Specifically, when HAT-sensitive myeloma cells are used for cell fusion, cells successfully fused with normal cells can be selectively proliferated in HAT medium. Cells other than the desired hybridoma (non-fusion cells) can be killed by continuing to culture them in the HAT medium for a sufficient period. Specifically, the desired hybridoma can generally be selected by culturing for several days to several weeks. Then, the hybridoma that produces the desired antibody is selected and monocloned by a conventional restriction dilution method.

[1041] Therefore, the manufactured monoclonal antibody-generating hybridoma can be subcultured in a conventional culture medium and stored in liquid nitrogen for a long period of time.

[1042] The above-mentioned hybridoma is cultured by conventional methods, and the desired monoclonal antibody can be prepared from the culture supernatant. Alternatively, the hybridoma is administered to a compatible mammal and grown, and the monoclonal antibody is prepared from the assortment. The aforementioned method is suitable for producing antibodies of high purity.

[1043] Antibodies encoded by antibody genes cloned from antibody-producing cells, such as the hybridoma mentioned above, may also preferably be used. The cloned antibody gene is inserted into a suitable vector, which is then introduced into a host to express the antibody encoded by the gene. Methods for isolating antibody genes, inserting genes into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192(3), 767-775). Methods for generating recombinant antibodies are also known as described below.

[1044] Preferably, the present invention provides a nucleic acid encoding an antigen-binding molecule or a multispecific antigen-binding molecule of the present invention. The present invention also provides a vector into which the nucleic acid encoding the antigen-binding molecule or a multispecific antigen-binding molecule is introduced, i.e., a vector containing the nucleic acid. Furthermore, the present invention provides a cell containing the nucleic acid or the vector, and the present invention also provides a method for generating an antigen-binding molecule or a multispecific antigen-binding molecule by culturing the cell. The present invention further provides an antigen-binding molecule or a multispecific antigen-binding molecule generated by the above method.

[1045] For example, cDNA encoding the variable region (V region) of an anti-PDGF-B or PDGF-D antibody is prepared from hybridoma cells expressing the anti-PDGF-B or PDGF-D antibody. For this purpose, total RNA is first extracted from the hybridoma. Methods used to extract mRNA from cells include, for example, the following:

[1046] - Guanidine ultracentrifugation method (Biochemistry (1979) 18(24), 5294-5299), and

[1047] - AGPC method (Anal. Biochem. (1987) 162(1), 156-159).

[1048] The extracted mRNA can be purified using an mRNA purification kit (GE Healthcare Bioscience), etc. Alternatively, kits for extracting total mRNA directly from cells, such as the QuickPrep mRNA purification kit (GE Healthcare Bioscience), are also commercially available. mRNA can be produced from hybridomas using these kits. cDNA encoding the antibody V region can be synthesized from the produced mRNA using reverse transcriptase. cDNA can be synthesized using an AMV reverse transcriptase 1st strand cDNA synthesis kit (Seikagaku Co.), etc. Additionally, the SMART RACE cDNA amplification kit (Clontech) and PCR-based 5'-RACE method (Proc. Natl. Acad. Sci. USA (1988) 85(23), 8998-9002; Nucleic Acids Res. (1989) 17(8), 2919-2932) can be appropriately used to synthesize and amplify cDNA. In this cDNA synthesis process, appropriate restriction enzyme sites described below can be introduced at both ends of the cDNA.

[1049] The cDNA fragment of interest is purified from the generated PCR product and then ligated to the vector DNA. Thus, a recombinant vector is constructed and introduced into E. coli, etc. After colony selection, the desired recombinant vector can be prepared from colony-forming E. coli. Then, whether the recombinant vector has the cDNA nucleotide sequence of interest is tested by a known method, such as the dideoxynucleotide chain termination method.

[1050] The 5'-RACE method, which uses primers to amplify variable region genes, is conveniently used to isolate genes encoding variable regions. First, a 5'-RACE cDNA library is constructed by cDNA synthesis using RNA extracted from hybridoma cells as a template. A 5'-RACE cDNA library is synthesized using a commercially available kit, such as the SMART RACE cDNA amplification kit, appropriately.

[1051] Antibody genes are amplified by PCR using a 5'-RACE cDNA library prepared as a template. Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. The nucleotide sequences of the primers vary depending on the immunoglobulin subclass. Therefore, it is desirable to determine the subclass in advance using a commercially available kit, such as the Iso Strip Mouse Monoclonal Antibody Isomorphism Kit (Roche Diagnostics).

[1052] Specifically, for example, primers that enable the amplification of genes encoding gamma-1, gamma-2a, gamma-2b, and gamma-3 heavy chains, as well as kappa and lambda light chains, are used to isolate mouse IgG-encoding genes. Generally, primers that anneale the constant region near the variable region are used as 3'-side primers for amplifying IgG variable region genes. Meanwhile, primers attached to the 5' RACE cDNA library construction kit are used as 5'-side primers.

[1053] The PCR product amplified in this way is used to reconstitute immunoglobulin composed of a combination of heavy and light chains. The desired antibody can be selected using the PDGF-B or PDGF-D binding activity of the reconstituted immunoglobulin as an indicator. For example, when the objective is to isolate antibodies against PDGF-B or PDGF-D, it is more desirable that the binding of the antibody against PDGF-B or PDGF-D be specific. PDGF-B or PDGF-D binding antibodies can be screened, for example, by the following steps:

[1054] (1) A step of contacting PDGF-B or PDGF-D with an antibody containing a V region encoded by cDNA isolated from a hybridoma;

[1055] (2) A step of detecting the binding of an antibody to PDGF-B or PDGF-D;

[1056] (3) a step of selecting an antibody that specifically binds to PDGF-B or PDGF-D; and preferably

[1057] (4) A step of selecting an antibody that exhibits strong binding to PDGF-B or PDGF-D.

[1058] A preferred antibody screening method using binding activity as an indicator also includes a panning method using phage vectors. Screening methods using phage vectors are advantageous when antibody genes are isolated from heavy and light chain subclass libraries from a population of polyclonal antibody-expressing cells. Genes encoding heavy and light chain variable regions can be linked by appropriate linker sequences to form single-stranded Fvs (scFvs). Phages presenting scFvs on their surface can be generated by inserting the gene encoding the scFv into a phage vector. The phage is brought into contact with the antigen of interest. Then, DNA encoding scFvs with the binding activity of interest can be isolated by collecting phages bound to the antigen. This process can be repeated as needed to enrich scFvs with the desired binding activity.

[1059] After isolating the cDNA encoding the V region of the anti-PDGF-B or PDGF-D antibody of interest, the cDNA is cleaved with a restriction enzyme that recognizes restriction sites introduced at both ends of the cDNA. A preferred restriction enzyme recognizes and cleaves nucleotide sequences occurring at a low frequency in the nucleotide sequence of the antibody gene. Additionally, a restriction site for an enzyme that generates adhesive ends is preferably introduced into the vector to insert a single-copy cleaved fragment in the correct orientation. The cDNA encoding the V region of the anti-PDGF-B or PDGF-D antibody is cleaved as described above and inserted into a suitable expression vector to construct an antibody expression vector. In this case, a chimeric antibody is obtained when the gene encoding the antibody constant region (C region) and the gene encoding the V region are fused in-frame. In this invention, "chimeric antibody" means that the origin of the constant region differs from the origin of the variable region. Accordingly, in addition to mouse / human heterochimeric antibodies, human / human allochimeric antibodies are included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already has an invariant region. Specifically, for example, a recognition sequence for a restriction enzyme that cleaves the V region gene can be appropriately placed on the 5' side of an expression vector carrying DNA encoding the desired antibody invariant region (C region). A chimeric antibody expression vector is constructed by fusing two genes cleaved by the same restriction enzyme combination in-frame.

[1060] To generate an anti-PDGF-B or PDGF-D monoclonal antibody, an antibody gene is inserted into an expression vector so that the gene is expressed under the control of an expression regulatory region. The expression regulatory region for antibody expression includes, for example, an enhancer and a promoter. Additionally, an appropriate signal sequence may be attached to the amino terminus to allow the expressed antibody to be secreted outside the cell. Meanwhile, other appropriate signal sequences may be attached. The expressed polypeptide is cleaved at the carboxyl terminus of the sequence, and the resulting polypeptide is secreted outside the cell as a mature polypeptide. Then, an appropriate host cell is transformed with the expression vector, and a recombinant cell expressing anti-PDGF-B or PDGF-D antibody-encoding DNA is obtained.

[1061] DNA encoding the antibody heavy chain (H chain) and light chain (L chain) is separately inserted into different expression vectors to express the antibody gene. Antibody molecules having H and L chains can be expressed by co-transfecting the same host cell with a vector into which the H-chain and L-chain genes are inserted, respectively. Alternatively, the host cell can be transformed with a single expression vector into which the DNA encoding the H and L chains is inserted (Ref. WO 94 / 11523).

[1062] There are various known host cell / expression vector combinations for antibody production by introducing the isolated antibody gene into a suitable host. All of these expression systems are applicable to the isolation of the domain containing the antibody variable region of the present invention. Suitable eukaryotic cells used as host cells include animal cells, plant cells, and fungal cells. Specifically, animal cells include, for example, the following cells.

[1063] (1) Mammalian cells: CHO, COS, multiple myeloma, baby hamster kidney (BHK), HeLa, or Vero, etc.;

[1064] (2) Amphibian cells: Xenopus oocytes, etc.; and

[1065] (3) Insect cells: sf9, sf21, or Tn5, etc.

[1066] In addition, an antibody gene expression system is known that uses plant cells derived from the genus Nicotiana, such as Nicotiana tabacum. Cells cultured in a color can be appropriately used to transform plant cells.

[1067] In addition, the following cells can be used as fungal cells:

[1068] Yeast: genus Saccharomyces, e.g., Saccharomyces cerevisiae, and genus Pichia, e.g., Pichia pastoris; and

[1069] Filamentous fungi: genus Aspergillus, e.g. Aspergillus niger.

[1070] In addition, antibody gene expression systems utilizing prokaryotic cells are also known. For example, when using bacterial cells, E. coli cells and Bacillus subtilis cells, etc., may be suitably used in the present invention. An expression vector containing the antibody gene of interest is introduced into these cells by transfection. The transfected cells are cultured in vitro, and the desired antibody can be produced from the culture of the transfected cells.

[1071] In addition to the host cells described above, genetically modified animals may also be used to generate recombinant antibodies. That is, antibodies may be obtained from animals into which a gene encoding the antibody of interest has been introduced. For example, the antibody gene may be constructed as a fusion gene by being inserted in-frame into a gene encoding a protein that is produced and specifically secreted in milk. Goat beta-casein, for example, may be used as the protein secreted in milk. A DNA fragment containing a fusion gene containing the introduced antibody gene is injected into a goat embryo, and subsequently, this embryo is introduced into a female goat. The desired antibody may be obtained as a protein fused with a milk protein from milk produced by a transgenic goat born from the embryo-receiving goat (or its offspring). Additionally, to increase the volume of milk containing the desired antibody produced by the transgenic goat, hormones may be administered to the transgenic goat as needed (Ebert, KM et al., Bio / Technology (1994) 12 (7), 699-702).

[1072] Method for generating humanized antibodies

[1073] When the antigen-binding molecule described herein is administered to a human, a domain derived from a genetically recombinant antibody that has been artificially modified to reduce heterogenicity to humans, etc., can be suitably used as a domain of the antigen-binding molecule comprising an antibody variable region. Such genetically recombinant antibodies include, for example, humanized antibodies. These modified antibodies are suitably produced by known methods. Additionally, generally, the binding specificity of a specific antibody can be introduced into another antibody by CDR transplantation.

[1074] Specifically, humanized antibodies prepared by transplanting a CDR of a non-human animal antibody, such as a mouse antibody, into a human antibody are known. Genetic engineering techniques for obtaining such humanized antibodies are also generally known. Specifically, for example, nested extension PCR is known as a method for transplanting a mouse antibody CDR into a human FR. In nested extension PCR, the nucleotide sequence encoding the mouse antibody CDR to be transplanted is added to primers for synthesizing the human antibody FR. Primers are prepared for each of the four FRs. Generally, when transplanting a mouse CDR into a human FR, selecting a human FR that has high identity with the mouse FR is considered advantageous for maintaining CDR function. That is, it is generally desirable to use a human FR containing an amino acid sequence that has high identity with the amino acid sequence of the FR adjacent to the mouse CDR to be transplanted.

[1075] The nucleotide sequences to be ligated are designed to be in-frame connected to each other. Human FRs are synthesized individually using primers. As a result, a product is obtained in which mouse CDR-encoding DNA is attached to individual FR-encoding DNA. The nucleotide sequences encoding the mouse CDR of each product are designed to overlap each other. Then, a complementary strand synthesis reaction is performed to anneale the overlapping CDR regions of the synthesized products using a human antibody gene as a template. Human FRs are ligated through the mouse CDR sequences by this reaction.

[1076] The full-length V region gene, ligated with three CDRs and four FRs, is amplified using primers that anneale at its 5'- or 3'-terminus to which a suitable restriction enzyme recognition sequence is added. An expression vector for the humanized antibody can be generated by inserting the DNA obtained as previously described and the DNA encoding the human antibody C region into the expression vector, whereby these DNAs will be ligated in-frame. After the recombinant vector is transfected into a host to establish recombinant cells, the recombinant cells are cultured, and the DNA encoding the humanized antibody is expressed to generate the humanized antibody in the cell culture (cf. European Patent Publication No. EP 239400 and International Patent Publication No. WO 1996 / 002576).

[1077] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody generated as described above, a suitable human antibody FR can be selected that enables the CDR to form a favorable antigen-binding site when ligated through the CDR. Amino acid residues within the FR can be substituted as necessary to ensure that the CDR of the reformed human antibody forms an appropriate antigen-binding site. For example, amino acid sequence mutations can be introduced into the FR by applying the PCR method used to transplant mouse CDRs into human FRs. More specifically, partial nucleotide sequence mutations can be introduced into the primers used to anneale the FR. Nucleotide sequence mutations are introduced into the FR synthesized using these primers. A mutant FR sequence having desired characteristics can be selected by measuring and evaluating the activity of the amino acid-substituted mutant antibody binding to the antigen by the method mentioned above (Sato, K. et al., Cancer Res. (1993) 53: 851-856).

[1078] Method for generating human antibodies.

[1079] Alternatively, the desired human antibody can be obtained by immunizing a genetically modified animal (WO 1993 / 012227; WO 1992 / 003918; WO 1994 / 002602; WO 1994 / 025585; WO 1996 / 034096; WO 1996 / 033735) having the entire repertoire of human antibody genes by DNA immunization.

[1080] In addition, a technique for producing human antibodies by panning using a human antibody library is also known. For example, the V region of a human antibody is expressed as a single-stranded antibody (scFv) on the surface of a phage by a phage display method. A phage expressing an scFv that binds to an antigen can be selected. The DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined by analyzing the gene of the selected phage. The DNA sequence of the scFv that binds to the antigen is determined. An expression vector is prepared by fusing the V region sequence within a frame with the C region sequence of the desired human antibody and inserting it into a suitable expression vector. The expression vector is introduced into a cell suitable for expression as described above. A human antibody can be produced by expressing the human antibody encoding gene in a cell. These methods are already known (cf. WO 1992 / 001047; WO 1992 / 020791; WO 1993 / 006213; WO 1993 / 011236; WO 1993 / 019172; WO 1995 / 001438; WO 1995 / 015388).

[1081] vector

[1082] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is connected. This term includes vectors as self-replicating nucleic acid constructs and vectors integrated into the genome of a host cell into which they are introduced. Additionally, certain vectors may direct the expression of operably connected genes. Such vectors are referred to herein as "expression vectors."

[1083] Host cell

[1084] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and include the offspring of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their offspring regardless of the number of passages. Offspring may not be exactly identical to their parent cells in nucleic acid content and may contain mutations. Mutant offspring having the same function or biological activity as those selected or chosen from the original transformed cells are included herein.

[1085] Epitope

[1086] "Epitope" means an antigenic determinant within an antigen and refers to an antigenic site to which the antigen-binding domain of the antigen-binding molecule or antibody disclosed herein binds. Thus, for example, an epitope may be defined according to its structure. Alternatively, an epitope may be defined according to the antigen-binding activity of the antigen-binding molecule or antibody that recognizes the epitope. If the antigen is a peptide or polypeptide, the epitope may be specified by the amino acid residues forming the epitope. Alternatively, if the epitope is a sugar chain, the epitope may be specified by a specific sugar chain structure.

[1087] A linear epitope is an epitope containing an epitope whose primary amino acid sequence is recognized. Such a linear epitope typically contains at least 3 and most commonly at least 5, for example, about 8 to 10 or 6 to 20 amino acids in its specific sequence.

[1088] In contrast to linear epitopes, a "structural epitope" is an epitope in which the primary amino acid sequence containing the epitope is not the sole determinant of the recognized epitope (e.g., the primary amino acid sequence of a structural epitope is not necessarily recognized by the epitope-defining antibody). Structural epitopes can contain a greater number of amino acids compared to linear epitopes. The antigen-binding domain for recognizing structural epitopes recognizes the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds and forms a three-dimensional structure, the amino acids and / or polypeptide main chains forming the structural epitope align, and the epitope becomes recognizable by the antigen-binding domain. Methods for determining the epitope shape include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance, site-specific spin labeling, and electron magnetic resonance. For example, see Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[1089] Examples of methods for evaluating epitope binding by a test antigen-binding molecule or an antibody containing an anti-PDGF-B or PDGF-D antigen-binding domain are described below. According to the following examples, a method for evaluating epitope binding by a test antigen-binding molecule or an antibody containing an antigen-binding domain for antigens other than PDGF-B or PDGF-D may also be suitably carried out.

[1090] For example, whether a test antigen-binding molecule or antibody containing an anti-PDGF-B or PDGF-D antigen-binding domain recognizes a linear epitope in a PDGF-B or PDGF-D molecule can be determined, for example, as mentioned below. For this purpose, a linear peptide containing the amino acid sequence of PDGF-B or PDGF-D is synthesized. The peptide may be chemically synthesized or obtained by genetic engineering techniques utilizing a region of PDGF-B or PDGF-D cDNA encoding the amino acid sequence. Then, the test antigen-binding molecule or antibody containing the anti-PDGF-B or PDGF-D antigen-binding domain is evaluated for its binding activity to the linear peptide containing the amino acid sequence. For example, the immobilized linear peptide may be used as an antigen in an ELISA to evaluate the binding activity of a polypeptide complex to the peptide. Alternatively, the binding activity to the linear peptide may be evaluated based on the level to which the linear peptide inhibits the binding of the antigen-binding molecule or antibody to PDGF-B or PDGF-D. These tests can demonstrate the binding activity of antigen-binding molecules or antibodies to linear peptides.

[1091] Whether a test antigen-binding molecule or antibody containing an anti-PDGF-B or PDGF-D antigen-binding domain recognizes a structural epitope can be evaluated as follows. A test antigen-binding molecule or antibody containing an anti-PDGF-B or PDGF-D antigen-binding domain may be determined to recognize a structural epitope if, upon contact, it binds strongly to PDGF-B or PDGF-D but does not substantially bind to an immobilized linear peptide containing the amino acid sequence of PDGF-B or PDGF-D. In this invention, "substantially not binding" means that the binding activity is 80% or less, generally 50% or less, preferably 30% or less, and particularly preferably 15% or less compared to the binding activity for PDGF-B or PDGF-D.

[1092] A method for testing the binding activity of a test antigen-binding molecule or antibody containing an anti-PDGF-B or PDGF-D antigen-binding domain to PDGF-B or PDGF-D includes, for example, the method described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420).

[1093] In the ELISA format, the binding activity of a test antigen-binding molecule or antibody containing an anti-PDGF-B or PDGF-D antigen-binding domain to PDGF-B or PDGF-D can be quantitatively evaluated by comparing the levels of signals generated by an enzymatic reaction. Specifically, a test antigen-binding molecule or antibody containing an anti-PDGF-B or PDGF-D antigen-binding domain is added to an ELISA plate immobilized with PDGF-B or PDGF-D. Then, the test antigen-binding molecule or antibody bound to PDGF-B or PDGF-D is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule or antibody. Alternatively, when FACS is used, serial dilutions of the test antigen-binding molecule or antibody are prepared, and antibody binding titers to PDGF-B or PDGF-D can be determined to compare the binding activity of the test antigen-binding molecule or antibody toward PDGF-B or PDGF-D.

[1094] Whether a test antigen-binding molecule or antibody containing an anti-PDGF-B or PDGF-D antigen-binding domain shares a common epitope with another antigen-binding molecule or antibody can be evaluated based on competition between the two antigen-binding molecules or antibodies for the same epitope. Competition between antigen-binding molecules or antibodies can be detected through cross-blocking assays, etc. For example, a competitive ELISA assay is a preferred cross-blocking assay.

[1095] Specifically, in a cross-blocking assay, PDGF-B or PDGF-D proteins immobilized in the wells of a microtiter plate are pre-incubated in the presence or absence of candidate competing antigen-binding molecules or antibodies, and subsequently, test antigen-binding molecules or antibodies are added thereto. The amount of test antigen-binding molecules or antibodies bound to PDGF-B or PDGF-D proteins in the wells is indirectly correlated with the binding ability of candidate competing antigen-binding molecules or antibodies competing for binding to the same epitope. That is, the higher the affinity of the competing antigen-binding molecules or antibodies for the same epitope, the lower the binding activity of the test antigen-binding molecules or antibodies to the PDGF-B or PDGF-D protein-coated wells.

[1096] The amount of test antigen-binding molecules or antibodies bound to the wells via PDGF-B or PDGF-D proteins can be easily determined by pre-labeling the antigen-binding molecules or antibodies. For example, biotin-labeled antigen-binding molecules or antibodies are measured using an avidin / peroxidase conjugate and an appropriate substrate. In particular, cross-blocking assays using enzyme labels such as peroxidase are called "competitive ELISA assays." Antigen-binding molecules or antibodies may also be labeled with other labeling substances that enable detection or measurement. Specifically, radioactive labels and fluorescent labels are known.

[1097] If a candidate competing antigen-binding molecule or antibody can block binding by a test antigen-binding molecule or antibody containing an anti-PDGF-B or PDGF-D antigen-binding domain by at least 20%, preferably at least 20 to 50%, more preferably at least 50%, compared to binding activity in a control experiment conducted in the absence of the competing antigen-binding molecule or antibody, the test antigen-binding molecule or antibody is determined to substantially bind to the same epitope bound by the competing antigen-binding molecule or antibody, or to compete for binding to the same epitope.

[1098] If the structure of an epitope bound by a test antigen-binding molecule or an antibody containing an anti-PDGF-B or PDGF-D antigen-binding domain has already been identified, whether the test and control antigen-binding molecules or antibodies share a common epitope can be evaluated by comparing the binding activity of the two antigen-binding molecules or antibodies to a peptide prepared by introducing an amino acid mutation into the peptide forming the epitope.

[1099] To measure the above binding activity, for example, the binding activity of test and control antigen-binding molecules or antibodies to a linear peptide into which a mutation has been introduced is compared in the form of the above ELISA. In addition to the ELISA method, the binding activity to the mutant peptide bound to the column can be determined by flowing test and control antigen-binding molecules or antibodies through the column and then quantifying the antigen-binding molecules or antibodies eluted from the elution solution. For example, a method of adsorbing a mutant peptide onto a column in the form of a GST fusion peptide is known.

[1100] Specificity

[1101] "Specific" means that a molecule that specifically binds to one or more binding partners does not exhibit any significant binding to molecules other than the partners. Additionally, "specific" is used when the antigen-binding domain is specific to a specific epitope among multiple epitopes contained in the antigen. When the epitope bound by the antigen-binding domain is contained in multiple different antigens, the antigen-binding molecule containing the antigen-binding domain can bind to various antigens having epitopes.

[1102] monospecific antigen-binding molecule

[1103] The term "monospecific antigen-binding molecule" is used to refer to an antigen-binding molecule that specifically binds to only one type of antigen. A favorable example of a monospecific antigen-binding molecule is an antigen-binding molecule containing a single type of antigen-binding domain. A monospecific antigen-binding molecule may contain a single antigen-binding domain or multiple antigen-binding domains of the same type. A favorable example of a monospecific antigen-binding molecule is a monospecific antibody. If the monospecific antigen-binding molecule is a monospecific antibody in the form of an IgG, the monospecific antibody contains two antibody variable fragments having the same antigen-binding specificity.

[1104] antibody fragment

[1105] “Antibody fragment” refers to a molecule that is not a non-defective antibody but contains a portion of a non-defective antibody and binds to an antigen to which the non-defective antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[1106] The terms “full-length antibody,” “indestructible antibody,” and “full-length antibody” are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein.

[1107] Variable fragment (Fv)

[1108] In this document, the term “variable fragment (Fv)” refers to the smallest unit of an antibody-derived antigen-binding domain consisting of a pair of antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH). In 1988, Skerra and Pluckthun discovered that homogeneous and active antibodies could be produced from the peripheral cytoplasmic fraction of Escherichia coli (E. coli) by inserting antibody genes downstream of bacterial signal sequences and inducing gene expression in Escherichia coli (E. coli) (Science (1988) 240(4855), 1038-1041). In the Fv produced from the peripheral cytoplasmic fraction, VH associates with VL in a manner that allows it to bind to the antigen.

[1109] scFv, single-chain antibody, and sc(Fv) 2

[1110] In this document, the terms “scFv”, “single-chain antibody”, and “sc(Fv)2” all refer to antibody fragments of a single polypeptide chain that include a variable region derived from the heavy chain and the light chain, but do not include a constant region. Generally, single-chain antibodies also include a polypeptide linker between the VH and VL domains, which enables the formation of a desired structure thought to allow antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in “The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 269-315 (1994)”. See also International Patent Publication WO 1988 / 001649; U.S. Patent Nos. 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies may be bispecific and / or humanized.

[1111] scFv is an antigen-binding domain in which VH and VL are linked together by a peptide linker to form Fv (Proc. Natl. Acad. Sci. USA (1988) 85(16), 5879-5883). VH and VL can be kept close by a peptide linker.

[1112] sc(Fv)2 is a single-chain antibody in which four variable regions, namely two VLs and two VHs, are linked by a linker to form a single chain (J Immunol. Methods (1999) 231(1-2), 177-189). The two VHs and two VLs may be derived from different monoclonal antibodies. Such sc(Fv)2 preferably comprises a bispecific sc(Fv)2 that recognizes two epitopes present within a single antigen, for example, as disclosed in Journal of Immunology (1994) 152(11), 5368-5374. sc(Fv)2 may be produced by methods known to those skilled in the art. For example, sc(Fv)2 may be produced by linking scFv with a linker such as a peptide linker.

[1113] In the present invention, the form of the antigen-binding domain forming sc(Fv)2 comprises an antibody in which two VH units and two VL units are arranged in the order VH, VL, VH, and VL, starting from the N-terminus of a single-strand polypeptide ([VH]-linker-[VL]-linker-[VH]-linker-[VL]). The order of the two VH units and two VL units is not limited to the above form and may be arranged in any order. Examples of the form are listed below.

[1114] [VL]-Linker-[VH]-Linker-[VH]-Linker-[VL]

[1115] [VH]-Linker-[VL]-Linker-[VL]-Linker-[VH]

[1116] [VH]-Linker-[VH]-Linker-[VL]-Linker-[VL]

[1117] [VL]-Linker-[VL]-Linker-[VH]-Linker-[VH]

[1118] [VL]-Linker-[VH]-Linker-[VL]-Linker-[VH]

[1119] The molecular form of sc(Fv)2 is also described in detail in WO 2006 / 132352. According to these descriptions, a person skilled in the art can appropriately prepare the desired sc(Fv)2 to produce the polypeptide complex disclosed herein.

[1120] Additionally, the antigen-binding molecule or antibody of the present invention may be conjugated to a carrier polymer such as PEG or an organic compound such as an anticancer agent. Alternatively, a sugar chain addition sequence is preferably inserted into the antigen-binding molecule or antibody so that the sugar chain produces a desired effect.

[1121] Linkers used to link variable regions of antibodies include any peptide linker that can be introduced by genetic engineering, synthetic linker, and, for example, the linker disclosed in Protein Engineering, 9(3), 299-305, 1996. However, peptide linkers are preferred in the present invention. The length of the peptide linker is not particularly limited and can be appropriately selected by a person skilled in the art according to the purpose. The length is preferably 5 amino acids or more (without special limitation, the upper limit is generally 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. If sc(Fv)2 contains three peptide linkers, their lengths may all be the same or different.

[1122] For example, these peptide linkers include the following:

[1123] Ser

[1124] Gly Ser

[1125] Gly Gly Ser

[1126] Ser Gly Gly

[1127] Gly Gly Gly Ser (Sequence No. 187)

[1128] Ser Gly Gly Gly (Sequence No. 188)

[1129] Gly Gly Gly Gly Ser (Sequence No. 189)

[1130] Ser Gly Gly Gly Gly (Sequence No. 190)

[1131] Gly Gly Gly Gly Gly Ser (Sequence No. 191)

[1132] Ser Gly Gly Gly Gly Gly (Sequence No. 192)

[1133] Gly Gly Gly Gly Gly Gly Ser (Sequence No. 193)

[1134] Ser Gly Gly Gly Gly Gly Gly (Sequence No. 194)

[1135] (Gly Gly Gly Gly Ser (Sequence No. 189))n

[1136] (Ser Gly Gly Gly Gly (Sequence No. 190))n

[1137] Here, n is an integer greater than or equal to 1. The length or sequence of the peptide linker can be appropriately selected by a person skilled in the art depending on the purpose.

[1138] Synthetic linkers (chemical crosslinkers) are routinely used to crosslink peptides, examples of which are as follows:

[1139] N-hydroxy succinimide (NHS),

[1140] Disuccinimidyl suberate (DSS),

[1141] Bis(sulfosuccinimidyl) suberate (BS3),

[1142] Dithiobis (succinimidyl propionate) (DSP),

[1143] Dithiobis(sulfosuccinimidyl propionate)(DTSSP),

[1144] Ethylene glycol bis(succinimidyl succinate) (EGS),

[1145] Ethylene glycol bis(sulfosuccinimidyl succinate)(sulfo-EGS),

[1146] Disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),

[1147] Bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (BSOCOES) and

[1148] Bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[1149] These crosslinking agents are commercially available.

[1150] Generally, three linkers are required to link four antibody variable regions together. The linkers used may be of the same type or different types.

[1151] Fab, F(ab') 2 , and Fab'

[1152] "Fab" consists of a single light chain, and a CH1 domain and variable region from a single heavy chain. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[1153] "F(ab')2" or "Fab" refers to an antibody fragment produced by treating immunoglobulin (monoclonal antibody) with proteolytic enzymes, such as pepsin and papain, and cleaving the immunoglobulin (monoclonal antibody) near the disulfide bonds located between the hinge regions of each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bonds located between the hinge regions of each of the two H chains to produce two homologous antibody fragments, wherein the L chain containing VL (L-chain variable region) and CL (L-chain constant region) is connected to an H-chain fragment containing VH (H-chain variable region) and CH gamma 1 (gamma 1 region of the H-chain constant region) via disulfide bonds at its C-terminal region. Each of these two homologous antibody fragments is called Fab'.

[1154] "F(ab')2" consists of two light chains and two heavy chains, and includes an invariant region of the CH1 domain and a portion of the CH2 domain so that a disulfide bond is formed between the two heavy chains. The F(ab')2 disclosed herein can preferably be produced as follows: a whole monoclonal antibody, etc. containing the desired antigen-binding domain, is partially cleaved with a protease such as pepsin; and the Fc fragment is removed by adsorption onto a Protein A column. The protease is not particularly limited as long as it can selectively cleave the whole antibody to produce F(ab')2 under appropriate enzymatic reaction conditions, such as pH. Such proteases include, for example, pepsin and fisin.

[1155] Fc area

[1156] In this document, the terms “Fc region” or “Fc domain” are used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The terms include natural sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or Pro230, to the carboxyl-terminus of the heavy chain. However, C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) in the Fc region may or may not be present. Unless explicitly stated otherwise in this document, the numbering of amino acid residues in the Fc region or constant region follows the so-called EU index as described in the EU numbering system, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[1157] Fc receptor

[1158] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a natural human FcR. In some embodiments, the FcR is a receptor of the Fc gamma RI, Fc gamma RII, and Fc gamma RIII subclasses that bind to an IgG antibody (gamma receptor) and include allelic variants and alternatively spliced ​​forms of these receptors. The Fc gamma RII receptor includes Fc gamma RIIA ("activating receptor") and Fc gamma RIIB ("inhibitory receptor"), which have different similar amino acid sequences, primarily in their cytoplasmic domains. The activating receptor Fc gamma RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor Fc gamma RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are included in the term "FcR" herein.

[1159] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transmission of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); see WO 2004 / 92219 (Hinton et al.)).

[1160] In vivo binding of human FcRn and the plasma half-life of human FcRn high-affinity binding polypeptides can be tested, for example, in genetically modified mice expressing human FcRn or transfected human cell lines, or in primates administered polypeptides having a variant Fc region. WO 2000 / 42072 (Presta) describes antibody variants having increased or decreased binding to FcR. For example, see also Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[1161] Fc gamma receptor

[1162] The Fc gamma receptor refers to a receptor capable of binding to the Fc domain of a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody, and includes all members belonging to the family of proteins substantially encoded by the Fc gamma receptor gene. In humans, said family includes Fc γ RI (CD64), comprising homologous Fc γ RIa, Fc γ RIb, and Fc γ RIc; Fc γ RII (CD32), comprising homologous Fc γ RIIa (including allotypes H131 and R131), Fc γ RIIb (including homologous Fc γ RIIb-1 and Fc γ RIIb-2), and Fc γ RIIc; Fc γ RIII (CD16), including isoforms Fc γ RIIIa (including alloforms V158 and F158) and Fc γ RIIIb (including alloforms Fc γ RIIIb-NA1 and Fc γ RIIIb-NA2); as well as all unidentified human Fc γ receptors, Fc γ receptor isoforms, and their alloforms. However, Fc gamma receptors are not limited to these examples. Without being limited thereto, Fc gamma receptors include those derived from humans, mice, rats, rabbits, and monkeys. Fc gamma receptors may be derived from any organism. Mouse Fc gamma receptors include, but are not limited to, Fc gamma RI (CD64), Fc gamma RII (CD32), Fc gamma RIII (CD16), and Fc gamma RIII-2 (CD16-2), as well as all unidentified mouse Fc gamma receptors, Fc gamma receptor isoforms, and allotypes thereof. These preferred Fc gamma receptors include, for example, human Fc gamma RI (CD64), Fc gamma RIIA (CD32), Fc gamma RIIB (CD32), Fc gamma RIIIA (CD16), and / or Fc gamma RIIIB (CD16). The polynucleotide sequence and amino acid sequence of Fc gamma RI are SEQ ID NOs 178 (NM_000566.3) and 173 (NP_000557), respectively.1) is presented; the polynucleotide and amino acid sequences of Fc gamma RIIA are presented in SEQ No. 179 (BC020823.1) and 174 (AAH20823.1), respectively; the polynucleotide and amino acid sequences of Fc gamma RIIB are presented in SEQ No. 180 (BC146678.1) and 175 (AAI46679.1), respectively; the polynucleotide and amino acid sequences of Fc gamma RIIIA are presented in SEQ No. 181 (BC033678.1) and 176 (AAH33678.1), respectively; and the polynucleotide and amino acid sequences of Fc gamma RIIIB are presented in SEQ No. 182 (BC128562.1) and 177 (AAI28563.1), respectively (the RefSeq accession number is presented in parentheses for each). Whether the Fc gamma receptor has binding activity to the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies can be evaluated by ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR)-based BIACORE method, and other methods (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010) in addition to the FACS and ELISA formats described above.

[1163] Meanwhile, "Fc ligand" or "effector ligand" refers to a molecule, preferably a polypeptide, that binds to an antibody Fc domain to form an Fc / Fc ligand complex. The molecule may be derived from any organism. The binding of the Fc ligand to Fc preferably induces one or more effector functions. Such Fc ligands include, but are not limited to, Fc receptors, Fc gamma receptors, Fc alpha receptors, Fc beta receptors, FcRn, C1q and C3, mannan-binding lectins, mannose receptors, Staphylococcus protein A, Staphylococcus protein G, and viral Fc gamma receptors. Fc ligands also include Fc receptor homologs (FcRH) (Davis et al., (2002) Immunological Reviews 190, 123-136), which is a family of Fc receptors homologous to the Fc gamma receptor. The Fc ligand also includes an unidentified molecule that binds to Fc.

[1164] Fc gamma receptor binding activity

[1165] The impaired binding activity of the Fc domain to any one of the Fc gamma receptors Fc gamma RI, Fc gamma RIIA, Fc gamma RIIB, Fc gamma RIIIA, and / or Fc gamma RIIIB can be evaluated using the FACS and ELISA formats described above, as well as the ALPHA screen (amplified luminescence proximity uniformity assay) and surface plasmon resonance (SPR)-based BIACORE method (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[1166] Alpha screens are implemented using two types of beads: donor and acceptor beads, based on the principles described below. Luminescence signals are detected only when the two beads are located very close together, through biological interactions between molecules connected to the donor and acceptor beads. When stimulated by a laser beam, the photosensitizer in the donor bead converts oxygen around the bead into stimulated singlet oxygen. When singlet oxygen diffuses around the donor bead and reaches the adjacent acceptor bead, a chemiluminescence reaction is induced within the acceptor bead. This reaction ultimately produces luminescence. If the molecule connected to the donor bead does not interact with the molecule connected to the acceptor bead, the singlet oxygen generated by the donor bead does not reach the acceptor bead, and thus no chemiluminescence reaction occurs.

[1167] For example, a biotin-labeled antigen-binding molecule or antibody is immobilized on a donor bead, and a glutathione S-transferase (GST)-tagged Fc gamma receptor is immobilized on a receptor bead. In the absence of an antigen-binding molecule or antibody containing a competitive mutant Fc domain, the Fc gamma receptor interacts with an antigen-binding molecule or antibody containing a wild-type Fc domain, thereby inducing a signal of 520 to 620 nm. When an antigen-binding molecule or antibody having an untagged mutant Fc domain competes with an antigen-binding molecule or antibody containing a wild-type Fc domain for interaction with the Fc gamma receptor, a decrease in fluorescence will be observed as a result of the competition, and the decrease can be quantified, thereby allowing the relative binding affinity to be determined. Methods for biotinizing antibodies or antigen-binding molecules, such as antibodies using sulfo-NHS-biotin, etc., are known. A suitable method for adding a GST tag to an Fc gamma receptor comprises the steps of fusing the Fc gamma receptor and a gene encoding GST in-frame, expressing the fused gene using cells into which a vector containing the fused gene has been introduced, and subsequently purifying the fused protein using a glutathione column. The induced signal can preferably be analyzed by fitting a 1-site competitive model based on non-linear regression analysis using software, e.g., GRAPHPAD PRISM (GraphPad; San Diego).

[1168] One of the materials to observe their interaction is immobilized as a ligand on a gold foil layer of the sensor chip. When light is shone on the back surface of the sensor chip to generate total reflection at the interface between the gold foil and the glass, the intensity of the reflected light is partially reduced at specific locations (SPR signal). Another material to observe their interaction is injected as an analyte on the surface of the sensor chip. When the analyte binds to the ligand, the mass of the immobilized ligand molecule increases. This changes the refractive index of the solvent on the surface of the sensor chip. The change in refractive index causes a shift in the position of the SPR signal (conversely, dissociation shifts the signal back to its original position). In the Biacore system, the amount of the aforementioned change (i.e., the change in mass on the sensor chip surface) is plotted on the vertical axis, and thus the change in mass over time is represented as measured data (sensorgram). The kinetic parameters (association rate constant (ka) and dissociation rate constant (kd)) are determined from the curve of the sensorgram, and the affinity (KD) is determined from the ratio between these two constants. Suppression tests are preferably used in the BIACORE method. An example of such suppression tests is described in Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.

[1169] Fc region having reduced Fc gamma receptor binding activity

[1170] In this document, "reduced Fc gamma receptor binding activity" means, for example, that based on the analysis method described above, the competitive activity of the test antigen-binding molecule or antibody is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, particularly preferably 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.

[1171] Antigen-binding molecules or antibodies containing the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies may be appropriately used as control antigen-binding molecules or antibodies. The structures of the Fc domain are shown in SEQ No. 183 (A added to the N-terminus of RefSeq Accession No. AAC82527.1), 184 (A added to the N-terminus of RefSeq Accession No. AAB59393.1), 185 (RefSeq Accession No. CAA27268.1), and 186 (A added to the N-terminus of RefSeq Accession No. AAB59394.1). Additionally, when an antigen-binding molecule or antibody containing an Fc domain mutant of a specific homozygous antibody is used as a test substance, the effect of the mutation of the corresponding mutant on Fc gamma receptor binding activity is evaluated using an antigen-binding molecule or antibody containing the same homozygous Fc domain as a control. As described above, an antigen-binding molecule or antibody containing an Fc domain mutant determined to have reduced Fc gamma receptor binding activity is appropriately prepared.

[1172] These known mutants include, for example, a mutant having a deletion of amino acids 231A-238S (EU numbering) (WO 2009 / 011941), as well as mutants C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11); C226S and C229S (Hum. Antibod. Hybridomas (1990) 1(1), 47-54); C226S, C229S, E233P, L234V, and L235A (Blood (2007) 109, 1185-1192).

[1173] Specifically, a preferred antigen-binding molecule or antibody comprises an Fc domain having a mutation (e.g., substitution) of at least one amino acid selected from the following amino acid positions within the amino acid forming the Fc domain of a specific isomorphic antibody: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, or 332 (EU numbering). The isoform of the antibody from which the Fc domain is derived is not particularly limited, and a suitable Fc domain derived from a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody may be used. It is preferable to use an Fc domain derived from an IgG1 antibody.

[1174] Desired antigen-binding molecules or antibodies include, for example, those comprising an Fc domain having any one of the substitutions shown below, the position thereof being designated according to EU numbering in the amino acids forming the Fc domain of the IgG1 antibody (each number indicates the position of an amino acid residue in EU numbering; the single-character amino acid symbol preceding the number indicates the amino acid residue before substitution, and the single-character amino acid symbol following the number indicates the amino acid residue after substitution):

[1175] (a) L234F, L235E, P331S;

[1176] (b) C226S / C229S, P238S;

[1177] (c) C226S, C229S; or

[1178] (d) C226S, C229S, E233P, L234V, L235A;

[1179] It also includes those having an Fc domain with a deleted amino acid sequence at positions 231 to 238.

[1180] Additionally, the preferred antigen-binding molecules or antibodies include those comprising an Fc domain having any one of the substitutions listed below, the position of which is specified according to EU numbering in the amino acids forming the Fc domain of the IgG2 antibody:

[1181] (e) H268Q, V309L, A330S and P331S;

[1182] (f) V234A;

[1183] (g) G237A;

[1184] (h) V234A and G237A;

[1185] (i) A235E and G237A; or

[1186] (j) V234A, A235E and G237A. Each number indicates the position of the amino acid residue in the EU numbering; the one-letter amino acid symbol before the number indicates the amino acid residue before substitution, and the one-letter amino acid symbol after the number indicates the amino acid residue after substitution.

[1187] Additionally, the preferred antigen-binding molecules or antibodies include those comprising an Fc domain having one of the substitutions listed below, the position of which is specified according to EU numbering in the amino acids forming the Fc domain of the IgG3 antibody:

[1188] (k) F241A;

[1189] (l) D265A; or

[1190] (m) V264A. Each number indicates the position of an amino acid residue in EU numbering; the one-letter amino acid symbol before the number indicates the amino acid residue before substitution, and the one-letter amino acid symbol after the number indicates the amino acid residue after substitution.

[1191] Additionally, the preferred antigen-binding molecules or antibodies include those comprising an Fc domain having one of the substitutions listed below, the position of which is specified according to EU numbering in the amino acids forming the Fc domain of the IgG4 antibody:

[1192] (n) L235A, G237A and E318A;

[1193] (o) L235E; or

[1194] (p) F234A and L235A. Each number indicates the position of the amino acid residue in the EU numbering; the 1-letter amino acid symbol before the number indicates the amino acid residue before substitution, and the 1-letter amino acid symbol after the number indicates the amino acid residue after substitution.

[1195] Other preferred antigen-binding molecules or antibodies include, for example, those comprising an Fc domain in which an amino acid at position 233, 234, 235, 236, 237, 327, 330, or 331 (EU numbering) in the amino acids forming the Fc domain of an IgG1 antibody is substituted with an amino acid at the corresponding position in the EU numbering of the corresponding IgG2 or IgG4. In some embodiments, the antigen-binding molecule or antibody of the present invention further comprises an Fc domain that exhibits reduced binding affinity for the human Fc gamma receptor compared to the natural human IgG1 Fc domain. In some embodiments, in the antigen-binding molecule or antibody of the present invention, the Fc domain comprises Arg at position 235 and Arg at position 236, wherein the amino acid positions are numbered according to the EU numbering.

[1196] Preferred antigen-binding molecules or antibodies also include those comprising an Fc domain in which one or more of the amino acids at positions 234, 235, and 297 (EU numbering) in the amino acids forming the Fc domain of, for example, an IgG1 antibody are substituted with other amino acids. The type of amino acid after substitution is not particularly limited, but an antigen-binding molecule or antibody comprising an Fc domain in which one or more of the amino acids at positions 234, 235, and 297 are substituted with alanine is particularly preferred.

[1197] Desired antigen-binding molecules or antibodies also include those comprising an Fc domain in which the amino acid at position 265 (EU numbering) in the amino acids forming the Fc domain of, for example, an IgG1 antibody is substituted with another amino acid. The type of amino acid after substitution is not particularly limited; however, an antigen-binding molecule or antibody comprising an Fc domain in which the amino acid at position 265 is substituted with alanine is particularly preferred.

[1198] In some embodiments, the preferred antigen-binding molecule or antibody also comprises an Fc domain that exhibits reduced binding affinity to the human Fc gamma receptor compared to the natural human IgG1 Fc domain, wherein the Fc region subunits included in the Fc domain comprise Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, wherein the amino acid positions are numbered according to EU numbering.

[1199] Antigen-binding domain that binds to PDGF-B

[1200] As used herein, the phrases “antigen-binding domain binding to PDGF-B” or “anti-PDGF-B antigen-binding domain” refer to an antigen-binding domain that specifically binds to an epitope on the aforementioned PDGF-B protein, or to a whole or part of a peptide of the PDGF-B protein, for example, human PDGF-B (an exemplary human PDGF-B is shown in SEQ ID NO. 172).

[1201] In certain embodiments, the antigen-binding domain that binds to PDGF-B comprises an antibody variable region (antibody light chain and heavy chain variable regions (VL and VH)). Suitable examples of a domain comprising antibody light chain and heavy chain variable regions include "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv2 (scFv2)", "Fab", "F(ab')2", etc. In certain embodiments, the antigen-binding domain that binds to PDGF-B comprises an antibody variable fragment. A domain comprising an antibody variable fragment may be provided from the variable domains of one or more antibodies.

[1202] In certain embodiments, the antigen-binding domain that binds to PDGF-B comprises three CDRs in the heavy chain variable region and three CDRs in the light chain variable region of the anti-PDGF-B antibody. In certain embodiments, the antigen-binding domain that binds to PDGF-B comprises the heavy chain variable region and the light chain variable region of the anti-PDGF-B antibody. In certain embodiments, the antigen-binding domain that binds to PDGF-B comprises a Fab structure.

[1203] Preferably, the anti-PDGF-B antibody comprises an H chain having an amino acid sequence (of the CDR of the H-chain variable region) as described in any one of embodiments (A-1)-(A-7) or in Table 5A below, and an L chain having an amino acid sequence (of the CDR of the L-chain variable region) as described in any one of embodiments (A-1)-(A-7) or in Table 5A below.

[1204] In certain embodiments, the antigen-binding domain that binds to PDGF-B comprises an antibody variable fragment that competes with any one antibody variable fragment shown in Table 5A for binding to human PDGF-B.

[1205] Alternatively, the antigen-binding domain that binds to PDGF-B comprises an antibody-variable fragment that competes for binding to human PDGF-B together with any one of the aforementioned antibody-variable fragments. Alternatively, the antigen-binding domain that binds to PDGF-B comprises an antibody-variable fragment that binds to the same epitope on human PDGF-B to which any one of the aforementioned antibody-variable fragments binds.

[1206] Antigen-binding domain that binds to PDGF-D

[1207] As used herein, the phrases “antigen-binding domain binding to PDGF-D” or “anti-PDGF-D antigen-binding domain” refer to an antigen-binding domain that specifically binds to an epitope on the aforementioned PDGF-D protein, or to a whole or part of a peptide of the PDGF-D protein, for example, human PDGF-D (an exemplary human PDGF-D is shown in SEQ No. 171).

[1208] In certain embodiments, the antigen-binding domain that binds to PDGF-D comprises an antibody variable region (antibody light chain and heavy chain variable regions (VL and VH)). Suitable examples of a domain comprising antibody light chain and heavy chain variable regions include "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv2 (scFv2)", "Fab", "F(ab')2", etc. In certain embodiments, the antigen-binding domain that binds to PDGF-D comprises an antibody variable fragment. A domain comprising an antibody variable fragment may be provided from the variable domains of one or more antibodies.

[1209] In certain embodiments, the antigen-binding domain that binds to PDGF-D comprises three CDRs in the heavy chain variable region of anti-PDGF-D and three CDRs in the light chain variable region. In certain embodiments, the antigen-binding domain that binds to PDGF-D comprises the heavy chain variable region and the light chain variable region of the anti-PDGF-D antibody. In certain embodiments, the antigen-binding domain that binds to PDGF-D comprises a Fab structure.

[1210] Preferably, the anti-PDGF-D antibody comprises an H chain having an amino acid sequence (of the CDR of the H-chain variable region) as described in any one of embodiments (A-1)-(A-7) or in Table 5B below, and an L chain having an amino acid sequence (of the CDR of the L-chain variable region) as described in any one of embodiments (A-1)-(A-7) or in Table 5B below.

[1211] In certain embodiments, the antigen-binding domain that binds to PDGF-D comprises any one of the antibody variable fragments presented in Table 5B below.

[1212] In certain embodiments, the antigen-binding domain that binds to PDGF-D comprises an antibody-variable fragment that binds to an epitope in human PDGF-D identical to any one of the antibody-variable fragments presented in Table 5B below.

[1213] Alternatively, the antigen-binding domain that binds to PDGF-D comprises an antibody-variable fragment that competes for binding to human PDGF-D together with any one of the aforementioned antibody-variable fragments. Alternatively, the antigen-binding domain that binds to PDGF-D comprises an antibody-variable fragment that binds to the same epitope on human PDGF-D to which any one of the aforementioned antibody-variable fragments binds.

[1214] In certain embodiments, the antigen binding domain that binds to PDGF-D comprises a heavy chain variable region comprising the amino acid sequence presented in SEQ ID NO. 165 and a light chain variable region comprising the amino acid sequence presented in SEQ ID NO. 166, and an antibody variable fragment that does not compete for binding to PDGF-D with the reference antibody.

[1215] Alternatively, the antigen-binding domain that binds to PDGF-D includes an antibody variable fragment that does not bind to the same epitope in the PDGF-D protein to which the reference antibody binds.

[1216] The amino acids included in the amino acid sequences described herein may undergo post-translational modification (e.g., the modification of N-terminal glutamine to pyroglutamic acid by pyroglutamide is generally known to those skilled in the art). Naturally, such sequences having post-translationally modified amino acids are also included in the amino acid sequences described herein.

[1217] Multispecific antigen-binding molecules that bind to PDGF-D and PDGF-B

[1218] "Multispecific antigen-binding molecule" means an antigen-binding molecule that specifically binds to more than one antigen. In a preferred embodiment, the multispecific antigen-binding molecule of the present invention comprises two or more antigen-binding domains, and different antigen-binding domains specifically bind to different antigens.

[1219] The multispecific antigen-binding molecule of the present invention, which binds to PDGF-D and PDGF-B, comprises a first antigen-binding domain that binds to PDGF-D and a second antigen-binding domain that binds to PDGF-B. A combination of an antigen-binding domain that binds to PDGF-B selected from those described in the "antigen-binding domain that binds to PDGF-B" and an antigen-binding domain that binds to PDGF-D selected from those described in the "antigen-binding domain that binds to PDGF-D" may be used.

[1220] For example, the first antigen-binding domain comprises antibody heavy chain and light chain variable regions, and the second antigen-binding domain comprises antibody heavy chain and light chain variable regions. Alternatively, the first antigen-binding domain comprises an antibody variable fragment, and the second antigen-binding domain comprises an antibody variable fragment. Alternatively, the first antigen-binding domain comprises a Fab structure, and / or the second antigen-binding domain comprises a Fab structure.

[1221] In specific embodiments, the present invention provides a multispecific antigen-binding molecule comprising a first antigen-binding domain comprising an antibody variable fragment that binds to PDGF-D, and a second antigen-binding domain comprising an antibody variable fragment that binds to PDGF-B. In specific embodiments, the present invention provides a bispecific antigen-binding molecule comprising a first antigen-binding domain that binds to PDGF-D, a second antigen-binding domain that binds to PDGF-B, and a third domain comprising an Fc region having reduced Fc gamma receptor binding activity. The Fc region may have reduced Fc gamma receptor binding activity compared to the Fc domain of an IgG1, IgG2, IgG3, or IgG4 antibody.

[1222] In specific embodiments, the present invention provides a bispecific antibody comprising a first antibody variable fragment that binds to human PDGF-D and a second antibody variable fragment that binds to human PDGF-B. In specific embodiments, the present invention provides a bispecific antibody comprising a first antibody variable fragment that binds to human PDGF-D, a second antibody variable fragment that binds to human PDGF-B, and an Fc region having reduced Fc gamma receptor binding activity. In specific embodiments, the present invention provides a bispecific antibody comprising a first antibody variable fragment that binds to human PDGF-D, a second antibody variable fragment that binds to human PDGF-B, and an Fc region having reduced Fc gamma receptor binding activity compared to a naturally occurring IgG Fc region.

[1223] Examples of preferred embodiments of the “multispecific antigen-binding molecule” of the present invention include multispecific antibodies. When an Fc region having reduced Fc gamma receptor binding activity is used as the Fc region of a multispecific antibody, an Fc region derived from a multispecific antibody may be appropriately used. Bispecific antibodies are particularly preferred as the multispecific antibodies of the present invention. In this case, the bispecific antibody is an antibody having two different specificities. IgG-type bispecific antibodies may be secreted from a hybridoma (quadromas) produced by fusing two types of hybridomas that produce IgG antibodies (Milstein et al., Nature (1983) 305, 537-540).

[1224] In addition, IgG type bispecific antibodies are secreted by introducing the genes for the L and H chains constituting the two types of IgG of interest—that is, a total of four genes—into a cell and co-expressing them. However, the number of combinations of IgG H and L chains that can be generated by these methods is theoretically 10. Consequently, it is difficult to purify IgG containing the desired combination of H and L chains from the 10 types of IgG. Furthermore, the amount of IgG with the theoretically desired combination will be significantly reduced, thus requiring large-scale culture and increasing production costs.

[1225] Therefore, a technique for promoting association between H chains having a desired combination and between L and H chains can be applied to the multispecific antigen-binding molecule of the present invention.

[1226] For example, a technique for inhibiting unwanted H-chain association by introducing electrostatic repulsion at the interface of the second constant region or the third constant region (CH2 or CH3) of the antibody H chain can be applied to multispecific antibody association (WO2006 / 106905).

[1227] In a technique for suppressing unintended H-chain association by introducing electrostatic repulsion at the CH2 or CH3 interface, examples of amino acid residues in contact at the interface of other constant regions of the H chain include regions corresponding to residues at EU numbering positions 356, 439, 357, 370, 399, and 409 in the CH3 region.

[1228] More specifically, examples include antibodies comprising two types of H-chain CH3 regions, wherein one to three pairs of amino acid residues of the first H-chain CH3 region selected from the amino acid residue pairs indicated in (1) to (3) below have the same type of charge: (1) amino acid residues contained in the H-chain CH3 region at EU numbering positions 356 and 439; (2) amino acid residues contained in the H-chain CH3 region at EU numbering positions 357 and 370; and (3) amino acid residues contained in the H-chain CH3 region at EU numbering positions 399 and 409.

[1229] Additionally, the antibody may be an antibody in which the amino acid residue pairs in the second H-chain CH3 region, which is different from the first H-chain CH3 region mentioned above, are selected from the amino acid residue pairs (1) to (3) mentioned above, wherein one to three amino acid residue pairs corresponding to the amino acid residue pairs (1) to (3) mentioned above, having the same type of charge in the first H-chain CH3 region mentioned above, have opposite charges to the corresponding amino acid residues in the first H-chain CH3 region mentioned above.

[1230] Each amino acid residue shown in (1) to (3) above comes into close proximity to each other during association. A person skilled in the art can identify the positions corresponding to the amino acid residues of (1) to (3) mentioned above in a desired H-chain CH3 region or H-chain invariant region through homology modeling using commercially available software, and can appropriately modify the amino acid residues at these positions.

[1231] In the antibody mentioned above, the "charged amino acid residue" is preferably selected from amino acid residues included, for example, in one of the following groups:

[1232] (a) glutamic acid (E) and aspartic acid (D); and

[1233] (b) Lysine (K), arginine (R), and histidine (H).

[1234] In the antibody mentioned above, the phrase “having the same charge” means, for example, that both of the two or more amino acid residues are selected from amino acid residues included in either of the groups (a) and (b) mentioned above. The phrase “having opposite charge” means, for example, that when at least one of the two or more amino acid residues is selected from amino acid residues included in either of the groups (a) and (b) mentioned above, the remaining amino acid residue is selected from amino acid residues included in the other group.

[1235] In a preferred embodiment, the aforementioned antibodies may have their first H-chain CH3 region and second H-chain CH3 region cross-linked by disulfide bonds.

[1236] In the present invention, the amino acid residues to be modified are not limited to the aforementioned amino acid residues in the antibody variable region or antibody constant region. Those skilled in the art can identify amino acid residues forming an interface in a mutant polypeptide or heteropolymer through homology modeling, etc., using commercially available software; and then amino acid residues at these positions can be modified to control association.

[1237] Other known techniques may also be used for the association of the multispecific antibodies of the present invention. Polypeptides containing Fc regions comprising different amino acids may be efficiently associated with each other by substituting an amino acid side chain present in one of the H-chain Fc regions of the antibody with a larger side chain (knob) and substituting an amino acid side chain present in the corresponding Fc region of the other H-chain with a smaller side chain (hole) so that the knob can be placed within the hole (WO1996 / 027011; Ridgway JB et al., Protein Engineering (1996) 9, 617-621; Merchant AM et al., Nature Biotechnology (1998) 16, 677-681; and US20130336973).

[1238] In addition, other known techniques may also be used for the formation of the multispecific antibodies of the present invention. The association of polypeptides having different sequences can be efficiently induced by the complementary association of strand-swapped domain CH3s generated by changing a portion of one H-chain CH3 of the antibody to a corresponding IgA-derived sequence and introducing the corresponding IgA-derived sequence into a complementary portion of another H-chain CH3 (Protein Engineering Design & Selection, 23; 195-202, 2010). These known techniques may also be used to efficiently form the multispecific antibodies of interest.

[1239] In addition, antibody production techniques using the association of antibody CH1 and CL and the association of VH and VL as described in WO 2011 / 028952, WO2014 / 018572, and Nat Biotechnol. 2014 Feb; 32(2):191-8; bispecific antibody production techniques combining separately prepared monoclonal antibodies (Fab arm exchange) as described in WO2008 / 119353, WO2011 / 131746, WO2015 / 046467, and WO2016159213; techniques for controlling the association between antibody heavy chain CH3s as described in WO2012 / 058768 and WO2013 / 063702; and bispecific antibody production techniques composed of two types of light chains and one type of heavy chain as described in WO2012 / 023053; A bispecific antibody production technique using two bacterial cell strains that individually express one of the antibody chains, including a single H chain and a single L chain, as described by Christoph et al. (Nature Biotechnology Vol. 31, p 753-758 (2013)); and such a technique can be used to form multiple specific antibodies.

[1240] Alternatively, even in cases where the multispecific antibody of interest cannot be efficiently formed, the multispecific antibody of the present invention can be obtained by isolating and purifying the multispecific antibody of interest from the generated antibody. For example, a method has been reported in which two types of homologous forms and a heterologous antibody of interest can be purified by ion exchange chromatography by introducing amino acid substitutions to variable regions of two types of H chains to impart a difference in isoelectric point (WO2007114325). To date, as a method for purifying heterologous antibodies, a method has been reported in which protein A is used to purify a heterodimeric antibody comprising a mouse IgG2a H chain that binds to protein A and a rat IgG2b H chain that does not bind to protein A (WO98050431 and WO95033844). In addition, heterodimeric antibodies can be efficiently purified by using an H chain in which the amino acid residues at EU numbering positions 435 and 436, which are IgG-protein A binding sites, are substituted with Tyr, His, or amino acids exhibiting different protein A affinities, or by using an H chain with different protein A affinities to change the interaction between each H chain and protein A, and then using a protein A column.

[1241] Additionally, an Fc region with improved C-terminal heterogeneity can be suitably used as the Fc region of the present invention. More specifically, the present invention provides an Fc region generated by deleting glycine at position 446 and lysine at position 447, as specified by the amino acid sequences of two polypeptides constituting the Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[1242] Multiple techniques, such as two or more of these techniques, may be used in combination. Additionally, these techniques may be applied appropriately and separately to two H chains to be associated. Furthermore, these techniques may be used in combination with the aforementioned Fc region having reduced binding activity to the Fc gamma receptor. Additionally, the antigen-binding molecule of the present invention may be a molecule separately produced to have the same amino acid sequence as the antigen-binding molecule that has undergone the modification described above.

[1243] Preferably, the antigen-binding molecule of the present invention is a multispecific antigen-binding molecule comprising a first antigen-binding domain that binds to PDGF-D and a second antigen-binding domain that binds to PDGF-B. More preferably, the antigen-binding molecule of the present invention specifically binds to PDGF-B (i.e., PDGF-B, PDGF-AB, and PDGF-BB) and PDGF-D (i.e., PDGF-D and PDGF-DD) and inhibits their interaction with PDGFR, thereby inhibiting the PDGF-B activity and PDGF-D activity defined above.

[1244] In a preferred embodiment, the multispecific antigen-binding molecule of the present invention comprises one or more polypeptide chains listed in Tables 5A to 5C and Tables 6A to 6C.

[1245] Note that in the multispecific antigen-binding molecule of the present invention, the first antigen-binding domain that specifically binds to PDGF-D does not include a heavy chain variable region containing the amino acid sequence presented in SEQ ID NO. 165 and a light chain variable region containing the amino acid sequence presented in SEQ ID NO. 166, and the second antigen-binding domain that specifically binds to PDGF-B does not include a heavy chain variable region containing the amino acid sequence presented in SEQ ID NO. 161 and a light chain variable region containing the amino acid sequence presented in SEQ ID NO. 162.

[1246] Pharmaceutical preparations

[1247] Pharmaceutical formulations of antigen-binding molecules (e.g., antibodies) binding to PDGF-D or multispecific antigen-binding molecules (e.g., bispecific antibodies) binding to PDGF-D and PDGF-B as described herein are prepared by mixing such antigen-binding molecules (e.g., antibodies) or multispecific antigen-binding molecules (e.g., bispecific antibodies) having a desired degree of purity in the form of lyophilized formulations or aqueous solutions with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are non-toxic to recipients at commonly used doses and concentrations and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; Preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohols; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; Metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).Exemplary pharmaceutically acceptable carriers herein include niche drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHAS...

Claims

Claim 1 As a multispecific antigen-binding molecule, comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to PDGF-D and comprises an antibody heavy chain variable region comprising a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence presented in SEQ ID NO. 292, a heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 293, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 294, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 300, a light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 301, and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 302; Herein, a multispecific antigen-binding molecule comprising any one of the following (a1) to (a5), wherein the second antigen-binding domain specifically binds to PDGF-B and comprises: (a1) an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 236, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 258, a light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 259, and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 260;(a2) an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 266, a light chain CDR 2 having the amino acid sequence presented in SEQ NO. 267, and a light chain CDR 3 having the amino acid sequence presented in SEQ NO. 268; (a3) ​​an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ NO. 236, and a light chain CDR 1 having the amino acid sequence presented in SEQ NO. 274, and an amino acid sequence presented in SEQ NO. 275 (a4) an antibody light chain variable region comprising a light chain CDR 2 and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 276; and a heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 242, a heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 243, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 244, and a light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 274, a light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 275, and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 276.and (a5) an antibody heavy chain variable region comprising a heavy chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 234, a heavy chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 235, and a heavy chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 236, and an antibody light chain variable region comprising a light chain CDR 1 having the amino acid sequence presented in SEQ ID NO. 282, a light chain CDR 2 having the amino acid sequence presented in SEQ ID NO. 283, and a light chain CDR 3 having the amino acid sequence presented in SEQ ID NO. 284.; Claim 2 In claim 1, the first antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 291, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 299; and a multispecific antigen-binding molecule comprising any one of the following (a1) to (a5): (a1) an amino acid sequence having at least 90%, 91%, 92%, 93%, and the amino acid sequence presented in SEQ ID NO.

233. A heavy chain variable region comprising an amino acid sequence having 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ NO. 257, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ NO. 257; (a2) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ NO. 233, and at least 90%, 91%, A light chain variable region comprising an amino acid sequence having 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity;(a3) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ NO. 233, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ NO. 273; (a4) a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ NO. 241 A heavy chain variable region comprising an amino acid sequence having identity, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ ID NO. 273; and (a5) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 233, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence presented in SEQ No. 281.; Claim 3 A multispecific antigen-binding molecule according to claim 1 or 2, wherein the second antigen-binding domain comprises framework region 1 of human variable kappa subgroup 2. Claim 4 A multispecific antigen-binding molecule according to any one of claims 1 to 3, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant regions of the first antigen-binding domain and the second antigen-binding domain associate to form an antibody Fc domain. Claim 5 In paragraph 4, the antibody Fc domain is a multispecific antigen-binding molecule in which the antibody Fc domain is a human Fc domain derived from IgG1, IgG2, IgG3, or IgG4, preferably IgG1. Claim 6 In claim 4 or 5, the multispecific antigen-binding molecule comprises one or more of the following amino acid residues (i) to (xviii): (i) glutamic acid or lysine at position 175 (EU numbering) in the heavy chain constant region; (ii) glutamic acid at position 147 (EU numbering) in the heavy chain constant region; (iii) glutamic acid or lysine at position 131 (EU numbering) in the light chain constant region; (iv) asparagine at position 145 (EU numbering) in the light chain constant region; (v) lysine at position 152 (EU numbering) in the light chain constant region; (vi) glutamic acid or lysine at position 160 (EU numbering) in the light chain constant region; (vii) lysine at position 196 (EU numbering) in the heavy chain constant region; (viii) position in the heavy chain constant region Arginine at 235 (EU numbering); (ix) Arginine at position 236 (EU numbering) within the heavy chain constant region; (x) Lysine at position 356 (EU numbering) within the heavy chain constant region; (xi) Lysine at position 399 (EU numbering) within the heavy chain constant region; (xii) Glutamic acid at position 409 (EU numbering) within the heavy chain constant region; (xiii) Glutamic acid at position 419 (EU numbering) within the heavy chain constant region; (xiv) Leucine at position 428 (EU numbering) within the heavy chain constant region; (xv) Alanine at position 434 (EU numbering) within the heavy chain constant region; (xvi) Arginine at position 438 (EU numbering) within the heavy chain constant region; (xvii) of at position 439 (EU numbering) within the heavy chain constant region Glutamic acid; (xviii) Glutamic acid at position 440 (EU numbering) in the heavy chain constant region. Claim 7 A multispecific antigen-binding molecule according to any one of claims 4 to 6, wherein the heavy chain constant region and light chain constant region of each of the first antigen-binding domain and the second antigen-binding domain comprise at least one of the following (a) to (d): (a) the heavy chain constant region of the first antigen-binding domain comprises at least one of Lys at position 175 and Lys at position 196 (EU numbering); (b) the light chain constant region of the first antigen-binding domain comprises at least one of Glu at position 131, Glu at position 160, and Lys at position 152 (EU numbering); (c) the heavy chain constant region of the second antigen-binding domain comprises at least one of Glu at position 147 and Glu at position 175 (EU numbering); and (d) the light chain constant region of the second antigen-binding domain comprises at least one of Lys at position 131 and Lys at position 160 (EU numbering). Claim 8 A multispecific antigen-binding molecule according to any one of claims 4 to 7, wherein the first heavy chain constant region comprises at least one of Lys at position 399 and Glu (EU numbering) at position 439, and the second heavy chain constant region comprises at least one of Lys at position 356, Glu at position 409, and Glu (EU numbering) at position 419. Claim 9 A multispecific antigen-binding molecule according to any one of claims 4 to 8, wherein the antibody Fc domain comprises at least one of Arg at position 235, Arg at position 236, Leu at position 428, Ala at position 434, Arg at position 438, and Glu (EU numbering) at position 440. Claim 10 A multispecific antigen-binding molecule comprising any one of the following (a) to (e): (a) a first heavy chain comprising the amino acid sequence presented in SEQ ID NO. 372, a first light chain comprising the amino acid sequence presented in SEQ ID NO. 373, a second heavy chain comprising the amino acid sequence presented in SEQ ID NO. 370, and a second light chain comprising the amino acid sequence presented in SEQ ID NO. 371; (b) a first heavy chain comprising the amino acid sequence presented in SEQ ID NO. 376, a first light chain comprising the amino acid sequence presented in SEQ ID NO. 377, a second heavy chain comprising the amino acid sequence presented in SEQ ID NO. 374, and a second light chain comprising the amino acid sequence presented in SEQ ID NO. 375; (c) a first heavy chain comprising the amino acid sequence presented in SEQ ID NO. 380, a first light chain comprising the amino acid sequence presented in SEQ ID NO. 381, a second heavy chain comprising the amino acid sequence presented in SEQ ID NO. 378, and a second heavy chain comprising the amino acid sequence presented in SEQ ID NO. 379 Light chain; (d) a first heavy chain comprising the amino acid sequence presented in SEQ ID NO. 384, a first light chain comprising the amino acid sequence presented in SEQ ID NO. 385, a second heavy chain comprising the amino acid sequence presented in SEQ ID NO. 382, ​​and a second light chain comprising the amino acid sequence presented in SEQ ID NO. 383; and (e) a first heavy chain comprising the amino acid sequence presented in SEQ ID NO. 388, a first light chain comprising the amino acid sequence presented in SEQ ID NO. 389, a second heavy chain comprising the amino acid sequence presented in SEQ ID NO. 386, and a second light chain comprising the amino acid sequence presented in SEQ ID NO.

387. Claim 11 A pharmaceutical composition comprising a multispecific antigen-binding molecule according to any one of claims 1 to 10. Claim 12 Optionally, fibrosis (e.g., cardiomyopathy, hepatic fibrosis, renal fibrosis, cutaneous fibrosis, ocular fibrosis, and myelofibrosis), nephritis, chronic kidney disease, progressive kidney disease and / or related diseases, e.g., IgA nephropathy, glomerulonephritis, angioproliferative nephritis, angioproliferative glomerulonephritis, interstitial telomerulonephritis, systemic lupus erythematosus, lupus nephritis, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, nephrosclerosis, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, membranous nephropathy, non-alcoholic steatohepatitis (NASH), vascular disease, atherosclerosis, restenosis, stroke, eye disease, proliferative vitreoretinopathy (PVR), retinopathy of prematurity (ROP), neovascularization-related age-associated macular degeneration (NVAMD), glaucoma, and retinitis pigmentosa (RP), preferably chronic kidney A multispecific antigen-binding molecule of any one of claims 1 to 10 or a pharmaceutical composition of claim 11 for use in a therapy for the treatment of a disease (CKD), particularly glomerulonephritis accompanied by vascular interstitial cell proliferation, e.g., IgA nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, interstitial proliferative nephritis, vascular interstitial proliferative glomerulonephritis and / or lupus nephritis. Claim 13 An isolated polynucleotide or a plurality of polynucleotides encoding a multispecific antigen-binding molecule of any one of claims 1 to 10. Claim 14 A host cell comprising the polynucleotide of claim 13 or a plurality of polynucleotides. Claim 15 A method for generating a multispecific antigen-binding molecule according to any one of claims 1 to 10, comprising the steps of: (a) culturing a host cell of claim 14 under conditions suitable for the expression of the multispecific antigen-binding molecule; and (b) recovering the multispecific antigen-binding molecule.

Images