Ligand-binding molecules capable of modulating ligand-binding activity

Abstract

This invention relates to a ligand-binding molecule whose binding activity to a ligand is weakened by being cleaved at a cleavage site, a method for manufacturing the same, a complex formed by the ligand-binding molecule and a ligand, a fusion protein containing the ligand-binding molecule and a ligand, and a pharmaceutical composition containing the ligand-binding molecule or a fusion protein containing the ligand-binding molecule and a ligand.

Description

Technical Field

[0001] The present invention provides a ligand-binding molecule having at least one cleavage site and whose binding to a ligand is weakened when the cleavage site is cleaved, a method for manufacturing the ligand-binding molecule, and a pharmaceutical composition containing the ligand-binding molecule. Prior Technology

[0002] Antibodies exhibit high stability and few side effects in plasma, thus attracting significant attention as pharmaceutical products. Several IgG1 antibody drugs are already on the market, and many more are currently under development (Non-Patent Literature 1 and Non-Patent Literature 2).

[0003] As cancer treatments using antibody-based medicines, those currently recognized include Rituxan (anti-CD20 antigen), Cetuximab (anti-EGFR antigen), and Herceptin (anti-HER2 antigen) (Non-Patent Literature 3). These antibody molecules bind to antigens expressed on cancer cells and exert damaging activity against cancer cells through ADCC, signal blocking, and other mechanisms.

[0004] Furthermore, it is known that there are methods for delivering ligands such as interleukins to solid tumors by fusing antibody molecules that bind to cancer antigens highly expressed in cancer cells. These methods utilize immune interleukins delivered to solid tumors to exert anti-tumor effects through immune activation. Interleukins, primarily IL2, IL12, and TNF, are highly toxic and act locally on the tumor. Therefore, it is hoped that antibodies can be used to deliver these ligands to the tumor site, thereby reducing side effects and enhancing efficacy (Non-Patent Literature 4, 5, 6). However, these methods have drawbacks, including insufficient clinical efficacy when administered systemically, a narrow therapeutic window, and high toxicity preventing systemic administration. Consequently, they have not yet been approved as pharmaceutical products.

[0005] The main reasons for this include: although they are immunocytokines, systemically administered immunocytokines are exposed throughout the body, which may lead to systemic effects and toxicity, or they may be administered in very low doses to avoid toxicity. There are also reports that immunocytokines fused with IL2 and those fused with IL2 but not bound to cancer antigens have no difference in antitumor efficacy (Non-Patent Literature 7).

[0006] Regarding methods to avoid the aforementioned problems, some reports describe molecules formed by the binding of cytokines to their receptors via a linker that is cleaved by proteases highly expressed in cancer. Cytokines are inhibited by the linker-bound cytokines receptor, but if the linker is cleaved by the protease, the cytokines are released from the receptor and become active. For example, some reports describe molecules formed by the binding of TNF-alpha and TNF-R via a linker cleaved by uPA (Non-Patent Document 8), and molecules formed by the binding of IL2 and IL2R via a linker cleaved by MMP2 (Non-Patent Document 9). However, while these molecules show cytokine activity before linker cleavage, the increase in activity due to linker cleavage is only about 10-fold. Furthermore, some reports describe replacing IL2R with anti-IL2 scFv, forming a molecule formed by the binding of IL2 via a linker cleaved by MMP-2 (Non-Patent Document 9). [Previous Technical Documents] [Non-patent literature]

[0007] [Non-Patent Literature 1] Monoclonal antibody successes in the clinic. Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nat. Biotechnol. (2005) 23, 1073-1078 [Non-Patent Literature 2] The therapeutic antibodies market to 2008. Pavlou AK, Belsey MJ., Eur. J. Pharm. Biopharm. (2005) 59 (3), 389-396 [Non-Patent Literature 3] Monoclonal antibodies: versatile platforms for cancer immunotherapy. Weiner LM, Surana R, Wang S., Nat. Rev. Immunol. (2010) 10 (5), 317-327 [Non-Patent Document 4] Cyclophosphamide and tucotuzumab (huKS-IL2) following first-line chemotherapy in responding patients with extensive-disease small-cell lung cancer. Gladkov O, Ramlau R, Serwatowski P, Milanowski J, Tomeczko J, Komarnitsky PB, Kramer D, Krzakowski MJ. Anticancer Drugs. 2015 Nov;26(10):1061-8. [Non-Patent Document 5] Defining the Pharmacodynamic Profile and Therapeutic Index of NHS-IL12 Immunocytokine in Dogs with Malignant Melanoma. Paoloni M, Mazcko C, Selting K, Lana S, Barber L, Phillips J, Skorupski K, Vail D, Wilson H, Biller B, Avery A, Kiupel M, LeBlanc A, Bernhardt A, Brunkhorst B, Tighe R, Khanna C. PLoS One. 2015 Jun 19;10(6):e0129954. [Non-patent Document 6] Isolated limb perfusion with the tumor-targeting human monoclonal antibody-cytokine fusion protein L19-TNF plus melphalan and mild hyperthermia in patients with locally advanced extremity melanoma. Papadia F, Basso V, Patuzzo R, Maurichi A, Di Florio A, Zardi L, Ventura E, Gonzalez-Iglesias R, Lovato V, Giovannoni L, Tasciotti A, Neri D, Santinami M, Menssen HD, De Cian F. J Surg Oncol. 2013 Feb;107(2):173-9. [Non-patent Document 7] Antigen specificity can be irrelevant to immunocytokine efficacy and biodistribution. Tzeng A, Kwan BH, Opel CF, Navaratna T, Wittrup KD. Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3320-5. [Non-patent Document 8] Cancer Immunol Immunother. 2006 Dec;55(12):1590-600. Epub 2006 Apr 25. Target-selective activation of a TNF prodrug by urokinase-type plasminogen activator (uPA) mediated proteolytic processing at the cell surface. Gerspach J1, Nemeth J, Munkel S, Wajant H, Pfizenmaier K. [Patent Document 9] Immunology. 2011 Jun;133(2):206-20. doi: 10.1111 / j.1365-2567.2011.03428.x. Epub 2011 Mar 23. Development of an attenuated interleukin-2 fusion protein that can be activated by tumour-expressed proteases. Puskas J1, Skrombolas D, Sedlacek A, Lord E, Sullivan M, Frelinger J. Summary of the Invention

[0008] (The problem the invention aims to solve)

[0009] The present invention was made in view of the following circumstances, and one of its objectives is to provide a ligand-binding molecule that selectively activates ligands such as intercytokines or chemokines in a target tissue, a pharmaceutical composition containing the ligand-binding molecule, the pharmaceutical composition, and a method for manufacturing the active ingredient. (Methods for solving problems)

[0010] The inventors of this invention have diligently researched and developed a ligand-binding molecule that weakens the binding activity to ligands by cleaving the cleavage site. Furthermore, the inventors have found that this ligand-binding molecule, or a pharmaceutical composition containing this ligand-binding molecule, is useful in treating diseases using the ligand, and is useful in treating diseases including the administration of the ligand-binding molecule, and in manufacturing medicines for treating diseases. The inventors have also developed a method for manufacturing this ligand-binding molecule, thus completing this invention.

[0011] The present invention is based on such knowledge, and specifically includes the following exemplary states. (1) A ligand-binding molecule, which is a molecule capable of binding to a ligand, wherein the molecule is a polypeptide having at least one cleavage site, and the binding to the ligand is weakened when the molecule is cleaved at at least one cleavage site. (2) The ligand-binding molecule as in (1), wherein the ligand is freed from the ligand-binding molecule when the aforementioned cleavage site is cleaved. (3) A ligand-binding molecule such as (1) or (2), wherein the cleavage site includes a protease cleavage sequence. (4) The ligand-binding molecule as in (3), wherein the protease is a tissue-specific protease. (5) The ligand-binding molecule as in (4), wherein the target tissue is cancer tissue and the protease specific to the target tissue is a protease specific to cancer tissue. (6) The ligand-binding molecule as in (4), wherein the target tissue is an inflamed tissue and the target tissue-specific protease is an inflamed tissue-specific protease. (7) The ligand-binding molecule of any one of (3) to (6), wherein the aforementioned protease is selected from at least one protease selected from Matriptase, urokinase (uPA), and metalloproteinase. (8) The ligand-binding molecule as in (3), wherein the aforementioned protease cleavage sequence contains a sequence selected from the sequences recorded in sequence numbers 3, 34, 66, 70, 71, 72, 73, 35, 75, 76, and 345. (9) A ligand-binding molecule of any one of (3) to (8), wherein a first mobile linker is further attached to one end of the aforementioned protease cleavage sequence. (10) The ligand-binding molecule as in (9), wherein a second mobile linker is attached to the other end of the aforementioned protease cleavage sequence. (11) The ligand-binding molecule as in (9), wherein the aforementioned first mobile linker is a mobile linker composed of a glycine-serine polymer. (12) The ligand-binding molecule as in (10), wherein the aforementioned second mobile linker is a mobile linker composed of a glycine-serine polymer. (13) The ligand-binding molecule of any one of (1) to (12), wherein the aforementioned ligand-binding molecule includes antibody VH, antibody VL, and antibody invariant region. (14) The ligand-binding molecule as in (13), wherein the aforementioned cleavage site, or the aforementioned protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence and the first mobile linker and the second mobile linker, is located within the aforementioned antibody invariant region. (15) The ligand-binding molecule as in (14), wherein the aforementioned cleavage site, or the aforementioned protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence and the first mobile linker and the second mobile linker, is inserted at any position in the sequence from amino acid 118 (EU number) to amino acid 140 (EU number) in the antibody heavy chain invariant region. (16) The ligand-binding molecule as in (14), wherein the aforementioned cleavage site, or the aforementioned protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence and the first mobile linker and the second mobile linker, is inserted at any position in the sequence from amino acid 108 (EU number) (Kabat number 108) to amino acid 131 (EU number) (Kabat number 131) in the antibody light chain invariant region. (17) The ligand-binding molecule as in (13), wherein the aforementioned cleavage site, or the aforementioned protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence and the first mobile linker and the second mobile linker, is located within the aforementioned antibody VH or the aforementioned antibody VL. (18) The ligand-binding molecule as in (17), wherein the aforementioned cleavage site, or the aforementioned protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence and the first mobile linker and the second mobile linker, is inserted at any position in the sequence of the group consisting of amino acids VH7 (Kabat number) to 16 (Kabat number), amino acids 40 (Kabat number) to 47 (Kabat number), amino acids 55 (Kabat number) to 69 (Kabat number), amino acids 73 (Kabat number) to 79 (Kabat number), amino acids 83 (Kabat number) to 89 (Kabat number), amino acids 95 (Kabat number) to 99 (Kabat number), and amino acids 101 (Kabat number) to 113 (Kabat number). (19) The ligand-binding molecule as in (17), wherein the aforementioned cleavage site, or the aforementioned protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence and the first mobile linker and the second mobile linker, is inserted at any position in the sequence of the group consisting of antibody VL7 amino acids (Kabat number) to 19 amino acids (Kabat number), 39 amino acids (Kabat number) to 46 amino acids (Kabat number), 49 amino acids (Kabat number) to 62 amino acids (Kabat number), and 96 amino acids (Kabat number) to 107 amino acids (Kabat number). (20) The ligand-binding molecule as in (13), wherein the aforementioned cleavage site, or the aforementioned protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence and the first mobile linker and the second mobile linker, is located near the boundary between the aforementioned antibody invariant region and the aforementioned antibody VH, or / and near the boundary between the aforementioned antibody invariant region and the aforementioned antibody VL. (21) The ligand-binding molecule as in (20), wherein the aforementioned cleavage site, or the aforementioned protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence and the first mobile linker and the second mobile linker, is inserted at any position in the sequence from amino acid (Kabat number) of antibody VH109 to amino acid (EU number) of antibody heavy chain invariant region 122. (22) The ligand-binding molecule as in (20), wherein the aforementioned cleavage site, or the aforementioned protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence and the first mobile linker and the second mobile linker, is inserted at any position in the sequence from amino acid (Kabat number) at antibody VL104 to amino acid (EU number) at antibody light chain invariant region 113 (Kabat number 113). (23) The ligand-binding molecule of any one of (13) to (22), wherein, The ligand binds to the antibody VL in the molecule and the antibody VH assembles together. This assembly is discontinued either by the cleavage site being cleaved or by the protease cleavage sequence being cleaved by the protease. (24) A ligand-binding molecule as in any of (1) to (23), wherein the aforementioned ligand is a biologically active molecule, and the aforementioned ligand-binding molecule inhibits the biological activity of the aforementioned ligand by binding to the aforementioned ligand. (25) A ligand-binding molecule of any one of (1) to (24), wherein the aforementioned ligand is a cytokine or a chemokine. (26) The ligand-binding molecule of any one of (1) to (24), wherein the aforementioned ligand system is selected from interleukins, interferons, hematopoietic factors, TNF superfamily, chemokines, cell proliferation factors and TGF-β family ligands. (27) A ligand-binding molecule as in any of (1) to (24), wherein the aforementioned ligand is CXCL10, IL12, PD1, or IL6R. (28) The ligand-binding molecule as in (27), wherein the aforementioned ligand is CXCL10, and the aforementioned ligand-binding molecule contains antibody VH and antibody VL, the ligand-binding molecule: (a) Having antibody VH containing H-CDR1 (sequence number 374), H-CDR2 (sequence number 375), and H-CDR3 (sequence number 376), and antibody VL containing L-CDR1 (sequence number 377), L-CDR2 (sequence number 378), and L-CDR3 (sequence number 379); or (b) Having antibody VH containing H-CDR1 (sequence number 380), H-CDR2 (sequence number 381), and H-CDR3 (sequence number 382), and antibody VL containing L-CDR1 (sequence number 383), L-CDR2 (sequence number 384), and L-CDR3 (sequence number 385); or (c) Having antibody VH and antibody VL that compete with (a) or (b); or (d) Having antibodies VH and VL that bind to the same antigenic determinant as (a) or (b). (29) The ligand-binding molecule as in (28), wherein the aforementioned ligand-binding molecule contains an antibody heavy chain selected from the sequences shown in sequence numbers 4-14, 23-27, 33, 59, 60, 356, and 367, or an antibody light chain selected from the sequences shown in sequence numbers 15-22. (30) The ligand-binding molecule as in (27), wherein the aforementioned ligand is IL12, and the aforementioned ligand-binding molecule contains antibody VH and antibody VL, the ligand-binding molecule: (a) Having antibody VH containing H-CDR1 (sequence number 386), H-CDR2 (sequence number 387), and H-CDR3 (sequence number 388), and antibody VL containing L-CDR1 (sequence number 389), L-CDR2 (sequence number 390), and L-CDR3 (sequence number 391); or (b) Possesses antibodies VH and VL that compete with (a); or (c) and (a) are antibodies VH and VL that bind to the same antigenic determinant. (31) The ligand-binding molecule as in (30), wherein the aforementioned ligand-binding molecule contains an antibody heavy chain represented by sequence number 146. (32) The ligand-binding molecule as in (27), wherein the aforementioned ligand is PD1, and the aforementioned ligand-binding molecule contains antibody VH and antibody VL, the ligand-binding molecule: (a) Having antibody VH containing H-CDR1 (sequence number 392), H-CDR2 (sequence number 393), and H-CDR3 (sequence number 394), and antibody VL containing R3L-CDR1 (sequence number 395), L-CDR2 (sequence number 396), and L-CDR3 (sequence number 397); or (b) Possesses antibodies VH and VL that compete with (a); or (c) Having antibodies VH and VL that bind to the same antigenic determinant as (a). (33) The ligand-binding molecule as in (32), wherein the aforementioned ligand-binding molecule contains an antibody heavy chain selected from the sequences shown in sequence numbers 304 and 305, or an antibody light chain selected from the sequences shown in sequence numbers 306-315 and 322. (34) The ligand-binding molecule as in (27), wherein the aforementioned ligand is IL-6R (IL-6 receptor), and the aforementioned ligand-binding molecule contains antibody VH and antibody VL, the ligand-binding molecule: (a) Having antibody VH containing H-CDR1 (sequence number: 398), H-CDR2 (sequence number: 399), and H-CDR3 (sequence number: 400), and antibody VL containing L-CDR1 (sequence number: 401), L-CDR2 (sequence number: 402), and L-CDR3 (sequence number: 403); or (b) Possesses antibodies VH and VL that compete with (a); or (c) Having antibodies VH and VL that bind to the same antigenic determinant as (a). (35) The ligand-binding molecule as in (34), wherein the aforementioned ligand-binding molecule contains an antibody heavy chain selected from the sequences shown in sequence numbers 153-156, 157-159 and 404-470, or an antibody light chain selected from the sequences shown in sequence numbers 471-535. (36) Any of the ligand-binding molecules in (1) to (35), wherein the aforementioned ligand-binding molecule is an IgG antibody. (37) The ligand-binding molecule of any one of (1) to (36) is bound to the aforementioned ligand. (38) The ligand-binding molecule of any one of (1) to (36) is fused with the aforementioned ligand. (39) The ligand-binding molecule as in (38), wherein the aforementioned ligand-binding molecule will not further bind to other ligands in the state of already fused with the ligand. (40) Ligand-binding molecules such as (38) or (39), wherein the aforementioned ligand-binding molecules fuse with the aforementioned ligands via a linker. (41) The ligand-binding molecule as in (40), wherein the aforementioned linker does not contain a protease cleavage sequence. (42) The ligand-binding molecule of any one of (38) to (41), wherein the ligand is CXCL10, the ligand-binding molecule contains an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused to the ligand. (43) The ligand-binding molecule as in (42), wherein the aforementioned cleavage site is contained in the aforementioned antibody light chain or the aforementioned antibody heavy chain. (44) A ligand-binding molecule as in (42) or (43), wherein the aforementioned ligand is CXCL10, and the antibody light chain contained in the aforementioned ligand-binding molecule is fused with the aforementioned ligand, wherein the aforementioned ligand-binding molecule: (a) Having an antibody heavy chain containing H-CDR1 (Sequence No. 374), H-CDR2 (Sequence No. 375), and H-CDR3 (Sequence No. 376), and an antibody light chain containing L-CDR1 (Sequence No. 377), L-CDR2 (Sequence No. 378), and L-CDR3 (Sequence No. 379); or (b) Having an antibody heavy chain containing H-CDR1 (serial number 380), H-CDR2 (serial number 381), and H-CDR3 (serial number 382), and an antibody light chain containing L-CDR1 (serial number 383), L-CDR2 (serial number 384), and L-CDR3 (serial number 385). (45) The ligand-binding molecule of any one of (42) to (44), wherein the aforementioned ligand is the CXCL10 variant represented by sequence number 370. (46) The ligand-binding molecule of any one of (38) to (41), wherein the ligand is PD1, the ligand-binding molecule contains an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused to the ligand. (47) The ligand-binding molecule as in (46), wherein the aforementioned cleavage site is contained in the aforementioned antibody light chain or the aforementioned antibody heavy chain. (48) The ligand-binding molecule as in (46) or (47), wherein the aforementioned ligand is PD1, the aforementioned antibody light chain has R3L-CDR1 as sequence number 395, L-CDR2 as sequence number 396, and L-CDR3 as sequence number 397, and the aforementioned antibody heavy chain has H-CDR1 as sequence number 392, H-CDR2 as sequence number 393, and H-CDR3 as sequence number 394. (49) The ligand-binding molecule of any one of (46) to (48), wherein the aforementioned ligand is PD1 represented by sequence number 320. (50) A ligand-binding molecule as in any of (46) to (49), wherein the aforementioned ligand is PD1, the antibody heavy chain contained in the aforementioned ligand-binding molecule is fused with the aforementioned ligand, and a series of peptides obtained by fusing PD1 with the antibody heavy chain contains a sequence selected from the sequences shown in sequence numbers 323 and 324. (51) A ligand-binding molecule as in any of (46) to (49), wherein the aforementioned ligand is PD1, the antibody light chain contained in the aforementioned ligand-binding molecule is fused with the aforementioned ligand, and a series of peptides obtained by fusing PD1 with the antibody light chain contains sequences selected from the sequences shown in sequence numbers 325 to 334. (52) The ligand-binding molecule of any one of (38) to (41), wherein the ligand is IL12, the ligand-binding molecule contains an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused to the ligand. (53) The ligand-binding molecule as in (52), wherein the aforementioned cleavage site is contained in the aforementioned antibody light chain or the aforementioned antibody heavy chain. (54) The ligand-binding molecule as in (52) or (53), wherein the aforementioned ligand is IL12, the aforementioned antibody light chain has L-CDR1 as sequence number 389, L-CDR2 as sequence number 390, and L-CDR3 as sequence number 391, and the aforementioned antibody heavy chain has H-CDR1 as sequence number 386, H-CDR2 as sequence number 387, and H-CDR3 as sequence number 388. (55) A ligand-binding molecule as in any of (38) to (41), wherein the ligand is IL-6R, the ligand-binding molecule contains an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused to the ligand. (56) The ligand-binding molecule as in (55), wherein the aforementioned cleavage site is contained in the aforementioned antibody light chain or the aforementioned antibody heavy chain. (57) The ligand-binding molecule as in (55) or (56), wherein the ligand is IL-6R, the antibody light chain has L-CDR1 as sequence number 401, L-CDR2 as sequence number 402, and L-CDR3 as sequence number 403, and the antibody heavy chain has H-CDR1 as sequence number 398, H-CDR2 as sequence number 399, and H-CDR3 as sequence number 400. (58) A complex formed by the aforementioned ligand and a ligand-binding molecule of any one of (1) to (36) bound to the aforementioned ligand. (59) A fusion protein wherein the aforementioned ligand is fused to a ligand-binding molecule as described in any of (1) to (36). (60) As in (59) fusion proteins, in which the aforementioned ligand-binding molecules have fused with the ligands, they will not further bind to other ligands. (61) A fusion protein such as (59) or (60), wherein the aforementioned ligand is fused with the aforementioned ligand by binding to a molecular mediator linker. (62) The fusion protein as in (61), wherein the aforementioned linker does not contain a protease cleavage sequence. (63) The fusion protein as in (61) or (62), wherein the aforementioned linker is a linker composed of a glycine-serine polymer. (64) A fusion protein as in any of (59) to (63), wherein the aforementioned ligand is CXCL10, the aforementioned ligand-binding molecule contains an antibody light chain and an antibody heavy chain, and the aforementioned antibody light chain or the aforementioned antibody heavy chain is fused with the aforementioned ligand. (65) The fusion protein as in (64), wherein the aforementioned cleavage site is contained in the aforementioned antibody light chain or the aforementioned antibody heavy chain preceding the aforementioned ligand-binding molecule. (66) A fusion protein as in (64) or (65), wherein the aforementioned ligand is CXCL10, and the antibody light chain contained in the aforementioned ligand-binding molecule is fused to the aforementioned ligand, and the aforementioned ligand-binding molecule: (a) Having an antibody heavy chain containing H-CDR1 (Sequence No. 374), H-CDR2 (Sequence No. 375), and H-CDR3 (Sequence No. 376), and an antibody light chain containing L-CDR1 (Sequence No. 377), L-CDR2 (Sequence No. 378), and L-CDR3 (Sequence No. 379); or (b) Having an antibody heavy chain containing H-CDR1 (serial number 380), H-CDR2 (serial number 381), and H-CDR3 (serial number 382), and an antibody light chain containing L-CDR1 (serial number 383), L-CDR2 (serial number 384), and L-CDR3 (serial number 385). (67) The fusion protein of any one of (64) to (66), wherein the aforementioned ligand is the CXCL10 variant represented by sequence number 370. (68) A fusion protein as in any of (59) to (63), wherein the aforementioned ligand is PD1, the aforementioned ligand-binding molecule contains an antibody light chain and an antibody heavy chain, and the aforementioned antibody light chain or the aforementioned antibody heavy chain is fused with the aforementioned ligand. (69) The ligand-binding molecule as in (68), wherein the aforementioned cleavage site is contained in the aforementioned antibody light chain or the aforementioned antibody heavy chain. (70) The fusion protein as in (68) or (69), wherein the aforementioned ligand is PD1, the aforementioned antibody light chain has R3L-CDR1 as sequence number 395, L-CDR2 as sequence number 396, and L-CDR3 as sequence number 397, and the aforementioned antibody heavy chain has H-CDR1 as sequence number 392, H-CDR2 as sequence number 393, and H-CDR3 as sequence number 394. (71) The fusion protein of any one of (68) to (70), wherein the aforementioned ligand is PD1 represented by sequence number 320. (72) A fusion protein as in any of (68) to (71), wherein the aforementioned ligand is PD1, the antibody heavy chain contained in the aforementioned ligand-binding molecule is fused with the aforementioned ligand, and a series of peptides obtained by fusing PD1 with the antibody heavy chain contains sequences selected from those shown in sequence numbers 323 and 324. (73) A fusion protein as in any of (68) to (71), wherein the aforementioned ligand is PD1, the antibody light chain contained in the aforementioned ligand-binding molecule is fused with the aforementioned ligand, and a series of peptides obtained by fusing PD1 with the antibody light chain contains sequences selected from those shown in sequence numbers 325 to 334. (74) A fusion protein as in any of (59) to (63), wherein the aforementioned ligand is IL12, the aforementioned ligand-binding molecule contains an antibody light chain and an antibody heavy chain, and the aforementioned antibody light chain or the aforementioned antibody heavy chain is fused with the aforementioned ligand. (75) The fusion protein as in (74), wherein the aforementioned cleavage site is contained in the aforementioned antibody light chain or the aforementioned antibody heavy chain. (76) The fusion protein as in (74) or (75), wherein the aforementioned ligand is IL12, the aforementioned antibody light chain has L-CDR1 as sequence number 389, L-CDR2 as sequence number 390, and L-CDR3 as sequence number 391, and the aforementioned antibody heavy chain has H-CDR1 as sequence number 386, H-CDR2 as sequence number 387, and H-CDR3 as sequence number 388. (77) A fusion protein as in any of (59) to (63), wherein the aforementioned ligand is IL-6R, the aforementioned ligand-binding molecule contains an antibody light chain and an antibody heavy chain, and the aforementioned antibody light chain or the aforementioned antibody heavy chain is fused with the aforementioned ligand. (78) The fusion protein as in (77), wherein the aforementioned cleavage site is contained in the aforementioned antibody light chain or the aforementioned antibody heavy chain. (79) The fusion protein as in (77) or (78), wherein the aforementioned ligand is IL-6R, the aforementioned antibody light chain has L-CDR1 as sequence number 401, L-CDR2 as sequence number 402, and L-CDR3 as sequence number 403, and the aforementioned antibody heavy chain has H-CDR1 as sequence number 398, H-CDR2 as sequence number 399, and H-CDR3 as sequence number 400. (80) A pharmaceutical composition comprising a ligand-binding molecule as described in any one of (1) to (57). (81) A pharmaceutical composition comprising a ligand-binding molecule and a ligand as described in any one of (1) to (37). (82) A pharmaceutical composition comprising a complex as described in (58). (83) A pharmaceutical composition comprising a fusion protein of any one of (59) to (79). (84) A method for manufacturing a ligand-binding molecule as described in any one of (1) to (57). (85) The manufacturing method of (84) includes the step of introducing a protease cleavage sequence into a molecule that can bind to a ligand. (86) A method for manufacturing a fusion protein as described in any one of (59) to (79), comprising the step of fusing a ligand-binding molecule having a protease cleavage sequence with its ligand. (87) A polynucleotide encoding a ligand-binding molecule of any one of (1) to (57). (88) A carrier containing polynucleotides such as (87). (89) A host cell containing polynucleotides such as (87) or carriers such as (88). (90) A method for manufacturing a ligand-binding molecule as described in any one of (1) to (57), comprising the step of culturing a host cell as described in (89). (91) A polynucleotide encoding a fusion protein of any one of (59) to (79). (92) A carrier containing polynucleotides such as (91). (93) A host cell containing polynucleotides as in (91) or carriers as in (92). (94) A method for manufacturing a fusion protein as described in any one of (59) to (79), comprising the step of culturing a host cell as described in (93). Simple Explanation of the Diagram

[0012] Figure 1 illustrates a pattern of IgG antibody-ligand fusion protein containing a free ligand-linker-antiligand antibody VH molecule with target tissue specificity, and its activation. The ligand and antiligand system are linked by a linker. Figure 2 illustrates one pattern of IgG antibodies that specifically release ligands in the target tissue and their activation. Anti-ligand antibodies with protease-cleaving sequences inserted near the VH / CH1 junction are mixed with the ligand system and administered to the individual. Figure 3 illustrates one pattern of IgG antibodies that specifically release ligands in a target tissue and their activation. An anti-ligand antibody system with a protease-cleaving sequence inserted near the VH / CH1 junction is administered to the individual. The administered antibody binds to the ligand already present in the body, and the activation pattern is the same as in Figure 2. Figure 4 reveals the results of using Biacore to evaluate the interaction between MabCXCL10 and human CXCL10. Figure 5A shows a model of an antibody molecule created by inserting a protease cleavage sequence near the boundary between the variable and invariant regions of the MabCXCL10 antibody. Figure 5B reveals the names of the various heavy chain alterations produced, the locations where protease cleavage sequences were inserted, and the inserted amino acid sequences. Insertion sites are indicated by [insert]. Figure 5C reveals the names of the various light chain alterations produced, the locations of the inserted protease cleavage sequences, and the inserted amino acid sequences. The insertion sites are indicated by [insert]. Figure 6A reveals the results of Biacore evaluation of the interaction between antibody molecules prepared by inserting a protease cleavage sequence near the boundary between the variable and invariant regions of the heavy chain of MabCXCL10 and human CXCL10. Figure 6B reveals the results of Biacore evaluation of the interaction between antibody molecules prepared by inserting a protease cleavage sequence near the boundary between the variable and invariant regions of the light chain of MabCXCL10 and human CXCL10. Figure 7-1(A) illustrates the results of antibody molecules created by inserting a protease cleavage sequence near the boundary between the variable and invariant regions of the heavy chain of MabCXCL10. These molecules were then treated with the protease (MT-SP1), followed by SDS-PAGE electrophoresis and detection using Coomassie Brilliant Blue (CBB) to evaluate the degree of cleavage. Of the two new bands generated by protease treatment, the band appearing near 15 kDa originated from the VH region, while the band appearing between 25 and 50 kDa originated from the invariant region. Figure 7-2 shows the results of SDS-PAGE evaluation of antibody molecules (A) and (B) prepared by inserting protease cleavage sequences into the variable and invariant regions of the light chain of MabCXCL10, after treatment with the protease (MT-SP1). Two new bands appeared from the light chain cleaved by the protease treatment. Figure 7-3 continues (B). Figure 8 shows the names of the various heavy chain alterations created by inserting protease cleavage sequences and mobile linker sequences near the boundary between the variable and invariant regions of MabCXCL10, the locations of the inserted protease cleavage sequences and mobile linker sequences, and the inserted amino acid sequences. The insertion site is indicated by [insert]. Figure 9 reveals the results of Biacore evaluation of the interaction between antibody molecules and human CXCL10 by inserting protease cleavage sequences and mobile linker sequences near the boundary between the variable and invariant regions of the heavy chain of MabCXCL10. Figure 10A shows the results of antibody molecules prepared by inserting protease cleavage sequences and linker sequences near the boundary between the variable and invariant regions of the heavy chain of MabCXCL10. After protease treatment (uPA, MT-SP1), the degree of cleavage was evaluated by SDS-PAGE electrophoresis and CBB detection. Of the two new bands that appeared due to protease treatment, the band appearing near 15 kDa originated from the VH region, and the band appearing in the 25-50 kDa region originated from the invariant region. Figure 10B follows Figure 10A. Figure 11A reveals the results of evaluating whether CXCL10 is released by treating the complex of MabCXCL10a and CXCL10 with the protease (MT-SP1). Figure 11B reveals the results of evaluating whether CXCL10 can be released by treating the complex of EEIVHC006a / EEIVL and CXCL10 with the protease (MT-SP1). Figure 12 shows the names of the heavy chains created by replacing a portion of the amino acid sequence near the boundary between the variable and invariant regions of MabCXCL10 with protease cleavage sequences and mobile linker sequences, the sites of inserted and altered amino acids, the inserted sequences, and the inserted and altered amino acid sequences. Insertion sites are indicated by [insert]. In the "Insertion and Altered Positions" column, the amino acid residues represented by the cancel lines were removed during the insertion of the insert sequence, i.e., replaced by the amino acid at the C-terminus of the insert sequence. Figure 13 shows the results of antibody molecules prepared by replacing a portion of the amino acid sequence near the boundary between the variable and invariant regions of MabCXCL10 with protease cleavage sequences and mobile linkers. The molecules were then treated with a protease (uPA, MT-SP1), and the degree of cleavage was evaluated by reduction SDS-PAGE electrophoresis and CBB detection. Of the two new bands that appeared due to protease treatment, the band appearing near 15 kDa originated from the VH region, while the band appearing in the 25-50 kDa region originated from the invariant region. Figure 14 shows the activity (luminescence value) of luciferase. Figure 15 shows the SDS-PAGE results of the CXCL10-anti-CXCL10 antibody fusion protein before and after protease cleavage. Figure 16 shows the activity (luminescence value) of luciferase. Figure 17 shows an SDS-PAGE diagram of the reduction of anti-IL-12 neutralizing antibody due to protease cleavage, which was used to evaluate the incorporation of protease cleavage sequences and mobile linker sequences. Figure 18 shows the production of interferon gamma when IL-12 and antibody are added. NoAb is a sample with only IL-12 added without antibody, and NoIL-12 is a sample with neither IL-12 nor antibody added. Figure 19A shows that antibodies utilize proteases for cleavage. Figure 19B shows how antibodies utilize proteases for cleavage. Figure 20A shows the results of cleavage with various proteases. Figure 20B shows the results of cleavage with various proteases. Figure 21 shows the results of cleavage with various proteases. Figure 22A shows the results of protease cleavage of the MRA alteration. Figure 22B shows the results of protease cleavage of the MRA alteration. Figure 22C shows the results of MRA alteration by protease cleavage. Figure 22D reveals the results of protease cleavage of the MRA alteration. Figure 22E shows the results of protease cleavage of the MRA alteration. Figure 22F shows the results of MRA alteration by protease cleavage. Figure 22G reveals the results of protease cleavage of the MRA alteration. Figure 22H reveals the results of protease cleavage of the MRA alteration. Figure 22I shows the results of protease cleavage of the MRA alteration. Figure 23A shows the results of protease cleavage of the MRA alteration. Figure 23B shows the results of protease cleavage of the MRA altered body. Figure 23C shows the results of MRA alteration by protease cleavage. Figure 24A shows the results of MRA alteration by protease cleavage. Figure 24B shows the results of MRA alteration by protease cleavage. Figure 24C shows the results of MRA alteration by protease cleavage. Figure 24D reveals the results of protease cleavage of the MRA alteration. Figure 24E shows the results of protease cleavage of the MRA alteration. Figure 25A shows the results of protease cleavage of the MRA alteration. Figure 25B shows the results of protease cleavage of the MRA alteration. Figure 26 shows a real-time graph comparing the binding of PD1 to 5C4-bio in samples containing protease-treated antibodies, untreated antibodies, and PD1 binding evaluation samples. The thick black line represents the binding evaluation samples containing protease-treated antibodies, and the thin gray line represents the binding evaluation samples containing untreated antibodies. The X-axis represents the measurement time (seconds), with the measurement starting at 0 seconds. The Y-axis represents binding. The names of each axis represent the antibodies present in the evaluation samples; the "None" axis (antigen only) indicates that only the antigen was used as the evaluation sample without antibody mixing. Figure 27 shows the electrophoresis results of antibodies treated with protease and antibodies not treated with protease. The protease(+) lane represents antibodies treated with protease, and the protease(-) lane represents antibodies not treated with protease. Figure 28 shows a real-time graph comparing the binding of protease-treated antibodies and untreated antibodies to PD1. The thick black line represents protease-treated antibodies, and the thin gray line represents untreated antibodies. The X-axis represents the measurement time (seconds), with the measurement start time set to 0 seconds. The Y-axis represents binding. The names of each axis represent the antibodies used; the "None" axis indicates the use of only PBS buffer and no antibody. Figure 29 shows a real-time graph comparing the binding of free PD1 to 5C4-bio in samples treated with protease in the presence of PD1 and samples not treated with protease in the presence of PD1. The thick black line represents the protease-treated sample, and the thin gray line represents the untreated sample. The X-axis represents the measurement time (seconds), with the measurement start time set to 0 seconds. The Y-axis represents binding. The axis names represent the antibodies present in the sample; on the "Antigen and Protease" axis, the sample contains only PD1 and no antibodies. Figure 30 shows a real-time graph comparing the binding of free PD1 to 5C4-bio in protease-treated fusion protein and protease-untreated protein solutions. The thick black line represents the protease-treated sample, and the thin gray line represents the untreated sample. The X-axis represents the measurement time (seconds), with the measurement start set to 0 seconds. The Y-axis represents binding. The names of the axes indicate that for fusion proteins, the None (antigen only) axis does not use the fusion protein for evaluation, only the antigen PD1 is used. The 5C4H-G1T4 / 5C4L-KT0 axis does not use the fusion protein, only the 5C4H-G1T4 / 5C4L-KT0 antibody is used. Figure 31 shows the electrophoresis results of the antibody-PD1 fusion protein treated with protease. The protease(+) lanes contain the fusion protein treated with protease, while the protease(-) lanes contain the fusion protein that has not been treated with protease. Implementation

[0013] In this invention, peptides generally refer to peptides and proteins having a length of approximately four or more amino acids. Furthermore, in this invention, peptides are generally peptides composed of artificially designed sequences, but are not particularly limited; for example, they can also be bio-derived peptides. They can also be any of the following: natural peptides, synthetic peptides, recombinant peptides, etc. Moreover, fragments of the aforementioned peptides are also included in the peptides of this invention.

[0014] In this specification, amino acids such as Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, Val / V are represented by single-letter codes, three-letter codes, or both.

[0015] To alter the amino acid sequence of a polypeptide, site-specific mutation induction methods (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR, among other known methods, can be employed. Furthermore, several known methods exist for altering amino acids by replacing them with those other than natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, the cell-free translation system of tRNA (Clover Direct (Protein Express)) obtained by binding a complementary amber suppressor tRNA containing one of the stop codons, the UAG codon, to a non-natural amino acid is also ideal.

[0016] In this specification, the use of "and / or" to indicate changes in the amino acid composition includes various combinations of appropriate combinations of "and" and "or". Specifically, for example, "substitution of amino acids 37, 45, and / or 47" includes variations involving changes in the following amino acids; (a) No. 37, (b) No. 45, (c) No. 47, (d) No. 37 and No. 45, (e) No. 37 and No. 47, (f) No. 45 and No. 47, (g) No. 37, No. 45 and No. 47.

[0017] In this specification, regarding the expression of amino acid changes, a suitable method may be used, where single-letter or three-letter codes representing the amino acid before and after the change are added before and after the numbers representing specific positions. For example, when substituting amino acids contained in the variable region of an antibody, changes such as F37V or Phe37Val represent that Phe at position 37 of the Kabat number is replaced with Val. That is, the number represents the position of the amino acid indicated by the Kabat number, the single-letter or three-letter code of the amino acid before it represents the amino acid before substitution, and the single-letter or three-letter code of the amino acid after it represents the amino acid after substitution. Similarly, when substituting amino acids in the Fc region contained in the invariant region of an antibody, changes such as P238A or Pro238Ala represent that Pro at position 238 of the EU number is replaced with Ala. That is, the number represents the position of the amino acid indicated by the EU number. The single-letter code or three-letter code of the amino acid recorded before it represents the amino acid before substitution, and the single-letter code or three-letter code of the amino acid recorded after it represents the amino acid after substitution.

[0018] This invention relates to ligand-binding molecules that have a cleavage site and whose binding to a ligand is weakened when that cleavage site is cleaved. The ligand-binding molecules of this invention refer to peptide molecules capable of binding to ligands.

[0019] The ligand-binding molecule of this invention is a ligand-binding molecule, especially one that can bind to a ligand molecule in an uncleaved state. Here, "binding" generally refers to binding through interactions primarily composed of non-covalent bonds such as electrostatic forces, van der Waals forces, and hydrogen bonds. Ideal examples of the ligand-binding state of the ligand-binding molecule of this invention are not limited to this; for example, antigen-binding regions, antigen-binding molecules, antibodies, and antibody fragments can be listed as antigen-antibody reactions that bind to antigens.

[0020] Furthermore, "capable of binding to a ligand" means that even if the ligand-binding molecule and the ligand are different molecules, the ligand-binding molecule can still bind to the ligand. It does not mean that the ligand-binding molecule is covalently linked to the ligand using a covalent bond. For example, it does not mean that the ligand-binding molecule is covalently bonded to the ligand through a linker. Also, "weakened binding to a ligand" refers to a decrease in the aforementioned binding ability. For example, when the ligand-binding molecule is covalently bonded to the ligand through a linker, the cleavage of the linker is not considered a weakening of the binding to the ligand. Furthermore, in this invention, as long as the ligand-binding molecule can bind to the ligand, it can also be connected to the ligand through a linker, etc.

[0021] The ligand-binding molecule of this invention binds to the ligand only in an uncut state. As long as it can bind to the target ligand in an uncut state, molecules of any structure can be used. Ligand-binding molecules are not limited to the examples above, but also include: the heavy chain variable region (VH) and light chain variable region (VL) of antibodies; single-domain antibodies (sdAbs); the A domain module containing approximately 35 amino acids in the cell membrane protein Avimer (WO2004 / 044011, WO2005 / 040229); Adnectin (WO2002 / 032925), the protein-binding domain 10Fn3 of the glycoprotein fibronectin contained in the cell membrane; Affibody (WO1995 / 001937), which uses the IgG binding domain, consisting of three helical bundles composed of 58 amino acids of Protein A, as a scaffold; and ankyrin reflex, which has a structure of 33 amino acid residues, two antiparallel helices, and a reflexive subunit stacked structure. The exposed areas on the surface of repeat:AR molecules include DARPins (Designed Ankyrin Repeat proteins) (WO2002 / 020565), the four loop regions supported on one side of the highly conserved eight antiparallel, centrally twisted piston barrel structure in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL), and the hollow regions of the parallel sheet structure inside the horseshoe-shaped structure obtained by the repeated stacking of leucine-rich repeat (LRR) modules of the variable lymphocyte receptor (VLR) which does not have the structure of immunoglobulins in the acquired immune system of jawless eels such as hagfish and swamp eels (WO2008 / 016854).

[0022] In this specification, the term "antibody" is used in the broadest sense, referring to any antibody that can exhibit the desired antigen-binding activity, and is not limited to these, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments of various antibody structures.

[0023] Methods for preparing antibodies with the desired binding activity are well known to those skilled in the art. The following describes methods for preparing antibodies that bind to IL-6 (anti-IL-6R antibodies). Antibodies that bind to antigens other than IL-6R can also be prepared appropriately according to the following examples.

[0024] Anti-IL-6R antibodies can be obtained using publicly known methods in the form of polyclonal or monoclonal antibodies. Anti-IL-6R antibodies are preferably prepared as monoclonal antibodies derived from mammals. Monoclonal antibodies derived from mammals include those produced by fusion tumors and those produced by genetic engineering methods using host cells transgenic with an expression vector containing an antibody gene. The antibodies mentioned in this invention include "humanized antibodies" or "chimeric antibodies."

[0025] Monoclonal antibody-generating fusion tumors can be produced using known techniques, for example, as follows: Using the IL-6R protein as the antigen, mammals are immunized using standard immunization methods. The resulting immune cells are then fused with known parental cells using standard cell fusion methods. Next, monoclonal antibody-producing cells are screened using standard screening methods, selecting fusion tumors that produce anti-IL-6R antibodies.

[0026] Specifically, the preparation of monoclonal antibodies is carried out as follows: First, the IL-6R protein is obtained by expressing the IL-6R gene to be used as a antigen for antibody preparation. That is, appropriate host cells are transduced by inserting the gene sequence encoding IL-6R into a known expression vector. The desired human IL-6R protein is purified from the host cells or from the culture supernatant using known methods. To obtain a soluble form of IL-6R from the culture supernatant, for example, the soluble IL-6R of Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968) can be expressed. Furthermore, purified natural IL-6R protein can also be used as a antigen.

[0027] This purified IL-6R protein can be used as a susceptibility antigen for mammalian immunization. A partial peptide of IL-6R can also be used as a susceptibility antigen. In this case, the partial peptide can be chemically synthesized using the amino acid sequence of human IL-6R. Alternatively, a portion of the IL-6R gene can be incorporated into an expression vector for expression. Furthermore, the IL-6R protein can be obtained by breaking it down using a proteolytic enzyme; however, the region and size of the IL-6R peptide used as a partial peptide are not limited to a particular form. The number of amino acids constituting the peptide used as a susceptibility antigen is at least 5, preferably 6 or 7. More specifically, peptides with 8 to 50, preferably 10 to 30 residues, can be used as susceptibility antigens.

[0028] Furthermore, fusion proteins formed by fusing the desired portion of the IL-6R protein with different peptides can be used as antigens. To create fusion proteins for use as antigens, for example, the Fc fragment of an antibody or a peptide tag can be appropriately utilized. Vectors for expressing fusion proteins can be created by fusing genes encoding two or more desired peptide fragments in the same inframe and inserting the fusion gene into an expression vector as described above. Methods for creating fusion proteins are described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. press). The methods for obtaining IL-6R, which can be used as an antigen, and the methods for immunization with it are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0029] Mammals immunized with this antigen are not limited to specific animals; the suitability of parent cells for cell fusion should be considered. Common rodents such as mice, rats, hamsters, or rabbits and monkeys are preferred.

[0030] The aforementioned animals were immunized with the antigen according to known methods. For example, a common method is to administer the antigen intraperitoneally or subcutaneously to mammals. Specifically, the antigen, diluted appropriately with PBS (Phosphate-Buffered Saline) or physiological saline, is mixed with a common adjuvant such as Freud complete adjuvant and emulsified as desired. This antigen is then administered to mammals several times every 4 to 21 days. Furthermore, a suitable carrier can be used for antigen immunization. Especially when using low molecular weight partial peptides as antigens, it is sometimes ideal to use antigen peptides bound to carrier proteins such as albumin or keyhole lympet hemocyanin for immunization.

[0031] Furthermore, fusion tumors that produce the desired antibodies can be generated using DNA immunization and prepared as follows. DNA immunization refers to an immunization method in which a vector DNA, constructed from a gene encoding an antigen protein, is administered to an immunized animal. This provides immune stimulation through the expression of the antigen in the animal's vivo body. Compared to conventional immunization methods involving the administration of protein antigens to immunized animals, DNA immunization offers the following advantages. - It can maintain the structure of membrane proteins such as IL-6R and provide immune stimulation. -No need to refine immune antigens

[0032] To obtain the monoclonal antibody of this invention via DNA immunization, DNA encoding the IL-6R protein is first administered to an immunized animal. The DNA encoding IL-6R can be synthesized using known methods such as PCR. The obtained DNA is then inserted into a suitable expression vector and administered to the immunized animal. A commercially available expression vector, such as pcDNA3.1, is ideally suitable. The method of administering the vector to the animal in vivo can be any commonly used method. For example, DNA immunization can be performed by introducing gold particles adsorbed with the expression vector into the cells of the immunized animal using a gene gun. Furthermore, the antibody recognizing IL-6R can also be prepared using the method described in international publication WO2003 / 104453.

[0033] In this manner, mammalian immunization is performed. Once the titer of antibodies binding to IL-6R in the serum increases, immune cells are harvested from the mammal for cell fusion. Spleen cells are preferred as the preferred immune cell type.

[0034] Cells fused with the aforementioned immune cells can be mammalian myeloma cells. Myeloma cells should ideally possess appropriate selection markers for screening. Selection markers refer to morphologies that enable (or prevent) survival under specific culture conditions. Among selection markers, hypoxanthine-guanine phosphoribosyl transferase deficiency (hereinafter referred to as HGPRT deficiency) or thymidine kinase deficiency (hereinafter referred to as TK deficiency) are well-known. Cells with HGPRT or TK deficiency possess hypoxanthine-aminopterin-thymidine susceptibility (hereinafter referred to as HAT susceptibility). HAT-susceptible cells cannot synthesize DNA in HAT-selective medium and will die, but if fused with normal cells, they will continue to synthesize DNA using the normal cell's salvage pathway, thus proliferating in HAT-selective medium.

[0035] Cells deficient in HGPRT or TK can be selected using culture media containing thioguanine, 8-azoguanine (hereinafter referred to as 8AG), or 5'-bromodeoxyuracil. Normal cells with these pyrimidine analogs in their DNA will die. On the other hand, cells deficient in these enzymes but without these pyrimidine analogs can survive in the selective medium. Another selection marker, known as G418 tolerance, utilizes a neomycin tolerance gene to provide tolerance to streptamine-based antibiotics (gentamicin analogs). It is known for its application to various myeloma cell lines with ideal cell fusion.

[0036] Such myeloma cells can ideally be used, for example: P3 (P3x63Ag8.653) (J. Immunol.(1979)123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology(1978)81, 1-7), NS-1 (C. Eur. J. Immunol.(1976)6 (7), 511-519), MPC-11 (Cell(1976)8 (3), 405-415), SP2 / 0 (Nature(1978)276 (5685), 269-270), FO (J. Immunol. Methods(1980)35 (1-2), 1-21), S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323, R210 (Nature (1979) 277 (5692), 131-133), etc.

[0037] Basically, the aforementioned cell fusion of immune cells and myeloma cells can be carried out in accordance with well-known methods such as those of Keller and Milstein (Methods Enzymol. (1981) 73, 3-46). More specifically, the aforementioned cell fusion can be carried out in a normal nutrient culture medium in the presence of, for example, a cell fusion promoter. Fusion promoters may include, for example, polyethylene glycol (PEG), Sendai virus (HVJ), etc., and to further improve fusion efficiency, adjuvants such as dimethyl sulfoxide may be added as desired.

[0038] The ratio of immune cells to myeloma cells can be set arbitrarily. For example, it is better to set the ratio of immune cells to myeloma cells to 10:1. The culture medium used for the aforementioned cell fusion is, for example, RPMI 1640 medium or MEM medium suitable for the proliferation of the aforementioned myeloma cell line. In addition, the usual culture medium used for this type of cell culture can be used. Furthermore, it is ideal to add serum such as fetal bovine serum (FCS) for replenishment.

[0039] The cell fusion method involves thoroughly mixing the aforementioned immune cells and myeloma cells in a predetermined amount in the aforementioned culture medium, and adding a PEG solution preheated to approximately 37°C (e.g., with an average molecular weight of approximately 1000 to 6000) at a concentration typically of 30% to 60% (w / v). By slowly mixing the mixture, the desired fused cells (fusion tumors) are formed. Subsequently, appropriate culture media as listed above are added sequentially, and repeated centrifugation and removal of the supernatant are performed to remove cell fusion agents and other substances detrimental to the growth of fusion tumors.

[0040] Fusomonas obtained in this manner can be selected by culturing in a conventional selection medium, such as HAT medium (containing hypoxanthine, aminopterin, and thymine). To kill cells other than the desired fusomonas (non-fused cells), culturing in the aforementioned HAT medium can be continued for a sufficient time (usually several days to several weeks). Next, fusomonas producing the desired antibody are screened and single-celled using the conventional limiting dilution method.

[0041] Fusion tumors obtained in this manner can be selected using selective culture media containing selectable markers specific to the myeloma cells used for cell fusion. For example, cells with HGPRT or TK deficiencies can be selected by culturing in HAT medium (containing hypoxanthine, aminopterin, and thymine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have successfully fused with normal cells can be selectively proliferated in HAT medium. To kill off cells other than the desired fusion tumor (non-fused cells), culture in the aforementioned HAT medium can be continued for a sufficient time. Specifically, the desired fusion tumor can generally be selected through several to several weeks of culture. Next, the usual limiting dilution method can be used to screen and single-cell proliferate fusion tumors that produce the desired antibodies.

[0042] The desired antibody screening and monoclonal selection can be ideally implemented using screening methods based on known antigen-antibody reactions. For example, a monoclonal antibody binding to IL-6R can bind to IL-6R expressed on the cell surface. Such monoclonal antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS involves analyzing cells in contact with fluorescent antibodies using laser light and measuring the fluorescence emitted by each cell, thereby determining the antibody's binding system to the cell surface.

[0043] To screen fusion tumors that produce the monoclonal antibody of this invention using FACS, cells expressing IL-6R must first be prepared. Preferred cells for screening are mammalian cells that induce IL-6R expression. By using untransformed mammalian cells as a control, the binding activity of the antibody to IL-6R on the cell surface can be selectively detected. That is, by selecting fusion tumors that produce antibodies that bind to cells that induce IL-6R expression but not to the host cell, fusion tumors producing the monoclonal antibody IL-6R can be obtained.

[0044] Alternatively, the binding activity of antibodies to immobilized IL-6R-expressing cells can be evaluated based on the principles of ELISA. For example, IL-6R-expressing cells are immobilized in the wells of an ELISA plate. The culture supernatant of the fusion tumor is then brought into contact with the immobilized cells to detect antibodies bound to the immobilized cells. When the monoclonal antibody is of mouse origin, the antibody binding to the cells can be detected using anti-mouse immunoglobulin antibodies. Fusion tumors selected through such screening that produce the desired antibody with the ability to bind to the antigen can be selected using methods such as limiting dilution.

[0045] The fusion tumors that produce monoclonal antibodies in this way can be subcultured in conventional culture media. Furthermore, these fusion tumors can be stored long-term in liquid nitrogen.

[0046] The fusion tumor can be cultured using standard methods, and the desired monoclonal antibody can be obtained from its culture supernatant. Alternatively, the fusion tumor can be introduced into a suitable mammal to proliferate, and the monoclonal antibody can be obtained from its ascites. The former method is suitable for obtaining high-purity antibodies.

[0047] Antibodies encoded by antibody genes selected from antibody-producing cells such as fusion tumors can also be appropriately utilized. By incorporating the selected antibody gene into a suitable vector and introducing it into a host, the antibody encoded by that gene can be expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767-775). The methods for manufacturing recombinant antibodies described below are also well known.

[0048] For example, cDNA encoding the variable region (V region) of the anti-IL-6R antibody can be obtained from fusion tumor cells that produce anti-IL-6R antibodies. For this purpose, whole RNA is usually extracted from the fusion tumor first. Methods for extracting mRNA from cells include, for example, the following. -Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) -AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

[0049] The extracted mRNA can be purified using an mRNA Purification Kit (manufactured by GE Healthcare Bioscience). Alternatively, there are commercially available kits for directly extracting whole mRNA from cells, such as the QuickPrep mRNA Purification Kit (manufactured by GE Healthcare Bioscience). Using such kits, mRNA can be obtained from fusion tumors. The obtained mRNA can be used to synthesize cDNA encoding the antibody V region using reverse transcriptase. cDNA can be synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Biochemical Industry Co., Ltd.). Furthermore, for the synthesis and amplification of cDNA, the SMART RACE cDNA amplification kit (manufactured by Clontech) and the 5'-RACE method of PCR can be appropriately used (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002, Nucleic Acids Res. (1989) 17 (8), 2919-2932). Furthermore, during such cDNA synthesis, appropriate restriction enzyme sites can be introduced at both ends of the cDNA.

[0050] The target cDNA fragment is purified from the obtained PCR product and then ligated with vector DNA. A recombinant vector is prepared in this manner and introduced into bacteria such as *E. coli*. After selecting a colony, the desired recombinant vector is prepared from the *E. coli* colony. Whether the recombinant vector possesses the target cDNA sequence can be confirmed using known methods such as dideoxynucleotide chain termination.

[0051] To obtain genes encoding variable regions, the 5'-RACE method using primers for variable region gene amplification is a simple approach. First, RNA extracted from fusion tumor cells is used as a template to synthesize cDNA, obtaining a 5'-RACE cDNA library. Commercially available kits such as the SMART RACE cDNA amplification kit can be used for the synthesis of the 5'-RACE cDNA library.

[0052] Using the obtained 5'-RACE cDNA library as a template, antibody genes were amplified by PCR. Primers for mouse antibody gene amplification were designed based on known antibody gene sequences. These primers consist of base sequences that differ according to the subclass of immunoglobulin. Therefore, subclass determination is ideally achieved using commercially available kits such as the Iso Strip mouse monoclonal antibody isotype determination kit (Roche diagnostics).

[0053] Specifically, when the goal is to obtain, for example, the gene encoding mouse IgG, primers that amplify genes encoding γ1, γ2a, γ2b, and γ3 (heavy chains) and κ and λ chains (light chains) can be used. To amplify the variable region gene of IgG, a primer on the 3' side is generally used that attaches a portion of the invariant region corresponding to the variable region. On the other hand, the 5' side primer is the primer included with the kit created using the 5' RACE cDNA database.

[0054] Using the amplified PCR products, immunoglobulins composed of heavy and light chains can be reconstructed. The binding activity of the reconstructed immunoglobulins to IL-6R can be used as an indicator to screen for desired antibodies. For example, when the goal is to obtain antibodies against IL-6R, the antibody's binding to IL-6R should ideally be specific. Antibodies binding to IL-6R can be screened using methods such as the following: (1) Steps for contacting IL-6R-expressing cells with antibodies containing the V region encoded by cDNA obtained from fusion tumors. (2) Steps for detecting the binding between IL-6R-expressing cells and antibodies, and (3) Steps for selecting antibodies that bind to IL-6R-expressing cells.

[0055] Methods for detecting the binding of antibodies to IL-6R-expressing cells are well known. Specifically, methods such as FACS described above can be used to detect the binding of antibodies to IL-6R-expressing cells. To evaluate the binding activity of the antibody, fixed specimens of IL-6R-expressing cells can be appropriately used.

[0056] For antibody screening using binding activity as an indicator, phage vector-based screening is also suitable. When obtaining antibody genes from multiple antibody-expressing cell populations in the form of heavy and light chain subclasses, phage vector-based screening is advantageous. Genes encoding the variable regions of the heavy and light chains can be linked together with appropriate linker sequences to form single-stranded Fvs (scFvs). By inserting the gene encoding scFv into a phage vector, phages expressing scFv on their surface can be obtained. After the phage comes into contact with the desired antigen, the DNA encoding the scFv with the desired binding activity can be recovered by retrieving the antigen-bound phage. This process can be repeated as needed to concentrate the scFvs with the desired binding activity.

[0057] After obtaining the cDNA encoding the V region of the target anti-IL-6R antibody, the cDNA is digested with a restriction enzyme that recognizes the restriction enzyme sites at both ends of the cDNA. Preferably, the restriction enzyme recognizes and digests a low-frequency base sequence in the antibody gene. Furthermore, to ensure that one copy of the digested fragment is inserted into the vector in the correct orientation, a restriction enzyme providing attachment ends is inserted. By inserting the cDNA encoding the V region of the anti-IL-6R antibody, digested in the above manner, into a suitable expression vector, an antibody expression vector can be obtained. At this point, if the gene encoding the antibody invariant region (C region) and the gene encoding the aforementioned V region are fused in the same reading frame, a chimeric antibody can be obtained. Here, a chimeric antibody system refers to one where the invariant region and the variable region originate from different sources. Therefore, in addition to xenogeneic chimeric antibodies such as mouse-human, human-human allogeneic chimeric antibodies are also included in the chimeric antibodies of this invention. By pre-inserting the aforementioned V region gene into an expression vector containing the invariant region, a chimeric antibody expression vector can be constructed. Specifically, a restriction enzyme recognition sequence for digesting the aforementioned V region gene can be appropriately configured on the 5' side of the expression vector containing, for example, the DNA encoding the desired antibody-invariant region (C region). By fusing the two digested with the same combination of restriction enzymes into the same reading frame, a chimeric antibody expression vector can be constructed.

[0058] To produce monoclonal antibodies against IL-6R, the antibody gene can be incorporated into an expression vector in a manner controlled by an expression control region. The expression control region used to express the antibody includes, for example, an enhancer or a promoter. Alternatively, an appropriate signal sequence can be appended to the amino terminus to induce extracellular secretion of the expressed antibody. For example, a peptide with the amino acid sequence MGWSCIILFLVATATGVHS (sequence number: 536) can be used as the signal sequence, but other appropriate signal sequences can also be appended. The expressed peptide is cleaved at the carboxyl terminus of the above sequence, and the cleaved peptide can be secreted extracellularly in the form of a mature peptide. Next, by transforming appropriate host cells using this expression vector, recombinant cells expressing DNA encoding the anti-IL-6R antibody can be obtained.

[0059] "Antibody fragment" refers to a molecule other than the complete antibody that binds to an antigen bound to the complete antibody. Examples of antibody fragments are not limited to this, including Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody antibodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0060] The terms "full-length antibody," "complete antibody," and "all antibody" are used interchangeably in this specification and refer to antibodies that have a structure substantially similar to that of natural antibodies or that have a heavy chain containing the Fc region as defined in this specification.

[0061] The term "variable region" or "variable domain" refers to a domain of the heavy or light chain of an antibody that enables the antibody to bind to an antigen. The variable domains (VH and VL) of the antibody's heavy and light chains typically have a similar structure with four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) Sufficient antigen-binding specificity can be provided by a single VH or VL domain.

[0062] The terms "complementarity-determining region" or "CDR" as used in this specification refer to regions of the antibody's variable domains that are hypervariable and / or form structurally designated loops ("hypervariable loops") and / or antigen contact residues ("antigen contacts"). Antibodies typically include six CDRs: three VHs (H1, H2, H3) and three VLs (L1, L2, L3). The CDRs illustrated in this specification include the following: (a) The supervariable loop formed 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)); (b) CDRs generated 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)); (c) Antigen contacts formed 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)); and, (d) includes combinations of (a), (b), and / or (c) of HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise specified, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered in accordance with the above-mentioned Kabat et al.

[0063] "Frame" or "FR" refers to the variable domain residues outside the complementarity-determining region (CDR). A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of CDR and FR usually appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0064] In this specification, the terms "invariant region" or "fixed region" refer to the portion of an antibody other than its variable region. For example, an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy chain domain or heavy chain variable domain, followed by a heavy chain invariant region (CH) containing a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a fixed light chain (CL) domain. The light chains of native antibodies can be classified into one of two types, kappa (κ) and lambda (λ), based on the amino acid sequence of their fixed regions.

[0065] The "class" of an antibody refers to the type of fixed or invariant regions possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these can be further subdivided into isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The fixed regions of the heavy chain corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ.

[0066] In this specification, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the invariant region. This term includes both native and variant Fc regions. In the case of human IgG1, the heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present. Unless otherwise specified, the amino acid residues in the Fc region or invariant region are numbered according to the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0067] The ligand-binding molecule of this invention is a polypeptide containing a cleavage site. The cleavage site can be cleaved by an enzyme, but it can also be reduced by a reducing agent or photodecomposed. The cleavage site, as long as it can weaken the binding of the ligand-binding molecule to the ligand through cleavage, can be located at any position within the polypeptide. Furthermore, the polypeptide may contain one or more cleavage sites.

[0068] Furthermore, the ligand-binding molecule of the present invention exhibits weaker ligand binding (i.e., reduced binding) in the cleaved state compared to the uncleaved state. The reduced ligand binding in the antigen-antibody reaction can be evaluated using the ligand-binding activity of the ligand-binding molecule.

[0069] The binding activity of ligand-binding molecules can be evaluated using well-known methods such as FACS, ELISA, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method using surface plasma resonance (SPR) phenomenon, and BLI (Bio-Layer Interferometry) (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). The ALPHA screen utilizes ALPHA technology, employing two types of beads—a provider bead and a receiver bead—based on the following principle: Molecules near the provider bead interact with molecules bound to the receiver bead, and luminescence is detected only when the two beads are close together. A photosensitizer within the provider bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. This singlet oxygen diffuses to the vicinity of the provider bead, triggering a chemiluminescence reaction within the nearby receiver bead, ultimately emitting light. When the molecules bound to the provider bead and those bound to the receiver bead do not interact, the singlet oxygen produced by the provider bead does not reach the receiver bead, thus no chemiluminescence reaction occurs.

[0070] For example, a donor bead binds a ligand-binding molecule including a biotin-labeled Fc region, while a receiver bead binds a ligand tagged with glutathione S-transferase (GST). When competing unlabeled ligand-binding molecules are absent, the ligand-binding molecule interacts with the ligand, generating a signal at 520-620 nm. Unlabeled ligand-binding molecules compete with labeled ligand-binding molecules for ligand-binding interactions. Quantifying the fluorescence reduction resulting from the competition allows determination of relative binding affinity. It is known that ligand-binding molecules such as antibodies are biotinylated using systems such as Sulfo-NHS-biotin. For ligand tagging with GST, a suitable method is to fuse a polynucleotide encoding the ligand and a polynucleotide encoding GST within the same reading frame to obtain a fusion gene, express the GST fusion ligand in cells with a transmissible vector, and then purify it using a glutathione column. The obtained signals can be ideally analyzed by fitting a one-site competition model using software such as GraphPad PRISM (GraphPad, San Diego) with nonlinear regression analysis.

[0071] One of the interacting substances (ligands) is immobilized on a gold thin film of a sensing wafer. Light is shone from the back side of the wafer, causing total internal reflection at the interface between the gold film and glass. A portion of the reflected light forms a portion with reduced reflection intensity (SPR signal). The other interacting substance (analyte) flows over the surface of the sensing wafer, allowing the ligand to bind to the analyte. The mass of the immobilized ligand molecules increases, changing the refractive index of the solvent on the surface of the sensing wafer. This change in refractive index shifts the position of the SPR signal (conversely, if the binding dissociates, it returns to the signal position). The Biacore system uses this shift, i.e., the mass change on the surface of the sensing wafer, as the vertical axis, and represents the change in mass over time as measurement data (sensing graph). From the curve of the sensing graph, the kinetics are derived: the binding rate constant (ka) and the dissociation rate constant (kd). The ratio of these constants yields the dissociation constant (KD). The BIACORE method can also be ideally used for suppression assays and equilibrium value analysis. Inhibition assays, for example Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010, and an example of equilibrium value analysis are described in Methods Enzymol. 2000;323:325-40.

[0072] A weakened binding activity between the ligand-binding molecule and the ligand refers to, for example, a ligand binding amount per unit of the test ligand-binding molecule compared to the control ligand-binding molecule, which is less than 50%, preferably less than 45%, 40%, 35%, 30%, 20%, or 15%, and even more preferably less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. The binding activity can be assessed using any desired indicator, such as the dissociation constant (KD). When using the dissociation constant (KD) as an indicator of binding activity, if the dissociation constant (KD) of the test ligand-binding molecule is larger than that of the control ligand-binding molecule, it indicates that the binding activity of the test ligand-binding molecule is weaker than that of the control ligand-binding molecule. A weakened ability to bind to ligands means, for example, that the dissociation constant (KD) of the test ligand-binding molecule to the ligand is more than 2 times, preferably more than 5 times, more than 10 times, and even more than 100 times compared to the dissociation constant (KD) of the control ligand-binding molecule to the ligand. Compare with ligand-binding molecules, such as uncleaved ligand-binding molecules.

[0073] In one embodiment of the present invention, the ligand-binding molecule of the present invention is cleaved at a cleavage site, thereby releasing the ligand from the ligand-binding molecule. Here, the ligand binds to a portion of the ligand-binding molecule through a linker. When this linker has no cleavage site, the ligand is released while maintaining its connection with that portion of the ligand-binding molecule through the linker (see, for example, Figure 1). Thus, the ligand is released together with a portion of the ligand-binding molecule. As long as more than half of the ligand-binding molecule is released, it can be said that the ligand has been released from the ligand-binding molecule.

[0074] As a method for detecting the release of ligands from ligand-binding molecules due to cleavage at the cleavage site, there are methods for detecting ligands using ligand detection antibodies and other methods that recognize the ligands. When the ligand-binding molecule is an antibody fragment, it is better for the ligand detection antibody to bind to the same antigenic determinant as the ligand-binding molecule. The detection of ligands using ligand detection antibodies can be confirmed using well-known methods such as FACS, ELISA format, ALPHAscreen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method which utilizes the surface plasma resonance (SPR) phenomenon, and BLI (Bio-Layer Interferometry) (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). For example, when using Octet to detect ligand detachment, a ligand detection antibody that recognizes the ligand can be biotinylated, and after contacting the antibody with a biosensor, the binding of the ligand to the sample can be measured to detect ligand detachment. Specifically, for samples containing ligand-binding molecules and ligands before or after protease treatment, the amount of ligand is measured using a ligand detection antibody, and the amount of ligand detected in samples before and after protease treatment is compared to detect ligand detachment. Furthermore, for samples containing protease, ligand-binding molecules, and ligands, and samples containing ligand-binding molecules and ligands but without protease, the amount of ligand is measured using a ligand detection antibody, and the amount of ligand detected in samples with and without protease is compared to detect ligand detachment to detect ligand detachment. More specifically, the method described in the embodiments of this application can be used to detect ligand detachment. When a ligand-binding molecule fuses with a ligand to form a fusion protein, the amount of ligand is determined using a ligand detection antibody in samples containing the fusion protein before or after protease treatment. The amount of ligand detected in samples before and after protease treatment is compared to detect the free ligand. Furthermore, the amount of ligand is determined using a ligand detection antibody in samples containing both protease and the fusion protein, as well as in samples containing the fusion protein but without protease. The amount of ligand detected in samples with and without protease is compared to detect the free ligand. More specifically, the method described in the embodiments of this application can be used to detect the free ligand.

[0075] Furthermore, in samples where binding to ligand-binding molecules inhibits the physiological activity of the ligand, the release of the ligand from the ligand-binding molecule can be detected by measuring the physiological activity of the ligand in the sample. Specifically, for samples containing ligand-binding molecules and ligands before or after protease treatment, the physiological activity of the ligand is measured and compared to detect ligand release. Also, for samples containing protease, ligand-binding molecules, and ligands, and samples containing ligand-binding molecules and ligands but without protease, the physiological activity of the ligand is measured and compared to detect ligand release. When ligand-binding molecules and ligands fuse to form a fusion protein, for samples containing fusion proteins before or after protease treatment, the physiological activity of the ligand is measured and compared to detect ligand release. Also, for samples containing protease and fusion proteins, and samples containing fusion proteins but without protease, the physiological activity of the ligand is measured and compared to detect ligand release.

[0076] In one embodiment of the present invention, the cleavage site includes a protease cleavage sequence and is cleaved by a protease.

[0077] In this specification, the term "protease" refers to enzymes such as endopeptidases or exopeptidases that hydrolyze peptide bonds, commonly known as endopeptidases. The proteases used in this invention are limited to those capable of cleaving the protease-cleaving sequence, and their types are not particularly limited. In some embodiments, tissue-specific proteases are used. Tissue-specific proteases may, for example, refer to any of the following: (1) Proteases exhibiting higher levels of activity in target tissues compared to normal tissues. (2) Proteases exhibiting higher activity in target tissues compared to normal tissues. (3) Proteases exhibiting higher levels of expression in target cells compared to normal cells. (4) Proteases that have higher activity in target cells than in normal cells. In more specific implementations, proteases specific to cancer tissue or proteases specific to inflamed tissue are used.

[0078] In this specification, the term "target tissue" refers to a tissue containing at least one target cell. In some embodiments of the present invention, the target tissue is cancerous tissue. In some embodiments of the present invention, the target tissue is inflamed tissue.

[0079] The term "cancer tissue" refers to tissue containing at least one cancer cell. Therefore, it can refer to, for example, all cell types that contribute to the formation of a tumor mass, including cancer cells and endothelial cells, by containing cancer cells and blood vessels. In this specification, "tumor" refers to a foci of tumor tissue. The term "tumor" generally refers to either benign or malignant growths.

[0080] In this instruction manual, "inflamed tissue" may be listed as an example below. Joints in rheumatoid arthritis and osteoarthritis • Lungs (alveolar tissues) in bronchial asthma and COPD • Digestive organs in inflammatory bowel disease, Crohn's disease, and ulcerative colitis Fibrotic tissue in fibrosis of the liver, kidneys, and lungs • Tissues that cause rejection after organ transplantation • Blood vessels and heart (myocardium) in arteriosclerosis and heart failure Visceral fat in metabolic syndrome • Atopic dermatitis and other dermatitis involving skin tissue Spinal nerve involvement in herniated discs and chronic low back pain

[0081] In some types of target tissues, there are known proteases that exhibit specific expression or specific activation, or are considered to be associated with the disease state of the target tissue (target tissue-specific proteases). For example, International Publication WO2013 / 128194, International Publication WO2010 / 081173, and International Publication WO2009 / 025846 reveal proteases that exhibit specific expression in cancer tissues. Also, J Inflamm (Lond). 2010; 7: 45., Nat Rev Immunol. 2006 Jul;6(7):541-50., Nat Rev Drug Discov. 2014 Dec;13(12):904-27., Respir Res. 2016 Mar 4;17:23., Dis Model Mech. 2014 Feb;7(2):193-203., Biochim Biophys Acta. 2012 Jan;1824(1):133-45. revealed proteases that are thought to be associated with inflammation.

[0082] In addition to proteases that exhibit specific expression in target tissues, there are also proteases that are specifically activated in target tissues. For example, proteases sometimes exhibit an inactive form and then become active. In many tissues, there are substances that inhibit active proteases, and activity is controlled by activation treatment and the presence of inhibitors (Nat Rev Cancer. 2003 Jul;3(7):489-501.). In target tissues, sometimes active proteases escape from their inhibited state and become specifically activated. Methods for determining active proteases include using antibodies that recognize active proteases (PNAS 2013 Jan 2; 110(1): 93-98.) or labeling the peptides recognized by the protease with fluorescence and quenching them before cleavage, but allowing them to glow after cleavage (Nat Rev Drug Discov. 2010 Sep;9(9):690-701. doi: 10.1038 / nrd3053.).

[0083] From one perspective, the term "tissue-specific protease" can refer to any of the following: (i) Proteases exhibiting higher levels of activity in target tissues compared to normal tissues. (ii) Proteases that exhibit higher activity in target tissues compared to normal tissues. (iii) Proteases exhibiting higher levels of expression in target cells compared to normal cells. (iv) Proteases that have higher activity in target cells than in normal cells.

[0084] While not explicitly defined, specific proteases can be listed, including: cysteine ​​proteases (including the cathepsin family B, L, S, etc.), aspartic acid proteases (autolysins D, E, K, O, etc.), serine proteases (including mastriptases (including MT-SP1), autolysins A and G, thrombin, plasmain, urokinase (uPA), tissue plasminogen activating factor (tPA), elastase, protease 3, thrombin, kallikrein, and neutral proteins. Tryptase, chymase, metalloproteinases (including membrane-bound (MMP14-17 and MMP24-25) and secretory (MMP1-13, MMP18-23 and MMP26-28) metalloproteinases (MMP1-28), disintegrin and ADAM, metalloproteinases with disintegrin or thrombospondin functional domains (ADAMTS), meprin (Meprin α, Meprin β), CD10 (CALLA), prostate-specific antigen (PSA), Legumin, TMPRSS3, TMPRSS4, neutrophil elastase (HNE), beta-secretase (BACE), fibroblast activation protein alpha (FAP), Granzyme B. Guanidine-benzoatase (GB), pepsin, neprilysin, NS3 / 4A, HCV-NS3 / 4, calpain, ADAMDEC1, renin, autolysin C, autolysin V / L2, autolysin X / Z / P, cruzipain, otubain2, kallikrein-related peptidases (KLKs (KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14)), bone morphogenetic protein 1 (BMP-1), activated protein C, blood coagulation-related proteases (Factor VIIa, Factor IXa, Factor Xa, Factor XIa, Factor... XIIa), HtrA1, lactoferrin, Marapsin, PACE4, DESC1, dipeptidase-4 (DPP-4), TMPRSS2, autolysin F, autolysin H, autolysin L2, autolysin O, autolysin S, granzyme A, gepsin calpain2. Glutamic acid carboxypeptidase 2, AMSH-Like Proteases, AMSH, gamma secretase, A anti-cytoplasmic cleavage enzyme (APCE), Decysin 1, N-Acetylated Alpha-Linked Acidic Dipeptidase-Like 1 (NAALADL1), furin, etc.

[0085] From another perspective, tissue-specific proteases can refer to proteases specific to cancer tissues or proteases specific to inflamed tissues.

[0086] Cancer tissue-specific proteases, such as those disclosed in International Publication WO2013 / 128194, International Publication WO2010 / 081173, and International Publication WO2009 / 025846, exhibit specific expression of proteases in cancer tissues.

[0087] Regarding the types of cancer-specific proteases, the higher the specificity of the protease in the cancerous tissue of the treatment target, the greater the reduction in side effects. Ideally, the concentration of cancer-specific proteases in cancerous tissue should be at least 5 times higher than that in normal tissue; at least 10 times higher is better; at least 100 times higher is ideal; at least 500 times higher is particularly ideal; and at least 1000 times higher is most ideal. Furthermore, the activity of cancer-specific proteases in cancerous tissue should ideally be at least 2 times higher than that in normal tissue; at least 3 times, 4 times, 5 times, or 10 times higher is better; at least 100 times higher is more ideal; at least 500 times higher is particularly ideal; and at least 1000 times higher is most ideal. Furthermore, cancer-specific proteases can be those that bind to the cell membrane of cancer cells or those that are secreted extracellularly instead of binding to the cell membrane. When cancer-specific proteases are not bound to the cell membrane of cancer cells, the cell damage caused by immune cells is specific to cancer cells. Therefore, it is preferable that cancer-specific proteases be located inside or near cancer tissue. In this specification, "near cancer tissue" refers to the area within which the cleavage sequence of cancer-specific proteases is cleaved, thereby reducing their ligand-binding activity. However, it is best to minimize damage to normal cells. From another perspective, cancer-specific proteases are any of the following: (i) Proteases exhibiting higher levels of activity in cancerous tissues compared to normal tissues. (ii) Proteases that exhibit higher activity in cancerous tissues than in normal tissues. (iii) Proteases expressed at higher levels in cancer cells than in normal cells. (iv) Proteases that have higher activity in cancer cells than in normal cells. Cancer-specific proteases can be a single type or a combination of two or more. The number of cancer-specific proteases can be appropriately determined by those with ordinary knowledge in the art, taking into account the type of cancer being treated.

[0088] Based on the above viewpoints, the most ideal proteases specific to cancer tissue are serine proteases and metalloproteinases among the proteases listed above, with mastriptase (including MT-SP1), urokinase (uPA), and metalloproteinases being even better, and MT-SP1, uPA, MMP2, and MMP9 being the most desirable.

[0089] Regarding the types of proteases specific to inflamed tissues, the higher the specificity in the inflamed tissue of the treatment target, the greater the reduction in side effects. Ideally, the concentration of proteases specific to inflamed tissues should be at least 5 times higher than that in normal tissues, more than 10 times higher is better, more than 100 times higher is ideal, more than 500 times higher is particularly ideal, and more than 1000 times higher is most ideal. Furthermore, the activity of proteases specific to inflamed tissues should ideally be at least 2 times higher than that in normal tissues, more than 3 times, 4 times, 5 times, or 10 times higher is better, more than 100 times higher is more ideal, more than 500 times higher is particularly ideal, and more than 1000 times higher is most ideal. Furthermore, tissue-specific proteases can bind to the cell membrane of inflamed tissue or be secreted extracellularly instead of binding to the cell membrane. When tissue-specific proteases are not bound to the cell membrane of inflamed tissue, the cell damage caused by immune cells is specific to inflamed tissue; therefore, it is preferable that tissue-specific proteases be located inside or near the inflamed tissue. In this specification, "near the inflamed tissue" refers to the area within which the cleavage sequence of tissue-specific proteases is cleaved, thereby reducing their ligand-binding activity. However, it is best to minimize damage to normal cells. According to other viewpoints, the protease specific to inflamed tissues is any of the following: (i) Proteases exhibiting higher levels of activity in inflamed tissues compared to normal tissues. (ii) Proteases that exhibit higher activity in inflamed tissues than in normal tissues (iii) Proteases that are expressed at a higher level in inflammatory cells than in normal cells (iv) Proteases that have higher activity in inflammatory cells than in normal cells. Inflammatory tissue-specific proteases can be a single type or a combination of two or more. The number of types of inflammatory tissue-specific proteases can be appropriately determined by someone with ordinary knowledge in the art, taking into account the symptoms of the patient being treated.

[0090] Based on the above points, the most ideal proteases for inflamed tissues are metalloproteinases among the proteases listed above, and among metalloproteinases, ADAMTS5, MMP2, MMP7, MMP9, and MMP13 are even more ideal.

[0091] A protease cleavage sequence is a specific amino acid sequence that is specifically recognized by a tissue-specific protease when a polypeptide is hydrolyzed in aqueous solution by the tissue-specific protease. From the perspective of reducing side effects, it is preferable to use amino acid sequences that are specifically hydrolyzed by protease cleavage to utilize proteins that are specifically expressed in or activated in the target tissues / cells of the treatment subject. Specific protease cleavage sequences, such as those disclosed in International Publications WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846, are target sequences specifically hydrolyzed by proteases exhibiting cancer-specific behavior or inflammatory tissue-specific behavior as exemplified above. Sequences artificially altered by introducing appropriate amino acid variations into known protease-specific hydrolysis target sequences can also be used. Furthermore, protease cleavage sequences can be identified using methods known to those skilled in the art, such as those described in Nature Biotechnology 19, 661-667 (2001). Alternatively, naturally occurring protease cleavage sequences can be used. For example, the protease-cleavage-receiving sequence can be used in proteins whose molecular shape is altered due to protease cleavage, such as TGFβ, which changes to its latent form upon acceptance of protease cleavage.

[0092] While the protease cleavage sequence is not limited to these examples, the following international publications may be used: WO2015 / 116933, WO2015 / 048329, WO2016 / 118629, WO2016 / 179257, WO2016 / 179285, WO2016 / 179335, WO2016 / 179003, WO2016 / 046778, WO2016 / 014974, US Patent Publication US2016 / 0289324, US Patent Publication US2016 / 0311903, PNAS (2000) 97: 7754-7759, Biochemical Journal (2010) 426: 219-228, and Beilstein J. The sequence is illustrated in Nanotechnol. (2016) 7: 364-373. The amino acid sequence for protease cleavage, as described above, is preferably an amino acid sequence that can be specifically hydrolyzed by a protease with ideal target tissue specificity. Among the amino acid sequences that can be specifically hydrolyzed by a protease with target tissue specificity, the following amino acid sequences are preferred. LSGRSDNH (Sequence number: 3, MT-SP1, can be cleaved using uPA) PLGLAG (Sequence number: 34, can be cut using MMP2 and MMP9) VPLSLTMG (Sequence number: 35, can be cut using MMP7) The following sequences can also be used for protease cleavage sequences. TSTSGRSANPRG (Serial Number: 66, MT-SP1, can be cut using uPA) ISSGLLSGRSDNH (Sequence number: 67, MT-SP1, can be cut using uPA) AVGLLAPPGGLSGRSDNH (Serial No.: 68, MT-SP1, can be cut using uPA) GAGVPMSMRGGAG (Serial number: 69, can be cut using MMP1) GAGIPVSLRSGAG ​​(Serial No.: 70, can be cut using MMP2) GPLGIAGQ (Serial Number: 71, can be cut using MMP2) GGPLGMLSQS (Serial Number: 72, can be cut using MMP2) PLGLWA (Sequence number: 73, can be cut using MMP2) GAGRPFSMIMGAG (Serial Number: 74, can be cut using MMP3) GAGVPLSLTMGAG (Serial No.: 75, can be cut using MMP7) GAGVPLSLYSGAG (Serial No.: 76, can be cut using MMP9) AANLRN (Sequence number: 77, can be cut using MMP11) AQAYVK (Serial Number: 78, can be cut using MMP11) AANYMR (Sequence number: 79, can be cut using MMP11) AAALTR (Sequence number: 80, can be cut using MMP11) AQNLMR (Sequence number: 81, can be cut using MMP11) AANYTK (Serial Number: 82, can be cut using MMP11) GAGPQGLAGQRGIVAG (Serial number: 83, can be cut using MMP13) PRFKIIGG (Sequence number: 84, can be cleaved using prourokinase) PRFRIIGG (Sequence number: 85, can be cleaved using prourokinase) GAGSGRSAG (Serial Number: 86, can be cut using uPA) SGRSA (Sequence number: 87, can be cut using uPA) GSGRSA (Sequence number: 88, can be cut using uPA) SGKSA (Sequence number: 89, can be cut using uPA) SGRSS (Sequence number: 90, can be cut using uPA) SGRRA (Sequence number: 91, can be cut using uPA) SGRNA (Sequence number: 92, can be cleaved using uPA) SGRKA (Sequence number: 93, can be cut using uPA) QRGRSA (Sequence number: 94, can be cut using tPA) GAGSLLKSRMVPNFNAG (Sequence number: 95, can be cleaved by autolysin B) TQGAAA (Sequence number: 96, can be cleaved using autolysin B) GAAAAA (Sequence number: 97, can be cleaved using autolysin B) GAGAAG (Sequence number: 98, can be cleaved by autolysin B) AAAAAG (Sequence number: 99, can be cleaved using autolysin B) LCGAAI (Sequence number: 100, can be cleaved by autolysin B) FAQALG (Sequence number: 101, can be cleaved using autolysin B) LLQANP (Sequence number: 102, can be cleaved by autolysin B) LAAANP (Sequence number: 103, can be cleaved using autolysin B) LYGAQF (Sequence number: 104, can be cleaved by autolysin B) LSQAQG (sequence number: 105, cleavable by autolysin B) ASAASG (Sequence number: 106, can be cleaved by autolysin B) FLGASL (Sequence number: 107, can be cleaved by autolysin B) AYGATG (Sequence number: 108, can be cleaved by autolysin B) LAQATG (Sequence number: 109, can be cleaved by autolysin B) GAGSGVVIATVIVITAG (Sequence number: 110, can be cleaved by autolysin L) APMAEGGG (Sequence number: 111, can be cleaved using Meprinα and Meprinβ) EAQGDKII (Sequence number: 112, can be cleaved using Meprinα and Meprinβ) LAFSDAGP (Sequence number: 113, can be cleaved using Meprinα and Meprinβ) YVADAPK (Sequence number: 114, can be cleaved using Meprinα and Meprinβ) RRRRR (Sequence number: 115, can be cut using furin) RRRRRR (Sequence number: 116, can be cut using furin) GQSSRHRRAL (Sequence number: 117, can be cut using furin) SSRHRRALD (Serial Number: 118) RKSSIIIRMRDVVL (Sequence number: 119, can be cleaved using plasmagen) SSSFDKGKYKKGDDA (Sequence number: 120, can be cleaved using Staphylokinase) SSSFDKGKYKRGDDA (Sequence number: 121, can be cleaved using Staphylokinase) IEGR (Sequence number: 122, can be cut using FactorIXa) IDGR (Sequence number: 123, can be cut using FactorIXa) GGSIDGR (serial number: 124, can be cut using FactorIXa) GPQGIAGQ (Sequence number: 125, can be cleaved using collagenase) GPQGLLGA (Sequence number: 126, can be cleaved by collagenase) GIAGQ (Sequence number: 127, can be cleaved by collagenase) GPLGIAG (Sequence number: 128, can be cleaved by collagenase) GPEGLRVG (Sequence number: 129, can be cleaved by collagenase) YGAGLGVV (Sequence number: 130, can be cleaved using collagenase) AGLGVVER (Sequence number: 131, can be cleaved by collagenase) AGLGISST (Sequence number: 132, can be cleaved using collagenase) EPQALAMS (Sequence number: 133, can be cleaved using collagenase) QALAMSAI (Sequence number: 134, can be cleaved by collagenase) AAYHLVSQ (Sequence number: 135, can be cleaved using collagenase) MDAFLESS (Sequence number: 136, can be cleaved by collagenase) ESLPVVAV (Sequence number: 137, can be cleaved using collagenase) SAPAVESE (Sequence number: 138, can be cleaved by collagenase) DVAQFVLT (Sequence number: 139, can be cleaved using collagenase) VAQFVLTE (Sequence number: 140, can be cleaved using collagenase) AQFVLTEG (Sequence number: 141, can be cleaved using collagenase) PVQPIGPQ (Sequence number: 142, can be cleaved using collagenase) LVPRGS (Sequence number: 143, can be cleaved using thrombin) TSTSGRSANPRG (Serial Number: 345)

[0093] In one embodiment of the present invention, a movable linker may be added to one or both ends of the protease cleavage sequence. The movable linker at one end of the protease cleavage sequence may be referred to as a first movable linker, and the movable linker at the other end may be referred to as a second movable linker. In a specific embodiment, the protease cleavage sequence and the movable linker include one of the following formulas. (Protein cleavage sequence) (First mobile linker) - (Protein cleavage sequence) (Protein cleavage sequence) - (Second mobile linker) (First mobile linker) - (Protein cleavage sequence) - (Second mobile linker) In this embodiment, the mobile linker is preferably a peptide linker. The first and second mobile linkers are independent and arbitrary mobile linkers containing at least one flexible amino acid (Gly, etc.), which may be the same or different. For example, they contain a sufficient number of residues (amino acids of any choice from Arg, Ile, Gln, Glu, Cys, Tyr, Trp, Thr, Val, His, Phe, Pro, Met, Lys, Gly, Ser, Asp, Asn, Ala, etc., especially Gly, Ser, Asp, Asn, Ala, and further Gly and Ser, especially Gly, etc.) that would yield the desired protease accessibility from the protease cleavage sequence.

[0094] Movable linkers suitable for use at both ends of protease cleavage sequences typically improve the proximity of the protease to the cleavage sequence and enhance the cleavage efficiency of the protease. Ideal movable linkers can be easily selected from various lengths, primarily ranging from 1 amino acid (Gly, etc.) to 20 amino acids, 2 to 15 amino acids, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, with a preference for lengths from 3 to 12 amino acids. In some embodiments of the present invention, the movable linkers are peptide linkers of 1 to 7 amino acids.

[0095] Movable connectors, such as but not limited to, include: glycine polymers (G)n, glycine-serine polymers (e.g., including (GS)n, (GSGGS: sequence number: 45)n and (GGGS: sequence number: 36)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other movable connectors known in the art. Among them, glycine and glycine-serine polymers have attracted attention because these amino acids are relatively unstructured and can easily act as neutral chains between components. Movable linkers composed of glycine-serine polymers, such as, but not limited to, the following: Ser Gly·Ser(GS) Ser·Gly (SG) Gly·Gly·Ser (GGS) Gly·Ser·Gly(GSG) Ser·Gly·Gly (SGG) Gly·Ser·Ser(GSS) Ser·Ser·Gly (SSG) Ser·Gly·Ser(SGS) Gly·Gly·Gly·Ser (GGGS, Serial Number: 36) Gly·Gly·Ser·Gly (GGSG, Serial Number: 37) Gly·Ser·Gly·Gly (GSGG, Serial Number: 38) Ser·Gly·Gly·Gly (SGGG, Serial Number: 39) Gly·Ser·Ser·Gly (GSSG, Serial Number: 40) Gly·Gly·Gly·Gly·Ser (GGGGS, Serial Number: 41) Gly·Gly·Gly·Ser·Gly (GGGSG, Serial Number: 42) Gly·Gly·Ser·Gly·Gly (GGSGG, Serial Number: 43) Gly·Ser·Gly·Gly·Gly (GSGGG, Serial Number: 44) Gly·Ser·Gly·Gly·Ser (GSGGS, Serial Number: 45) Ser·Gly·Gly·Gly·Gly (SGGGG, Serial Number: 46) Gly·Ser·Ser·Gly·Gly (GSSGG, Serial Number: 47) Gly·Ser·Gly·Ser·Gly (GSGSG, Serial Number: 48) Ser·Gly·Gly·Ser·Gly (SGGSG, Serial Number: 49) Gly·Ser·Ser·Ser·Gly (GSSSG, Serial Number: 50) Gly·Gly·Gly·Gly·Gly·Ser(GGGGGS, Serial Number: 51) Ser·Gly·Gly·Gly·Gly (SGGGGG, Serial Number: 52) Gly·Gly·Gly·Gly·Gly·Ser(GGGGGGS, Serial Number: 53) Ser·Gly·Gly·Gly·Gly·Gly (SGGGGGG, Serial Number: 54) (Gly·Gly·Gly·Gly·Ser(GGGGS, Serial Number: 41))n (Ser·Gly·Gly·Gly·Gly(SGGGG, Serial Number: 46))n [n is an integer greater than or equal to 1] etc. However, the length and sequence of the peptide linker can be appropriately selected by those with general knowledge in this field, depending on the purpose.

[0096] In some embodiments of the present invention, the ligand-binding molecules include antibody VH and antibody VL. Ligand-binding molecules including VH and VL include, but are not limited to, Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, complete antibodies, etc.

[0097] In some embodiments of the present invention, the ligand-binding molecule includes an Fc region. When using the Fc region of an IgG antibody, its type is not limited; Fc regions such as IgG1, IgG2, IgG3, and IgG4 can be used. For example, an Fc region including one of the amino acid sequences represented by sequence numbers 55, 56, 57, and 58, or an Fc region variant with alterations applied to such Fc regions, can be used. Furthermore, in some embodiments of the present invention, the ligand-binding molecule includes an antibody-invariant region.

[0098] In some more specific embodiments of the present invention, the ligand-binding molecule is an antibody. When an antibody is used as the ligand-binding molecule, ligand binding is achieved by a variable region. In some more specific embodiments, the ligand-binding molecule is an IgG antibody. When using an IgG antibody as the ligand-binding molecule, the type is not limited, and IgG1, IgG2, IgG3, IgG4, etc., can be used. When using an IgG antibody as the ligand-binding molecule, ligand binding is also achieved by a variable region; one or both of the two variable regions of the IgG antibody can achieve ligand binding.

[0099] In some embodiments of the present invention, by cleaving the cleavage site / protease cleavage sequence in the ligand-binding molecule, the ligand-binding active domain of the ligand-binding molecule is segmented and cleaved, thereby weakening the binding to the ligand. For example, when using an IgG antibody as the ligand-binding molecule, a cleavage site / protease cleavage sequence is provided in the antibody variable region. In the cleaved state, the antibody variable region cannot be fully formed, resulting in a weakened binding to the ligand.

[0100] In this specification, "assembly" can be used to refer to, for example, the interaction of two or more peptide regions. Generally, the target peptide regions form assemblies by forming hydrophobic bonds, hydrogen bonds, ionic bonds, etc. A common example of assembly is known in antibodies, represented by natural antibodies, where the heavy chain variable region (VH) and the light chain variable region (VL) maintain their paired structure using non-covalent bonds between them.

[0101] In some embodiments of the present invention, the VH and VL groups contained in the ligand-binding molecule are assembled. Furthermore, the assembly of antibody VH and antibody VL can be dismantled, for example, by cleavage at a cleavage site / protease cleavage sequence. Dismantling of assembly can, for example, be replaced by the complete or partial dismantling of the interaction between two or more peptide regions. The dismantling of VH and VL assembly can be the complete dismantling of the VH and VL interaction, or it can be the partial dismantling of the VH and VL interaction. The ligand-binding molecule of the present invention includes an assembly of antibody VL or a portion thereof and antibody VH or a portion thereof, which is released by cleaving the cleavage site or by protease cleaving the protease cleavage sequence.

[0102] In some embodiments of the present invention, the ligand-binding molecule includes antibody VH and antibody VL. In the state where the cleavage site / protease cleavage sequence of the ligand-binding molecule is not cleaved, antibody VH and antibody VL in the ligand-binding molecule are assembled. Cleavage by the cleavage site / protease cleavage sequence disassembles the assembly of antibody VH and antibody VL in the ligand-binding molecule. The cleavage site / protease cleavage sequence in the ligand-binding molecule can be positioned at any position in the ligand-binding molecule, as long as cleavage weakens the binding ability of the ligand-binding molecule to the ligand.

[0103] In further embodiments of the present invention, the ligand-binding molecule includes antibody VH, antibody VL, and antibody invariant region. According to Rothlisberger et al. (J Mol Biol. 2005 Apr 8;347(4):773-89.), the VH and VL, CH and CL of antibodies are known to interact through the separation of multiple amino acid side chains between their domains. VH-CH1 and VL-CL are known to form stable structures with respect to the Fab domain, but as some have reported, the amino acid side chains between VH and VL generally interact with a dissociation constant in the range of 10⁻⁵ M to 10⁻⁸ M, and it is believed that the proportion of assembled states formed when only the VH and VL domains are present is small.

[0104] In some embodiments of the present invention, a ligand-binding molecule containing antibodies VH and VL is designed with a cleavage site / protease cleavage sequence. Before cleavage, the heavy-light chain interaction in the Fab structure exists between the two peptides. However, when the cleavage site / protease cleavage sequence is cleaved, the interaction between the peptide including VH (or a portion of VH) and the peptide including VL (or a portion of VL) is weakened, and the assembly of VH and VL is released.

[0105] In one embodiment of the present invention, the cleavage site / protease cleavage sequence is located within the antibody-invariant region. More specifically, in another embodiment, the cleavage site / protease cleavage sequence is located closer to the variable region side than amino acid 140 (EU number) in the antibody heavy chain invariant region, preferably closer to the variable region side than amino acid 122 (EU number) in the antibody heavy chain invariant region. In some specific embodiments, the cleavage site / protease cleavage sequence is inserted at any position within the sequence of amino acids 118 (EU number) to 140 (EU number) in the antibody heavy chain invariant region. In other more specific embodiments, the cleavage site / protease cleavage sequence is located closer to the variable region side than amino acid 130 (EU number) (Kabat number 130) in the antibody light chain invariant region, preferably closer to the variable region side than amino acid 113 (EU number) (Kabat number 113) in the antibody light chain invariant region, and closer to the variable region side than amino acid 112 (EU number) (Kabat number 112) in the antibody light chain invariant region. In some specific embodiments, the cleavage site / protease cleavage sequence is inserted at any position in the sequence from amino acid 108 (EU number) (Kabat number 108) to amino acid 131 (EU number) (Kabat number 131) in the antibody light chain invariant region.

[0106] In one embodiment of the present invention, the cleavage site / protease cleavage sequence is located within antibody VH or antibody VL. More specifically, in another embodiment, the cleavage site / protease cleavage sequence is located closer to the antibody-invariant region than amino acid 7 (Kabat number) of antibody VH, preferably closer to the antibody-invariant region than amino acid 40 (Kabat number) of antibody VH, more preferably closer to the antibody-invariant region than amino acid 101 (Kabat number) of antibody VH, even more preferably closer to the antibody-invariant region than amino acid 109 (Kabat number) of antibody VH, and closer to the antibody-invariant region than amino acid 111 (Kabat number) of antibody VH. In a more specific embodiment, the cleavage site / protease cleavage sequence is located closer to the antibody-invariant region than amino acid 7 (Kabat number) of antibody VL, preferably closer to the antibody-invariant region than amino acid 39 (Kabat number) of antibody VL, more preferably closer to the antibody-invariant region than amino acid 96 (Kabat number) of antibody VL, and even more preferably closer to the antibody-invariant region than amino acid 104 (Kabat number) of antibody VL, and closer to the antibody-invariant region than amino acid 105 (Kabat number) of antibody VL. In some more specific embodiments, the cleavage site / protease cleavage sequence is inserted into the residues forming the loop structure in antibody VH or antibody VL, and at positions close to the loop structure. The loop structure in antibody VH or antibody VL refers to the portion of antibody VH or antibody VL that has not formed secondary structures such as α-helices or β-lamellae. Specifically, the residues forming the loop structure and the positions close to the loop structure can refer to amino acids 7 to 16 (Kabat numbers), 40 to 47 (Kabat numbers), 55 to 69 (Kabat numbers), 73 to 79 (Kabat numbers), 83 to 89 (Kabat numbers), and 95 (Kabat number). The range of amino acids (Kabat numbers) from 99 to 113, from 7 to 19, from 39 to 46, from 49 to 62, and from 96 to 107. In some more specific embodiments, the cleavage site / protease cleavage sequence is inserted at any position in the sequence of amino acids VH7 (Kabat number) to 16 (Kabat number), amino acids 40 (Kabat number) to 47 (Kabat number), amino acids 55 (Kabat number) to 69 (Kabat number), amino acids 73 (Kabat number) to 79 (Kabat number), amino acids 83 (Kabat number) to 89 (Kabat number), amino acids 95 (Kabat number) to 99 (Kabat number), and amino acids 101 (Kabat number) to 113 (Kabat number). In some more specific embodiments, the cleavage site / protease cleavage sequence is inserted at any position in the sequence of amino acids VL7 (Kabat number) to 19 (Kabat number), amino acids 39 (Kabat number) to 46 (Kabat number), amino acids 49 (Kabat number) to 62 (Kabat number), and amino acids 96 (Kabat number) to 107 (Kabat number).

[0107] In one embodiment of the present invention, the cleavage site / protease cleavage sequence is located near the boundary between antibody VH and the antibody invariant region. The boundary between antibody VH and the antibody heavy chain invariant region can refer to the area between amino acid 101 (Kabat number) of antibody VH and amino acid 140 (EU number) of antibody heavy chain invariant region, preferably between amino acid 109 (Kabat number) of antibody VH and amino acid 122 (EU number) of antibody heavy chain invariant region, or between amino acid 111 (Kabat number) of antibody VH and amino acid 122 (EU number) of antibody heavy chain invariant region. Furthermore, when antibody VH is linked to the antibody light chain invariant region, the boundary between antibody VH and the antibody light chain invariant region can refer to the area between amino acid number 101 (Kabat number) of antibody VH and amino acid number 130 (EU number (Kabat number 130)) of antibody light chain invariant region, preferably between amino acid number 109 (Kabat number) of antibody VH and amino acid number 113 (EU number (Kabat number 113)) of antibody light chain invariant region, or between amino acid number 111 (Kabat number) of antibody VH and amino acid number 112 (EU number (Kabat number 112)) of antibody light chain invariant region.

[0108] In one embodiment, the cleavage site / protease cleavage sequence is located near the boundary between the antibody VL and the antibody invariant region. The boundary between the antibody VL and the antibody light chain invariant region can refer to the area between amino acid 96 (Kabat number) of the antibody VL and amino acid 130 (EU number) (Kabat number 130) of the antibody light chain invariant region; more preferably, it can refer to the area between amino acid 104 (Kabat number) of the antibody VL and amino acid 113 (EU number) (Kabat number 113) of the antibody light chain invariant region, or between amino acid 105 (Kabat number) of the antibody VL and amino acid 112 (EU number) (Kabat number 112) of the antibody light chain invariant region. When antibody VL is linked to the antibody heavy chain invariant region, the boundary between antibody VL and antibody heavy chain invariant region can refer to the area between amino acid number 96 (Kabat number) of antibody VL and amino acid number 140 (EU number) of antibody heavy chain invariant region, preferably between amino acid number 104 (Kabat number) of antibody VL and amino acid number 122 (EU number) of antibody heavy chain invariant region, or between amino acid number 105 (Kabat number) of antibody VL and amino acid number 122 (EU number) of antibody heavy chain invariant region.

[0109] The cleavage site / protease cleavage sequence can be set in multiple locations within the ligand molecule, for example, at multiple locations selected from within the antibody invariant region, within antibody VH, within antibody VL, near the boundary between antibody VH and the antibody invariant region, and near the boundary between antibody VL and the antibody invariant region. Furthermore, for those skilled in the art, the antibody VH and antibody VL can be replaced, and the shape of the molecule containing antibody VH, antibody VL, and the antibody invariant region can be changed without departing from the scope of this invention.

[0110] In this specification, the term "ligand" refers to a biologically active molecule. Biologically active molecules typically interact with receptors on the cell surface to regulate biological stimulation, inhibition, or other modes of action, and are believed to typically involve intracellular signaling pathways that contain the aforementioned receptors.

[0111] In this specification, ligands include the desired molecules that exert biological activity through interaction with molecules in a living organism. For example, a ligand is not only a molecule that interacts with a receptor, but also a molecule that exerts biological activity through interaction with that molecule, such as the receptor interacting with that molecule, its binding fragments, etc., are also included in ligands. For example, the ligand-binding site of a known receptor protein, and proteins containing sites where the receptor interacts with other molecules, are included in the ligands of this invention. Specifically, soluble receptors, soluble fragments of receptors, extracellular domains of membrane-transfer receptors, and peptides containing them are included in the ligands of this invention.

[0112] The ligands of this invention typically exert their desired biological activity by binding to one or more binding partners. The binding partners of the ligand can be extracellular, intracellular, or membrane-bound proteins. In one embodiment, the binding partner of the ligand is an extracellular protein, such as a soluble receptor. In other embodiments, the binding partner of the ligand is a membrane-bound receptor. The ligands of the present invention can bind specifically to their binding partners with dissociation constants (KD) below 10 μM, 1 μM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 25 pM, 10 pM, 5 pM, 1 pM, 0.5 pM, or 0.1 pM.

[0113] As biologically active molecules, such as, but not limited to, interleukins, chemokines, multipeptide hormones, growth factors, apoptosis-inducing factors, PAMPs, DAMPs, nucleic acids, or fragments thereof. In more detailed embodiments, interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell proliferation factors, the TGF-β family, myokine, adipokine, or neurotrophic factors may be used as ligands. In even more detailed embodiments, CXCL10, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-β, IFN-g, MIG, I-TAC, RANTES, MIP-1a, or MIP-1b may be used as ligands.

[0114] Chemokines are a family of homogeneous serum proteins ranging from 7 to 16 kDa, characterized by their ability to induce leukocyte migration. Most chemokines have four characteristic cysteine ​​residues (Cys), classified into CXC, or alpha, CC, or beta, C, or gamma, and CX3C, or delta chemokine classes based on the functional domains indicated by the first two cysteine ​​residues. Two disulfide bonds are formed between the first and third cysteine ​​residues, and between the second and fourth cysteine ​​residues. Generally, disulfide cross-linking is required; Clark-Lewis et al. reported that, at least for CXCL10, the disulfide bond is decisive for chemokine activity (Clark-Lewis et al., J. Biol. Chem. 269:16075-16081, 1994). The only exception with four cysteine ​​residues is lymphotactin, which has only two cysteine ​​residues. Therefore, lymphotactin maintains a functional structure using only one disulfide bond. Furthermore, the CXC or alpha subfamily is classified into two groups due to the presence of an ELR functional domain (Glu-Leu-Arg) that precedes cysteine: ELR-CXC chemokines and non-ELR-CXC chemokines (e.g., Clark-Lewis, mentioned above, and Belperio et al., "CXC Chemokines in Angiogenesis", J. Leukoc. Biol. 68:1-8, 2000).

[0115] Interferon-inducible protein 10 (IP-10 or CXCL10) is induced by interferon-γ and TNF-α and produced by keratinocytes, endothelial cells, fibroblasts, and monocytes. IP-10 is thought to play a role in supporting the proliferation of activated T cells toward inflamed tissues (Dufour, et al., "IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking," J Immunol., 168:3195-204, 2002). Furthermore, IP-10 may be responsible for allergic reactions. Furthermore, it may also be responsible for the occurrence of inflammatory demyelinating neuropathies (Kieseier, et al., "Chemokines and chemokine receptors in inflammatory demyelinating neuropathies: a central role for IP-10", Brain 125:823-34, 2002).

[0116] Studies have shown that IP-10 may be useful for the engraftment of stem cells in subsequent transplants (Nagasawa, T., Int. J. Hematol. 72:408-11, 2000), the enhancement of stem cells (Gazitt, Y., J. Hematother Stem Cell Res 10:229-36, 2001; Hattori et al., Blood 97:3354-59, 2001), and the enhancement of anti-tumor immunity (Nomura et al., Int. J. Cancer 91:597-606, 2001; Mach and Dranoff, Curr. Opin. Immunol. 12:571-75, 2000). For example, reports known to those of ordinary skill in the art discuss the biological activity of chemokines (Bruce, L. et al., "Radiolabeled Chemokine binding assays," Methods in Molecular Biology (2000) vol. 138, pp129-134; Raphaele, B. et al., "Calcium Mobilization," Methods in Molecular Biology (2000) vol. 138, pp143-148; Paul D. Ponath et al., "Transwell Chemotaxis," Methods in Molecular Biology (2000) vol. 138, pp113-120 Humana Press. Totowa, New Jersey).

[0117] The biological activities of CXCL10 include binding to the CXCL10 receptor (CXCR3), CXCL10-induced calcium flow, CXCL10-induced cell chemotaxis, CXCL10 binding to glycosaminoglycans, and CXCL10 oligomerization. Methods for measuring the physiological activity of CXCL10 include methods for measuring the cell migration activity of CXCL10, the Reporter assay using CXCR3 stable expression cell lines (see PLoS One. 2010 Sep 13;5(9):e12700.), and the PathHunter™ β-Arrestin recruitment assay using β-Arrestin recruitment induced in the early stage of GPCR signal transduction.

[0118] Interleukin-12 (IL-12) is a heterodimeric cytokine composed of 30- and 40-kD glycosylated polypeptide chains linked by disulfide bonds. Cytokines are synthesized and secreted by antigen-inducing cells including dendritic cells, monocytes, macrophages, B cells, Lambda cells, keratinocytes, and natural killer (NK) cells. IL-12 mediates various biological processes and involves NK cell-stimulating factor (NKSF), T cell-stimulating factor, cell barrier T lymphocyte maturation factor, and EBV-transformed B cell lineage factor.

[0119] Interleukin-12 (IL-12) can alter biological processes (e.g., initiation, inhibition) by binding to IL-12 receptors expressed on the cell membranes of cells (e.g., T cells, NK cells). For example, IL-12 binding to its receptors stimulates the proliferation of pre-activated T cells and NK cells, enhances the cytolytic activity of cytotoxic T cells (CTLs), NK cells, and LAK (lymphokine-activated killer) cells, induces the production of interferon-gamma (IFNγ) by T cells and NK cells, and further induces the differentiation of naive Th0 cells into Th1 cells that produce IFNγ and IL-2. In particular, IL-12 is absolutely essential for setting the generation of cytolytic cells (e.g., NK, CTLs) and cellular immune responses (e.g., Th1 cell-mediated immune responses). In conclusion, IL-12 is absolutely important in the generation and regulation of both preventive immunity (e.g., eradication of infections) and pathological immune responses (e.g., autoimmunity).

[0120] Methods for measuring the physiological activity of IL12 include measuring the cell proliferation activity of IL12, analyzing the STAT4 reporter gene, measuring cell activation (cell surface marker expression, intercytokine production, etc.) using IL12, and promoting cell differentiation using IL12.

[0121] Programmed Death 1 (PD-1) is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and bone marrow cells (Okazaki et al. (2002) Curr. Opin. Immunol. 14:391779-82, Bennett et al. (2003) J Immunol 170:711-8). The original members of this family, CD28 and ICOS, were discovered due to their functional effects on the increased T cell proliferation following the addition of monoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266, Hansen et al. (1980) Immunogenics 10:247-260). PD-1 was discovered by screening for different behaviors in apoptotic cells (Ishida et al. (1992) EMBO J. 11:3887-95). Other members of this family, CTLA-4 and BTLA, were discovered by screening for different behaviors in noxious T lymphocytes and TH1 cells. CD28, ICOS, and CTLA-4 all contain unpaired cysteine ​​residues, which can homopolymerize. In contrast, PD-1 is believed to exist in monomeric form and does not possess the unpaired cysteine ​​residues characteristic of other CD28 family members.

[0122] The PD-1 gene is a 55 kDa type I membrane-bound protein, part of the Ig gene superfamily. PD-1 includes the proximal tyrosine inhibitory domain (ITIM) and the distal tyrosine-base switch domain (ITSM). PD-1 is structurally similar to CTLA-4, but lacks the MYPPPY domain (sequence number: 537), which is crucial for binding to B7-1 and B7-2. Two ligands targeting PD-1, PD-L1 and PD-L2, have been identified. If they bind to PD-1, they negatively control T cell activation (Freeman et al. (2000) J Exp Med 192:1027-34, Latchman et al. (2001) Nat Immunol 2:261-8, Carter et al. (2002) Eur J Immunol 32:634-43). Both PD-L1 and PD-L2 are B7 homologues of PD-1 that bind to PD-1 but not to other CD28 family members. PD-L1, a ligand of PD-1, is abundant in various human cancers (Dong et al. (2002) Nat. Med. 8:787-9). The interaction between PD-1 and PD-L1 results in a decrease in tumor-infiltrating lymphocytes, a reduction in T-cell receptor-mediated proliferation, and immune avoidance using cancer cells (Dong et al. (2003) J. Mol. Med. 81:281-7, Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314, Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immunosuppression can be reversed by inhibiting the local interaction between PD-L1 and PD-1, and when the interaction between PD-L2 and PD-2 is inhibited in the same way, the effect is additive (Iwai et al. (2002) Proc. Nat' l. Acad. Sci. USA 99:12293-7, Brown et al. (2003) J. Immunol. 170:1257-66).

[0123] PD-1 is a suppressor member of the CD28 family that is expressed on activated B cells, T cells, and bone marrow cells. PD-1 deficient animals develop autoimmune cardiomyopathy and various autoimmune manifestations of lupus syndrome, including arthritis and nephritis (Nishimura et al. (1999) Immunity 11:141-51, Nishimura et al. (2001) Science 291:319-22). Furthermore, PD-1 is known to play important roles in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type 1 diabetes, and rheumatoid arthritis (Salama et al. (2003) J Exp Med 198:71-78, Prokunia and Alarcon-Riquelme (2004) Hum Mol Genet 13:R143, Nielsen et al. (2004) Lupus 13:510). It is known that in mouse B-cell tumor cell lines, the ITSM of PD-1 is necessary for the inhibition of BCR-mediated Ca2+ flow and the inhibition of tyrosine phosphorylation of downstream effector molecules (Okazaki et al. (2001) PNAS 98:13866-71).

[0124] In some embodiments of the present invention, the ligand is a cytokine. Cytokines are a family of secretory cell signaling proteins involved in immune regulation and inflammatory processes. They are secreted by glial cells in the nervous system and most cells in the immune system. Cytokines can be classified as proteins, peptides, or glycoproteins, encompassing a large and diverse family of regulatory factors. Cytokines bind to cell surface receptors to induce intracellular signal transduction, thereby regulating enzyme activity, the upregulation or downregulation of some genes and their transcription factors, or feedback interference. In some embodiments, the interleukins of the present invention include immunomodulatory factors such as interleukins (ILs) and interferons (IFNs). Suitable interleukins may include proteins from one or more of the following types: four α-helical bundle families (including the IL-2 subfamily, the IFN subfamily, and the IL-10 subfamily); the IL-1 family (including IL-1 and IL-8); and the IL-17 family. Interleukins may also include type 1 interleukins classified as enhancing cellular immune responses (e.g., IFN-γ, TGF-β, etc.) or type 2 interleukins (e.g., IL-4, IL-10, IL-13, etc.) that act favorably on antibody responses.

[0125] In some embodiments of the present invention, the ligand is a chemokine. Chemokines are generally used as chemical inducing agents to transport immune effector cells to chemokine expression sites. They are believed to be beneficial in conjunction with specific chemokine genes, such as cytokine genes, to transport other immune system components to the treatment site. Such chemokines include CXCL10, RANTES, MCAF, MIP1-α, and MIP1-β. Those skilled in the art know that certain cytokines also have chemical inducing effects, and they are considered to be classified as chemokines.

[0126] Furthermore, in some embodiments of the present invention, the ligands may be modified forms of intercytokines, chemokines, etc. (e.g., Annu Rev Immunol. 2015;33:139-67.) or fusion proteins including them (e.g., Stem Cells Transl Med. 2015 Jan; 4(1): 66-73.).

[0127] In some embodiments of the present invention, the ligand system is selected from CXCL10, PD1, IL12, and IL6R. The aforementioned CXCL10, PD1, IL12, and IL6R may have the same sequence as naturally occurring CXCL10, PD1, IL12, and IL6R, or they may be modified versions with different sequences but retaining the physiological activity of the corresponding natural ligands. To obtain modified ligands, artificial ligand sequences can be added for various purposes, preferably by adding alterations that are not cleaved by proteases (protease resistance).

[0128] In some embodiments of the present invention, the ligand inhibits its biological activity by binding to an uncleaved ligand-binding molecule. Embodiments in which the biological activity of the ligand is inhibited are not limited; examples include embodiments in which the binding of the uncleaved ligand-binding molecule to the ligand substantially or significantly hinders or competes with the binding of the ligand to its binding partner. When using an antibody or fragment thereof with ligand-neutralizing activity as the ligand-binding molecule, the ligand-binding molecule bound to the ligand can inhibit the biological activity of the ligand by exerting its neutralizing activity.

[0129] In one embodiment of the present invention, it is preferable that the uncleaved ligand-binding molecule can sufficiently neutralize the biological activity of the ligand by binding to the ligand. That is, it is preferable that the biological activity of the ligand bound to the uncleaved ligand-binding molecule is lower than that of the ligand not bound to the uncleaved ligand-binding molecule. This is not a limitation, but for example, the biological activity of the ligand bound to the uncleaved ligand-binding molecule, compared to the biological activity of the ligand not bound to the uncleaved ligand-binding molecule, may be 90% or less, preferably 80%, 70%, 60%, 50%, 40%, or 30%, and even more preferably 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. By fully neutralizing the biological activity of the ligand, when the ligand-binding molecule is administered, it can be expected that the ligand will prevent it from exerting its biological activity before reaching the target tissue.

[0130] In one embodiment of the present invention, the binding activity of the cleaved ligand-binding molecule to the ligand is preferably lower than the binding activity of the natural binding partner (e.g., the natural receptor for the ligand) in vivo. This is not a limitation, but for example, the binding activity of the cleaved ligand-binding molecule to the ligand, compared to the binding amount (per unit binding partner) of the natural binding partner in vivo, is preferably 90% or less, more preferably 80%, 70%, 60%, 50%, 40%, or 30%, and even more preferably 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. The binding activity can be measured using a suitable and ideal indicator, such as the dissociation constant (KD). When using the dissociation constant (KD) as an indicator of binding activity, if the KD of the cleaved ligand-binding molecule is larger than that of its natural binding partner in vivo, then the binding activity of the cleaved ligand-binding molecule will be weaker than that of its natural binding partner. The KD of the cleaved ligand-binding molecule should ideally be at least 1.1 times larger than that of its natural binding partner in vivo, preferably at least 1.5 times, 2 times, 5 times, or 10 times larger, and most preferably at least 100 times larger. By ensuring that the cleaved ligand-binding molecule retains only low or almost no binding activity to the ligand, it ensures that the ligand is freed after cleavage and can be expected to rebind to other ligand molecules.

[0131] When a ligand-binding molecule is cleaved, it is desirable that the bioactivity of the inhibited ligand be restored. It is hoped that by weakening the binding of the cleaved ligand-binding molecule to the ligand, the inhibitory function of the ligand-binding molecule on the ligand's bioactivity will also be weakened. Those skilled in the art can use known methods, such as methods for detecting the binding of a ligand to its binding partner, to confirm the ligand's bioactivity.

[0132] In some embodiments of the present invention, the ligand-binding molecule in its uncleaved state forms a complex with the ligand via antigen-antibody binding. In more specific embodiments, the complex of the ligand-binding molecule and the ligand can be formed using non-covalent bonds between the ligand-binding molecule and the ligand, such as antigen-antibody binding.

[0133] In some embodiments of the present invention, after the ligand-binding molecule in its uncut state fuses with the ligand to form a fusion protein, the ligand-binding molecule portion and the ligand portion within the fusion protein further interact via antigen-antibody binding. The ligand-binding molecule and the ligand may fuse with or without a linker. Even when the ligand-binding molecule and the ligand in the fusion protein fuse with or without a linker, the non-covalent bonds between the ligand-binding molecule portion and the ligand portion still exist. In other words, in embodiments where the ligand-binding molecule and the ligand are fused, the non-covalent bonds between the ligand-binding molecule portion and the ligand portion are similar to those in embodiments where the ligand-binding molecule and the ligand are not fused. By cleaving the ligand-binding molecule, its non-covalent bonds are weakened. That is, the binding between the ligand-binding molecule and the ligand becomes weaker. In one ideal embodiment of the present invention, the ligand-binding molecule is fused with a ligand-interspersed linker. The linker used for the fusion of the ligand-binding molecule and the ligand can be any peptide linker that can be introduced via genetic engineering, or a linker of a synthetic compound (see, for example, Protein Engineering, 9 (3), 299-305, 1996), but in this embodiment, a peptide linker is preferred. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art according to the purpose. Examples, but not limited to, of peptide linkers include: Ser Gly·Ser(GS) Ser·Gly (SG) Gly·Gly·Ser (GGS) Gly·Ser·Gly(GSG) Ser·Gly·Gly (SGG) Gly·Ser·Ser(GSS) Ser·Ser·Gly (SSG) Ser·Gly·Ser(SGS) Gly·Gly·Gly·Ser (GGGS, Serial Number: 36) Gly·Gly·Ser·Gly (GGSG, Serial Number: 37) Gly·Ser·Gly·Gly (GSGG, Serial Number: 38) Ser·Gly·Gly·Gly (SGGG, Serial Number: 39) Gly·Ser·Ser·Gly (GSSG, Serial Number: 40) Gly·Gly·Gly·Gly·Ser (GGGGS, Serial Number: 41) Gly·Gly·Gly·Ser·Gly (GGGSG, Serial Number: 42) Gly·Gly·Ser·Gly·Gly (GGSGG, Serial Number: 43) Gly·Ser·Gly·Gly·Gly (GSGGG, Serial Number: 44) Gly·Ser·Gly·Gly·Ser (GSGGS, Serial Number: 45) Ser·Gly·Gly·Gly·Gly (SGGGG, Serial Number: 46) Gly·Ser·Ser·Gly·Gly (GSSGG, Serial Number: 47) Gly·Ser·Gly·Ser·Gly (GSGSG, Serial Number: 48) Ser·Gly·Gly·Ser·Gly (SGGSG, Serial Number: 49) Gly·Ser·Ser·Ser·Gly (GSSSG, Serial Number: 50) Gly·Gly·Gly·Gly·Gly·Ser(GGGGGS, Serial Number: 51) Ser·Gly·Gly·Gly·Gly (SGGGGG, Serial Number: 52) Gly·Gly·Gly·Gly·Gly·Ser(GGGGGGS, Serial Number: 53) Ser·Gly·Gly·Gly·Gly·Gly (SGGGGGG, Serial Number: 54) (Gly·Gly·Gly·Gly·Ser(GGGGS, Serial Number: 41))n (Ser·Gly·Gly·Gly·Gly(SGGGG, Serial Number: 46))n [n is an integer greater than or equal to 1] etc. However, the length and sequence of the peptide linker can be appropriately selected by those with ordinary knowledge in the field of the art according to the purpose.

[0134] Synthetic chemical linkers (chemical cross-linking agents) are commonly used cross-linking agents for peptides, such as N-hydroxysuccinimide (NHS), disuccinimide octanoate (DSS), di(thiosuccinimide) octanoate (BS3), dithiobis(succinimide) propionate (DSP), and dithiobis(thiosuccinimide) propionate (DTSSP). Ethylene glycol bis(succiniminosuccinate) (EGS), ethylene glycol bis(thiosucciniminosuccinate) (thio-EGS), disuccinimino tartrate (DST), dithiosuccinimino tartrate (thio-DST), bis[2-(succiniminooxycarbonyloxy)ethyl] ion (BSOCOES), bis[2-(thiosucciniminooxycarbonyloxy)ethyl] ion (thio-BSOCOES), etc., these crosslinking agents are commercially available.

[0135] This invention also relates to pharmaceutical compositions (pharmaceuticals) including the ligand-binding molecule of this invention and a pharmaceutically permissible support, pharmaceutical compositions (pharmaceuticals) including the ligand-binding molecule of this invention, a ligand, and a pharmaceutically permissible support, and pharmaceutical compositions (pharmaceuticals) including a fused protein obtained by fusing the ligand-binding molecule of this invention with a ligand and a pharmaceutically permissible support.

[0136] As used in this specification, "treatment" (and its grammatical derivatives, such as "treatment of," "the act of treatment," etc.) refers to a clinical intervention that attempts to alter the natural processes of the individual being treated, and can be implemented for prevention or during clinically pathological processes. Ideal therapeutic effects are not limited to this, but include preventing the onset or recurrence of symptoms, alleviating symptoms, reducing any direct or indirect pathological effects caused by the disease, preventing metastasis, slowing the progression of the disease, restoring or alleviating the disease state, and improving or resolving the prognosis. In some embodiments, the ligand-binding molecule of this invention can control the biological activity of the ligand, used to delay the onset of the disease or slow its progression.

[0137] In this invention, pharmaceutical composition generally refers to a medicine for the treatment or prevention of a disease, or for examination or diagnosis. Furthermore, in this invention, the term "pharmaceutical composition including ligand-binding molecules" can also be replaced with "a treatment method for a disease including the step of delivering ligand-binding molecules to a treatment subject," or "the use of ligand-binding molecules for manufacturing a medicine for treating a disease." "Pharmaceutical composition including ligand-binding molecules" can also be replaced with "the use of ligand-binding molecules for treating a disease." The term "pharmaceutical composition including ligand-binding molecules and ligands" can also be replaced with "a treatment method for a disease including the step of delivering ligand-binding molecules and ligands to a subject of treatment," or "the use of ligand-binding molecules and ligands for the purpose of manufacturing a medicine for treating a disease." The term "pharmaceutical composition including fusion protein" can also be replaced with "a treatment method for a disease including the step of administering the fusion protein to a patient," or "the use of the fusion protein for the manufacture of a medicine for treating a disease." The term "pharmaceutical composition including fusion protein" can also be replaced with "the use of the fusion protein for the treatment of a disease."

[0138] In some embodiments of the present invention, a composition including a ligand-binding molecule can be administered to an individual. The ligand-binding molecule administered to the individual will bind to a ligand already present in the individual, such as in the blood or tissues, and will be further transported into the body in a ligand-bound state. The ligand-binding molecule transported to the target tissue is cleaved at the target tissue, weakening the binding to the ligand and releasing the bound ligand. The released ligand will exert biological activity in the target tissue and treat diseases originating from the target tissue. In embodiments where the ligand-binding molecule is already bound to a ligand, it inhibits the biological activity of the ligand, and the ligand-binding molecule is specifically cleaved at the target tissue, the biological activity of the ligand is not exerted during delivery, but only after cleavage at the target tissue, and can treat diseases and suppress systemic side effects.

[0139] In some embodiments of the present invention, a composition comprising a ligand-binding molecule and a composition comprising a ligand may be administered to an individual, individually or simultaneously. Alternatively, a composition comprising both a ligand-binding molecule and a ligand may be administered to an individual. When a composition comprising both a ligand-binding molecule and a ligand is administered to an individual, the ligand-binding molecule and the ligand in the composition may form a complex. When both the ligand-binding molecule and the ligand are administered to an individual, the ligand-binding molecule binds to the ligand already administered to the individual and remains bound to the ligand during delivery into the organism. The ligand-binding molecule delivered to the target tissue is cleaved in the target tissue, the binding to the ligand is weakened, and the bound ligand may be released from the target tissue. The released ligand exerts biological activity in the target tissue and treats diseases originating from the target tissue. When a ligand-binding molecule is already bound to a ligand, it inhibits the biological activity of the ligand. Furthermore, the ligand-binding molecule is specifically cleaved at the target tissue, meaning that the biological activity of the ligand is not released during delivery but only after cleavage at the target tissue. This allows for the treatment of diseases and the suppression of systemic side effects. Ligand-binding molecules administered to an individual may bind not only to ligands already administered to the individual but also to ligands already present in the individual, maintaining their binding state with either pre-existing or administered ligands during delivery into the organism.

[0140] In some embodiments of the present invention, a fusion protein obtained by fusing a ligand-binding molecule with a ligand can be delivered to an individual. In these embodiments, the ligand-binding molecule and the ligand are separated by or without linkers to form the fusion protein, but the non-covalent bonds between the ligand-binding molecule and the ligand remain. When the fusion protein obtained by fusing the ligand-binding molecule with the ligand is delivered to an individual, the fusion protein is transported within the organism. In the target tissue, the ligand-binding molecule portion of the fusion protein is cleaved, thereby weakening the non-covalent bond between the ligand-binding molecule portion and the ligand, and releasing the ligand and a portion of the ligand-binding molecule from the fusion protein. The released ligand and a portion of the ligand-binding molecule exert the biological activity of the ligand in the target tissue, and can treat diseases originating from the target tissue. When the ligand-binding molecule has already bound to the ligand, it inhibits the biological activity of the ligand. Furthermore, the ligand-binding molecule is specifically cleaved in the target tissue. During delivery, the biological activity of the ligand in the fusion protein is not exerted. Instead, the biological activity of the ligand is exerted only after the ligand is cleaved in the target tissue. This can treat diseases and inhibit systemic side effects.

[0141] The pharmaceutical composition of this invention can be formulated using methods known to those skilled in the art. For example, it can be administered non-orally in the form of an injectable solution or suspension of water or other pharmaceutically permissible liquids. For example, a pharmacologically permissible support or medium, specifically, can be formulated by appropriately combining sterile water or saline solution, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, carrier, preservative, binder, etc., in a unit dosage form required by generally accepted pharmaceutical practices. The amount of active ingredient in such formulations can be set to an appropriate volume that yields an indicated range.

[0142] Sterile components for injection can be formulated according to standard pharmaceutical practices using a carrier such as distilled water for injection. Aqueous solutions for injection, such as physiological saline, glucose, or isotropic solutions containing other excipients (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride), can also be used. Appropriate dissolving agents, such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80(TM), HCO-50, etc.), can also be used.

[0143] Oily liquids, such as sesame oil and soybean oil, may be used with benzyl benzoate and / or benzyl alcohol as dissolving agents. Buffers (e.g., phosphate buffer and sodium acetate buffer), analgesics (e.g., procaine hydrochloride), tranquilizers (e.g., benzyl alcohol and phenol), and antioxidants may also be incorporated. The prepared injection solution is typically filled into appropriate ampoules.

[0144] The pharmaceutical composition of the present invention is preferably administered non-orally. For example, it can be formulated into injectable, nasal, pulmonary, or transdermal dosage forms. For example, it can be administered systemically or locally via intravenous, intramuscular, intraperitoneal, or subcutaneous injection.

[0145] The administration method can be appropriately selected based on the patient's age and symptoms. The dosage of pharmaceutical components containing ligand-binding molecules can, for example, be set in the range of 0.0001 mg to 1000 mg per kg of body weight. Alternatively, it can be set in the range of 0.001 to 100,000 mg per patient, but the invention is not necessarily limited to these values. The dosage and administration method vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can consider these conditions to set an appropriate dosage and administration method.

[0146] This invention also relates to a method for manufacturing ligand-binding molecules whose binding to ligands is weakened in a cleaved state, or fusion proteins obtained by fusing the ligand-binding molecule with a ligand. In one embodiment of the invention, a method for manufacturing a ligand-binding molecule or fusion protein is provided, including the step of introducing a protease cleavage sequence into a ligand-binding molecule.

[0147] Examples of methods for introducing a protease cleavage sequence into a ligand-binding molecule include inserting a protease cleavage sequence into the amino acid sequence of a ligand-binding peptide, and replacing a portion of the amino acid sequence of a ligand-binding peptide with a protease cleavage sequence.

[0148] One method for obtaining molecules capable of binding to ligands is, for example, obtaining a ligand-binding region capable of binding to ligands. The ligand-binding region can be obtained, for example, using known methods for preparing antibodies. The antibodies obtained using this method can be used directly on the ligand-binding region, or only on the Fv region of the obtained antibody. When the Fv region can recognize the antigen with a single chain (also called "sc"), only the single chain can be used. Alternatively, the Fab region, which includes the Fv region, can also be used.

[0149] The specific methods for producing antibodies are well known to those skilled in the art. For example, in the case of monoclonal antibodies, they can be produced using the fusion tumor method (Kohler and Milstein, Nature 256:495 (1975)) or the recombinant method (US Patent No. 4,816,567). Alternatively, they can be isolated from a phage antibody library (Clackson et al., Nature 352:624-628 (1991); Marks et al., J.Mol.Biol. 222:581-597 (1991)). Alternatively, they can be isolated from a single B cell colony (N. Biotechnol. 28(5): 253-457 (2011)).

[0150] Humanized antibodies can also be called reshaped human antibodies. Specifically, it is known to obtain humanized antibodies by transplanting the CDR of an animal other than a human, such as a mouse, into a human antibody. General gene recombination methods for obtaining humanized antibodies are also known. Specifically, methods for transplanting the CDR of a mouse antibody into a human FR, such as overlap extension PCR, are known.

[0151] A vector for expressing humanized antibodies can be created by fusing DNA encoding the variable region of an antibody (linked with 3 CDRs and 4 FRs) and DNA encoding the invariant region of a human antibody within a reading frame and inserting them into an expression vector. After introducing this recombinant vector into a host and establishing recombinant cells, culturing these cells allows the DNA encoding the humanized antibody to be expressed, thereby generating the humanized antibody in the culture of these cells (see European Patent Application Publication No. 239400 and International Patent Publication No. 1996 / 002576).

[0152] Alternatively, depending on the need, the amino acid residues of the FR can be replaced in a way that allows the CDR constituting the human antibody to form an appropriate antigen-binding site. For example, the PCR method used for transplanting mouse CDRs into human FRs can be applied to introduce amino acid sequence variations into the FR.

[0153] Genetically modified animals possessing the complete set of human antibody genes (refer to International Publication Nos. 1993 / 012227, 1992 / 003918, 1994 / 002602, 1994 / 025585, 1996 / 034096, and 1996 / 033735) can be used as immunized animals to obtain the desired human antibodies through DNA immunization.

[0154] Furthermore, techniques for panning to obtain human antibodies using human antibody libraries are also known. For example, the Fv region of a human antibody can be expressed on the surface of a bacteriophage as a single-chain antibody (also known as "scFv") using phage suggestion. Phages expressing scFv that bind to the antigen can be selected. By analyzing the genes of the selected bacteriophages, the DNA sequence encoding the Fv region of the human antibody that binds to the antigen can be determined. After determining the DNA sequence of the scFv that binds to the antigen, the Fv region sequence is fused with the desired human antibody C region sequence within a reading frame, and then inserted into an appropriate expression vector to create an expression vector. By introducing this expression vector into the ideal expression cells listed above and expressing the gene encoding the human antibody, the human antibody can be obtained. These methods are well known (see International Publication No. 1992 / 001047, International Publication No. 1992 / 020791, International Publication No. 1993 / 006213, International Publication No. 1993 / 011236, International Publication No. 1993 / 019172, International Publication No. 1995 / 001438, and International Publication No. 1995 / 015388).

[0155] A molecule that has been introduced with a protease cleavage sequence into a ligand-binding molecule becomes the ligand-binding molecule of this invention. It can be determined whether treatment with a protease corresponding to the protease cleavage sequence will cleave the ligand-binding molecule. For example, by contacting a molecule with a protease cleavage sequence into a ligand-binding molecule with a protease and confirming the molecular weight of the product after protease treatment using electrophoresis such as SDS-PAGE, it can be confirmed whether the protease cleavage sequence has been cleaved.

[0156] Furthermore, the present invention also relates to polynucleotides encoding ligand-binding molecules in a cleaved state that have weakened binding to ligands, or polynucleotides encoding fusion proteins obtained by fusing the ligand-binding molecule with the ligand.

[0157] Furthermore, in this invention, the polynucleotide is typically carried (inserted) into a suitable vector and introduced into a host cell. The vector is not particularly limited as long as it can stably hold the inserted nucleic acid; for example, if the host is *E. coli*, the preferred vector for colonization is the pBluescript vector (manufactured by Stratagene), and various commercially available vectors can be used. When a vector is used to produce the ligand-binding molecule or fusion protein of this invention, expression vectors are particularly useful. Expression vectors are not particularly limited as long as they express the ligand-binding molecule in a test tube, in *E. coli*, in cultured cells, or in an organism. For example, for expression in a test tube, the pBEST vector (manufactured by Promega) is preferred; for *E. coli*, the pET vector (manufactured by Invitrogen) is preferred; for cultured cells, the pME18S-FL3 vector (GenBank Accession No. AB009864) is preferred; and for an organism, the pME18S vector (Mol Cell Biol. 8:466-472(1988)) is preferred. The DNA insertion vector of the present invention can be carried out by conventional methods, such as using conjugation enzyme reactions using restriction enzyme sites (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).

[0158] The host cells mentioned above are not particularly limited, and various host cells can be used depending on the purpose. Cells used to express ligand-binding molecules or fusion proteins include: bacterial cells (e.g., streptococci, staphylococci, Escherichia coli, Streptomyces, Subtilis), fungal cells (e.g., yeast, Aspergillus), insect cells (e.g., Drosophila S2, Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells), and plant cells. The introduction of the vector into the host cells can be carried out using known methods such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), liposome (Lipofectamine) method (manufactured by GIBCO-BRL), and microinjection.

[0159] To induce the secretion of ligand-binding molecules or fusion proteins expressed in host cells into the microsomal lumen, pericellular lumen, or extracellular environment, appropriate secretion signals can be embedded into the target ligand-binding molecule or fusion protein. These signals can be intrinsic or heterogenic for the target ligand-binding molecule or fusion protein.

[0160] In the above manufacturing method, the ligand-binding molecules or fusion proteins are recovered when the ligand-binding molecules or fusion proteins of the present invention are secreted into the culture medium. When the ligand-binding molecules or fusion proteins of the present invention are generated inside cells, the cells are first dissolved and then the ligand-binding molecules or fusion proteins are recovered.

[0161] When recovering and purifying the ligand-binding molecules or fusion proteins of the present invention from recombinant cell cultures, known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography can be used.

[0162] Any invention derived from any combination of one or more embodiments described in this specification is included in this invention, provided that it is not technically contradictory to the common technical knowledge of those skilled in the art. Furthermore, any invention other than those derived from any combination of one or more embodiments described in this specification should also be considered an invention described in this specification, provided that it is not technically contradictory to the common technical knowledge of those skilled in the art. [Example]

[0163] The following are examples of the methods and compositions of the present invention. It will be understood that various other embodiments may be implemented based on the above general description.

[0164] Example 1: Reported Issues Regarding the Activation of Cytokines by Immune Interferons and Proteases Immunointerstitials targeting antigens expressed in cancerous tissue are generally produced by fusing targeted cytokines at the ends of IgG and scFv (Expert Opin Investig Drugs. 2009 Jul;18(7):991-1000., Curr Opin Immunol. 2016 Jun;40:96-102.). Due to their high toxicity, IL2, IL12, and TNF-based cytokines are delivered locally to cancerous sites using antibodies, with the aim of reducing side effects and enhancing efficacy (Non-Patent Literature 4, 5, 6). However, these cytokines have not shown sufficient clinical efficacy when administered systemically, have a narrow therapeutic window, and are too toxic for systemic administration. Major reasons for this include the fact that even with immunointerstitials, systemically administered cytokines are exposed to the entire body, thus potentially exhibiting toxicity, or requiring administration at extremely low doses to avoid toxicity. Furthermore, interleukins that bind to cancer antigens are internalized into cancer cells and disappear within the tumor, making it difficult to expose interleukins locally to the tumor. It has also been reported that there is no difference in antitumor effects between interleukins fused with IL2 on antibodies that bind to cancer antigens and interleukins fused with IL2 on antibodies that do not bind to cancer antigens (Non-Patent Literature 7).

[0165] Regarding methods to reduce the systemic effects of interleukins, a significant challenge in the immune system, molecules have been reported that bind to the interleukin-receptor linker using a protease that cleaves it. Interleukins are inhibited by the linker-bound receptor, but if the linker is cleaved by the protease, the receptor is released, and the interleukin becomes active. For example, molecules that bind TNFalpha and TNFR using a linker cleaved by uPA (Non-Patent Document 8) and molecules that bind IL2 and IL2R using a linker cleaved by MMP2 (Non-Patent Document 9) have been reported. However, even before linker cleavage, the interleukin remains biologically active in these molecules; the activity only increases by about 10-fold after linker cleavage. The reasons for this are as follows: the affinity between cytokines and their receptors is not strong, so cytokines still have some activity even before being cleaved by proteases; or even after the linker is cleaved by proteases, the receptors can still bind to the cytokines, thus inhibiting their biological activity.

[0166] It has been reported that IL2R can be replaced with anti-IL2 scFv, a molecule obtained by binding to IL2 through a linker cleaved by MMP-2 (Non-Patent Document 9). This molecule obtained by binding IL2 to the linker through the anti-IL2 scFv linker cleavage is similar to the molecule obtained by the binding of interleukins to interleukin receptors. Considering the possibility of IL2 being released during linker cleavage, anti-IL2 scFv, which has a low affinity for IL2, would naturally be used. Furthermore, the protease-activated cytokines reported in these studies, unlike the aforementioned IgG-IL2 fusions, lack an Fc region, resulting in a predicted short half-life and difficulty in maintaining high exposure. The pharmacodynamics of cytokines before and after activation by protease cleavage show no significant difference (both have short half-lifes), making it difficult to broaden the therapeutic window.

[0167] Example 2: The Application of Chemokine in Cancer Immunotherapy Chemokines (Nature Immunology 9, 949-952 (2008)) are basic proteins that exert their effects via G protein conjugated receptors and belong to a group of cytokines. They act on specific leukocytes that express the receptors and have the activity (chemotaxis) to cause leukocytes to migrate in the direction of the concentration gradient of the substance (Nat Cell Biol. 2016 Jan;18(1):43-53.). Chemokines are known to be produced in large quantities at sites of inflammation, causing leukocytes to migrate from blood vessels to inflamed tissues. By controlling chemokines, the migration of white blood cells can be controlled, and therefore it is believed that this technology can be applied to cancer immunotherapy. If T cells, antigen-suggesting cells, and M1 macrophages can migrate to the local area of ​​solid tumors, it is thought that an anti-tumor effect could be achieved. While systemic administration of cytokines can also be effective, chemokines migrate to tissues with higher concentrations due to concentration gradients, meaning that even systemic administration of chemokines may not achieve the desired effect. Therefore, cancer immunotherapy (chemokine therapy) based on systemic administration of chemokines is considered impractical.

[0168] Example 3: Concept of a ligand-binding molecule that enables the release of a ligand with specificity to a target tissue by introducing a protease cleavage sequence. As shown in Examples 1 and 2, the following issues have been reported with cytokine-chemokine therapy. 1. Immunotherapy: Even when using antibodies to target cytokines to solid tumors, the cytokines still have systemic effects, which can cause side effects. In order to avoid side effects, they can only be administered in low doses, so they cannot be highly exposed in the tumor. 2. Interleukin receptors (or antibodies) and interleukins are linked by linkers that can be cleaved by proteases. The activity of interleukins is not neutralized, and even before the protease cleavage, the interleukin still has a certain degree of activity. 3. In protease-activated cytokines, even after the linker is cleaved by the protease, the cytokinin receptor (or antibody) can still bind to the cytokines, thus inhibiting their biological activity. 4. Among protease-activated cytokines, the inactive type has a short half-life and a short blood retention time, therefore requiring a larger dosage.

[0169] To solve this problem, it is considered important to meet the following conditions. 1. Throughout the body, ligands such as intercytokines or chemokines are significantly inhibited (minimize biological activity) due to ligand binding molecules. 2. Due to the cleavage caused by the protease, the biological activity of the ligand is restored (becoming an active ligand). 3. Due to cleavage by the protease, the ligand-binding molecule loses its ligand-binding activity. 4. Compared to ligands that are already bound to ligand-binding molecules before being cleaved by proteases, ligands that become active after being cleaved by proteases have a shorter half-life.

[0170] Pharmaceutical compositions meeting the above conditions are considered to be molecules whose binding to ligands is weakened by cleavage at cleavage sites. Ligand-binding molecules can be prepared by first obtaining a ligand-binding molecule and then inserting a cleavage site into the binding molecule.

[0171] Example 4: An example of introducing an anti-ligand antibody with a protease cleavage sequence. Figures 1, 2, and 3 illustrate examples of using antibodies as molecules for ligand binding. In these examples, neutralizing antibodies against the ligand are first obtained. Then, a protease cleavage sequence is inserted near the boundary between the variable region (VH or VL) and the invariant region (CH1 or CL) of the anti-ligand neutralizing antibody. It is confirmed that the anti-ligand antibody retains its ligand-binding activity after the insertion of the protease cleavage sequence. The ligand is then confirmed to dissociate by protease cleavage while the ligand is bound to the anti-ligand neutralizing antibody. It has been confirmed that the dissociated ligands will exert biological activity. In Figure 1, the C-terminus of the ligand binds to the N-terminus of the anti-ligand antibody's VH via a linker, and a protease cleavage sequence is inserted near the VH / CH1 junction. If the anti-ligand antibody has a sufficiently strong affinity for the ligand, the ligand's biological activity will be sufficiently inhibited. Even with systemic administration, the ligand in this ligand-anti-ligand antibody fusion will be neutralized, thus failing to exert its biological activity. Furthermore, the ligand-anti-ligand antibody fusion possesses a long half-life due to the presence of an Fc region. If the systemically administered ligand-anti-ligand antibody fusion cleaves the protease cleavage sequence near the VH / CH1 junction due to the highly expressed protease in tumor tissue, the VH molecule of the ligand-linker-anti-ligand antibody will become free. Neither VH nor VL can bind to the ligand alone (both VH and VL are required for ligand binding), ligand neutralization will be deactivated, and the ligand can exert its biological function in tumor tissue. Furthermore, the released ligand-linker-antiligand antibody VH molecule does not have an Fc region and has a small molecular weight, so it has a very short half-life and will disappear rapidly from the whole body, thus minimizing systemic side effects caused by ligands. In Figure 2, the ligand and anti-ligand antibody do not bind via a linker as in Figure 1. Instead, the anti-ligand antibody, with a protease-cleaving sequence inserted near the VH / CH1 junction, is mixed with the ligand and administered. If the anti-ligand antibody has a sufficiently strong affinity for the ligand and its concentration relative to the ligand is adequate, the biological activity of the ligand can be sufficiently inhibited. Even with systemic administration of this ligand-anti-ligand antibody complex, the ligand can still be neutralized, thus its biological activity will not be exerted. Furthermore, the ligand-anti-ligand antibody complex has a long half-life due to the presence of an Fc region. If the systemically administered ligand-anti-ligand antibody complex cleaves the protease-cleaving sequence near the VH / CH1 junction due to the highly expressed protease in tumor tissue, the VH molecule of the anti-ligand antibody will become free. VH or VL alone cannot bind to the ligand (both VH and VL are required for ligand binding), the ligand neutralization is broken, and the ligand can exert its biological function in tumor tissue. Furthermore, the released ligand molecules lack an Fc region and have a small molecular weight, resulting in a very short half-life. They disappear rapidly from the body, thus minimizing systemic side effects caused by the ligand. In Figure 3, an anti-ligand antibody obtained by inserting a protease cleavage sequence near the boundary between VH and CH1 was administered systemically. The administered antibody bound to the ligand already present in the body, and then proceeded as described in Figure 2 above. In this manner, by using anti-ligand antibodies with protease cleavage sequences inserted near the VH and CH1 junction, the ligands are selectively released in tissues expressing the protease and exert their biological effects. If the ligand is a cytokine, it allows the cytokine to act selectively in tissues expressing the protease. If the ligand is a chemokine, the chemokine is present in high concentrations in tissues expressing the protease, while its concentration decreases in peripheral blood, thus enabling cells expressing chemokine receptors to migrate to tissues expressing the protease.

[0172] Example 5: Preparation and Evaluation of CXCL10-Releasing Antibody 5-1. For the introduction of anti-CXCL10 neutralizing antibody into a protease cleavage sequence CXCL10 is one of the chemokines that induce migration in effector T cells. An expression vector for the neutralizing antibody MabCXCL10 (heavy chain: EEIVH (sequence number: 1), light chain: EEIVL (sequence number: 2)) targeting human CXCL10 was prepared using methods known to those skilled in the art, and expressed and purified using FreeStyle 293 (Life Technology) using methods known to those skilled in the art. The CDR sequences contained in MabCXCL10 are as follows: H-CDR1 (NNGMH, sequence number: 380), H-CDR2 (VIWFDGMNKFYVDSVKG, sequence number: 381), H-CDR3 (EGDGSGIYYYYGMDV, sequence number: 382), L-CDR1 (RASQSVSSSYLA, sequence number: 383), L-CDR2 (GASSRAT, sequence number: 384), and L-CDR3 (QQYGSSPIFT, sequence number: 385). The interaction between MabCXCL10 and human CXCL10 (266-IP-010 / CF, R&D Systems) was evaluated using Biacore. Specifically, R PROTEIN A (SURE) (28-4018-60, GE Healthcare) was immobilized on a CM3 sensor chip (BR100536, GE Healthcare) using the NHS EDC amine coupling method. Electrophoresis buffer (20 mM ACES, 0.05% Tween 20, 200 mM NaCl, pH 7.4) was used. With the antibody captured, 1.563 nM of human CXCL10 was added as the analyte and allowed to flow. The binding of the antibody to the antigen at 37°C was evaluated. Figure 4 shows a sensor plot representing the difference in binding over time between the analyte and the control (blank). The starting point of the horizontal axis is the time when the analyte begins to flow. The vertical axis represents the response (binding amount) at each time point from the start of the analyte flow to zero. As shown in the sensor plot in Figure 4, the binding of MabCXCL10 to human CXCL10 was confirmed. This study investigated the insertion of protease cleavage sequences near the boundary between the variable and invariant regions of the heavy or light chain of MabCXCL10. The design incorporated peptide sequence A (sequence number: 3), which is reportedly cleaved by cancer-specific urokinase (uPA) and mastriptase (MT-SP1), into the heavy and light chains at seven locations near the boundary between the variable and invariant regions, as shown in Figure 5. Modifications that do not induce glycoaddition with the insertion of cleavage sequences were also designed. The representation vectors encoding heavy chain alterations EEIVHA (serial number: 4), EEIVHB (serial number: 5), EEIVHC (serial number: 6), EEIVHD (serial number: 7), EEIVHE (serial number: 8), EEIVHF (serial number: 9), EEIVHG (serial number: 10), EEIVHBG (serial number: 11), EEIVHCG (serial number: 12), EEIVHDG (serial number: 13), EEIVHEG (serial number: 14), and light chain alterations EEIVLA (serial number: 15), EEIVLB (serial number: 16), EEIVLC (serial number: 17), EEIVLD (serial number: 18), EEIVLE (serial number: 19), EEIVLF (serial number: 20), EEIVLG (serial number: 21), and EEIVLEG (serial number: 22) are created using methods known to those skilled in the art. The following IgG1 antibodies with protease cleavage sequences were inserted near the boundary between the variable and invariant regions of the heavy chain: EEIVHA / EEIVL (heavy chain sequence number: 4, light chain sequence number: 2), EEIVHB / EEIVL (heavy chain sequence number: 5, light chain sequence number: 2), EEIVHC / EEIVL (heavy chain sequence number: 6, light chain sequence number: 2), EEIVHD / EEIVL (heavy chain sequence number: 7, light chain sequence number: 2). Sequence number: 2), EEIVHE / EEIVL (heavy chain sequence number: 8, light chain sequence number: 2), EEIVHF / EEIVL (heavy chain sequence number: 9, light chain sequence number: 2), EEIVHG / EEIVL (heavy chain sequence number: 10, light chain sequence number: 2), EEIVHBG / EEIVL (heavy chain sequence number: 11, light chain sequence number: 2), EEIVHCG / EEIVL (heavy chain sequence number: 12, light chain sequence number: 2), EEIVHDG / EEIVL (heavy chain sequence number: 2) EEIVH / EEIVLA (heavy chain sequence number: 1, light chain sequence number: 2), EEIVH / EEIVL (heavy chain sequence number: 14, light chain sequence number: 2), and the following IgG1 antibodies with protease cleavage sequences inserted near the boundary between the variable and constant regions of the light chain: EEIVH / EEIVLA (heavy chain sequence number: 1, light chain sequence number: 15), EEIVH / EEIVLB (heavy chain sequence number: 1, light chain sequence number: 16), EEIVH / EEIVLC (heavy chain sequence number: 1, light chain sequence number: 17), EEIVH / EEIVLD (heavy chain sequence number: 1, light chain sequence number: 18), EEIVH / EEIVLE (heavy chain sequence number: 1, light chain sequence number: 19), EEIVH / EEIVLF (heavy chain sequence number: 1, light chain sequence number: 20), EEIVH / EEIVLG (heavy chain sequence number: 1, light chain sequence number: 21), and EEIVH / EEIVLEG (heavy chain sequence number: 1, light chain sequence number: 22) were presented using a temporary representation of FreeStyle 293 (Life Technologies) in accordance with methods known to those skilled in the art, and refined using methods known to those skilled in the art of Protein A.

[0173] 5-2. Evaluation of the binding activity of anti-CXCL10 neutralizing antibody with protease cleavage sequence introduced. The interaction between the antibody prepared in step 5-1 and human CXCL10 (266-IP-010 / CF, R&D Systems) was evaluated using Biacore, and the results are shown in Figure 6. Specifically, R PROTEIN A (SURE) (28-4018-60, GE Healthcare) was immobilized on a CM3 sensor chip (BR100536, GE Healthcare) using the NHS EDC amine coupling method. The antibody was then passed through 3.125, 1.563, and 0.781 nM human CXCL10 as analytes in a captured state using an electrophoresis buffer of 20 mM ACES, 0.05% Tween 20, 150 mM NaCl, and pH 7.4 to evaluate the antibody binding to the antigen at 25°C. Figure 6 shows a sensor plot representing the binding amount over time, comparing the analyte (using only the electrophoresis buffer) with the control (blank). The starting point of the horizontal axis is the time when the analyte began to flow through the buffer. The vertical axis represents the response at the time the analyte begins to flow, with the response (binding amount) at each time point from 0. As shown in the sensor diagram in Figure 6, any antibody will bind to human CXCL10. That is, it can retain its antigen-binding activity and insert a protease cleavage sequence near the boundary between the variable and constant regions of the antibody.

[0174] 5-3. Evaluation of the cleavage effect of the anti-CXCL10 neutralizing antibody with introduced protease cleavage sequence by the protease. The study examined whether the antibodies prepared in step 5-1 would be cleaved by proteases. Recombinant human Matriptase / ST14 catalyst domain (MT-SP1) (R&D Systems, 3946-SE-010) was used as the protease. After a 20-hour reaction at 20 nM protease, 60 or 100 μg / mL antibody, PBS, and 37°C, the cleavage was evaluated using reducing SDS-PAGE. The results are shown in Figure 7. The results showed that EEIVHA / EEIVL, EEIVHE / EEIVL, EEIVHF / EEIVL, EEIVHG / EEIVL, EEIVHEG / EEIVL, and EEIVHBG / EEIVL exhibited new bands between 25 kDa and 50 kDa due to protease treatment. Furthermore, EEIVH / EEIVLEG, EEIVH / EEIVLF, and EEIVH / EEIVLG exhibited bands below 25 kDa due to protease treatment. Therefore, it was confirmed that in EEIVHA / EEIVL, EEIVHE / EEIVL, EEIVHF / EEIVL, EEIVHG / EEIVL, EEIVHEG / EEIVL, EEIVHBG / EEIVL, EEIVH / EEIVLEG, EEIVH / EEIVLF, and EEIVH / EEIVLG, the antibodies were cleaved by proteases.

[0175] 5-4. A mobile linker sequence was inserted near the protease cleavage sequence of the anti-CXCL10 neutralizing antibody containing the protease cleavage sequence. This study explored the insertion of sequences including linkers composed of glycine-serine polymers near the protease cleavage sequence of EEIVHC / EEIVL, which was not cleaved by the recombinant human Matriptase / ST14 (MT-SP1) catalyst domain (R&D Systems, 3946-SE-010). Five heavy chains, as shown in Figure 8, were designed. Expression vectors encoding the heavy chain alterations EEIVHC002 (Sequence No.: 23), EEIVHC003 (Sequence No.: 24), EEIVHC004 (Sequence No.: 25), EEIVHC005 (Sequence No.: 26), and EEIVHC006 (Sequence No.: 27) were prepared using methods known to those skilled in the art. These heavy chain alterations were combined with native light chains, and the following IgG1 antibodies with protease cleavage sequences were inserted near the boundary between the variable and invariant regions of the heavy chain: EEIVHC002 / EEIVL (heavy chain sequence number: 23, light chain sequence number: 2), EEIVHC003 / EEIVL (heavy chain sequence number: 24, light chain sequence number: 2), EEIVHC004 / EEIVL (heavy chain sequence number: 25, light chain sequence number: 2), EEIVHC005 / EEIVL (heavy chain sequence number: 26, light chain sequence number: 2), EEIVHC006 / EEIVL (heavy chain sequence number: 27, light chain sequence number: 2). The antibodies were expressed using FreeStyle 293 (Life Technologies) according to methods known to those skilled in the art, and purified using Protein A according to methods known to those skilled in the art.

[0176] 5-5. Evaluation of the binding activity of anti-CXCL10 neutralizing antibodies with protease cleavage sequences and mobile linker sequences introduced. The interaction between the antibody prepared in step 5-4 and human CXCL10 (266-IP-010 / CF, R&D Systems) was evaluated using Biacore, and the results are shown in Figure 9. Specifically, R PROTEIN A (SURE) (28-4018-60, GE Healthcare) was immobilized on a CM3 sensor chip (BR100536, GE Healthcare) using the NHS-EDC amine coupling method. Using 20 mM ACES, 0.05% Tween 20, 300 mM NaCl, and pH 7.4 as the electrophoresis buffer, human CXCL10 at concentrations of 6.25, 3.125, 1.563, and 0.781 nM as analytes was passed through the antibody-captured state to evaluate the antibody binding to the antigen at 25°C. Figure 9 shows the sensor map representing the binding amount over time, comparing the analyte (using only the electrophoresis buffer) with the control (blank). The starting point of the analyte flow is set as the horizontal axis. The vertical axis represents the response (binding amount) at each time point from the start of analyte flow to zero. As shown in the sensor diagram in Figure 9, any antibody binds to human CXCL10. That is, without losing its antigen-binding activity, a protease cleavage sequence and a mobile linker sequence can be inserted near the boundary between the variable and constant regions of the antibody.

[0177] 5-6. Evaluation of the cleavage effect of anti-CXCL10 neutralizing antibodies containing protease cleavage sequences and mobile linker sequences on protease cleavage. The study examined whether the antibodies prepared in step 5-5 would be cleaved by proteases. Human urokinase (uPA) (R&D Systems, 1310-SE-010) and recombinant human Matriptase / ST14 catalyst domain (MT-SP1) (R&D Systems, 3946-SE-010) were used as proteases. Reactions were performed at 12.5 nM protease, 133 μg / mL antibody, PBS, and 37°C for 2 and 20 hours. The cleavage caused by the protease was evaluated using reducing SDS-PAGE, and the results are shown in Figure 10. The results showed that EEIVHC002 / EEIVL, EEIVHC003 / EEIVL, EEIVHC004 / EEIVL, EEIVHC005 / EEIVL, and EEIVHEC006 / EEIVL exhibited new bands between 25 kDa and 50 kDa due to protease treatment. Therefore, it was confirmed that the antibodies in EEIVHC002 / EEIVL, EEIVHC003 / EEIVL, EEIVHC004 / EEIVL, EEIVHC005 / EEIVL, and EEIVHEC006 / EEIVL were cleaved by proteases. These results demonstrate that even antibodies that are not cleaved by proteases, such as EEIVHC / EEIVL antibodies where the protease cleavage site is only introduced near the boundary between the variable and invariant regions, can still be cleaved by proteases by introducing a mobile linker sequence near the cleavage sequence. Therefore, this shows that protease-cleaved antibodies can be created by arbitrarily combining protease cleavage sequences and mobile linkers.

[0178] 5-7. Activation of CXCL10-anti-CXCL10 neutralizing antibodies by ligand cleavage via protease. Then, Biacore was used to evaluate whether human CXCL10 conjugated to the antibody prepared in 5-5 would be released due to protease treatment. Specifically, using the antibody EEIVHC006a / EEIVL (heavy chain sequence number: 33, light chain sequence number: 2) prepared in 5-5, analytes were prepared with antigen / protease, without antigen / protease, and with antigen / without protease. The analytes with antigen / protease were those that had been treated with 20 nM recombinant human Matriptase / ST14 catalyst domain (MT-SP1) (R&D Systems, 3946-SE-010) for 20 hours after antibody conjugation to human CXCL10. The analytes without antigen / protease were those that had only been treated with 20 nM recombinant human Matriptase / ST14 catalyst domain (MT-SP1) (R&D Systems, 3946-SE-010) for 20 hours. An analyte containing antigen / without protease was prepared, and an antibody was used to bind to human CXCL10. Additionally, an analyte containing only CXCL10 as an antigen was prepared as a control group to confirm the systemic response to CXCL10 binding. The anti-CXCL10 antibody was immobilized on a CM5 sensor chip (BR100530, GE Healthcare) using methods known to those skilled in the art. Electrophoresis buffer of 20 mM ACES, 0.05% Tween 20, pH 7.4 was used to pass the analyte through four analytes: one containing antigen / with protease, one without antigen / with protease, one with antigen / without protease, and one containing only antigen. The binding of the anti-CXCL10 antibody to human CXCL10 on the sensor chip at 25°C was evaluated. Furthermore, using MabCXCL10a (heavy chain: EEIVHa (sequence number: 65), light chain: EEIVL (sequence number: 2)) with the same Fab region as the antibody MabCXCL10 which does not have a protease cleavage sequence, analytes with antigen / protease, without antigen / protease, and with antigen / without protease were prepared in the same manner as the antibody EEIVHC006a / EEIVL. Similarly, the anti-CXCL10 antibody was immobilized on a CM5 sensor chip (BR100530, GE Healthcare) according to methods known to those skilled in the art. Using 20 mM ACES, 0.05% Tween 20, pH 7.4 as the electrophoresis buffer, four analytes were passed through: one with antigen / protease, one without antigen / protease, one with antigen / without protease, and one antigen-only analyte (CXCL10). The binding of the anti-CXCL10 antibody to human CXCL10 on the sensor chip at 25°C was evaluated. Figure 11 shows a sensor plot representing the binding amount over time between the flow cell containing the antibody and the unfixed anti-CXCL10 antibody. The starting point of the vertical axis is the time when the analyte begins to flow through. The vertical axis represents the response at various time points when the response at the start of analyte flow is set to 100. As shown in Figure 11(A), CXCL10 remains non-free even after protease treatment with MabCXCL10a without the inserted cleavage sequence. However, as shown in Figure 11(B), CXCL10 is confirmed to be free after protease treatment with EEIVHC006a / EEIVL.

[0179] 5-8. Preparation and utilization of anti-CXCL10 neutralizing antibodies obtained by substituting a portion of the amino acid sequence near the boundary between the variable and invariant regions of the antibody with a portion of the protease cleavage sequence and the sequence of the mobile linker; evaluation of protease cleavage. This study explored replacing a portion of the amino acid sequence near the boundary between the variable and invariant regions of the heavy chain of MabCXCL10 with a portion of the protease cleavage sequence and a mobile linker sequence. The heavy chain shown in Figure 12 was designed by replacing a portion of the amino acid sequence of the heavy chain with peptide sequence A (Sequence No.: 3), which is reportedly cleaved by urokinase (uPA) and Matriptase (MT-SP1) in cancer-specific expression, resulting in a heavy chain alteration vector EESVHA009 (Sequence No.: 59) and EESVHA012 (Sequence No.: 60). Expression vectors encoding the heavy chain alteration vectors EESVHA009 (Sequence No.: 59) and EESVHA012 (Sequence No.: 60) were fabricated using methods known to those skilled in the art. The heavy chain alterations were combined with the natural light chain, and the following IgG1 antibodies: EESVHA009 / EEIVL (heavy chain sequence number: 59, light chain sequence number: 2) and EESVHA012 / EEIVL (heavy chain sequence number: 60, light chain sequence number: 2) were expressed using FreeStyle 293 (Life Technologies) according to methods known to those skilled in the art, and purified using Protein A according to methods known to those skilled in the art. The study investigated whether EESVHA009 / EEIVL and EESVHA012 / EEIVL were cleaved by proteases. Human urokinase (uPA) (R&D Systems, 1310-SE-010) and recombinant human Matriptase / ST14 catalyst domain (MT-SP1) (R&D Systems, 3946-SE-010) were used as proteases. The reaction was carried out at 12.5 nM protease, 100 μg / mL antibody, PBS, and 37°C for 20 hours. The cleavage caused by the protease was evaluated using reducing SDS-PAGE, and the results are shown in Figure 13. The results showed that EESVHA009 / EEIVL and EESVHA012 / EEIVL exhibited new bands between 25 kDa and 50 kDa due to protease treatment. Therefore, it was confirmed that the antibody in EESVHA009 / EEIVL and EESVHA012 / EEIVL is cleaved by the protease.

[0180] Example 6: Investigation into the insertion of permissible sites into cleaved sequences that have lost their antigen-binding ability due to protease cleavage. A report has been published on the preparation and in vitro functional evaluation of an antibody with a protease cleavage sequence inserted directly before aspartic acid 216 of the heavy chain of human IgG1 antibody (International Publication WO2004 / 021861A2). Experimental data were not recorded, but it is argued that if this antibody is mixed with the antigen and treated with a culture medium containing the corresponding protease, the antigen will be released from the antigen-antibody complex. The report claims that the amino acid at position 216 of the heavy chain of the human IgG1 antibody, into which a protease cleavage sequence has been inserted, is not aspartic acid according to any of the numbering systems listed in Kabat, EU, or OU in Kabat, E. et al. Sequences of Proteins of Immunological Interest, 5th edition. However, according to different literature, amino acid at position 216 of the heavy chain of the human IgG1 antibody is considered to be aspartic acid immediately preceding cysteine, which forms a disulfide bond between the heavy and light chains (Nature 344, 667-670 (12 April 1990), Kabat, E. et al. Sequences of Proteins of Immunological Interest, 4th edition). When a protease cleavage sequence is inserted immediately preceding this aspartic acid position 216, it is believed that the same Fab region is formed as when the antibody is cleaved by papain in the hinge region of the antibody, similar to the region cleaved by the protease. It is generally believed that the hinge region of an antibody made with papain is not easily cleaved, thus it is thought that even if an antibody with a protease cleavage sequence inserted directly before aspartic acid at position 216 is cleaved by the corresponding protease, it will still not lose its antigen-binding ability. This study also explores the hypothesis that a protease cleavage sequence is inserted directly before amino acid 216 of the heavy chain of the human IgG1 antibody (Kabat number) as described in Kabat, E. et al. Sequences of Proteins of Immunological Interest, 5th edition. This site contains several amino acids further to the N-terminus than cysteine ​​(Kabat number), which forms a disulfide bond between the heavy and light chains. Therefore, the effect of protease cleavage of the heavy chain at this site is presumably similar to the effect of losing the disulfide bond between the heavy and light chains. According to previous literature, even Fab regions, which cannot form disulfide bonds between the heavy and light chains, are not prone to losing antigen binding (MAbs. 2014 Jan-Feb;6(1):204-18.). Therefore, the case described in Kabat, E. et al. Sequences of Proteins of Immunological Interest 5th edition, where a protease cleavage sequence is inserted directly before amino acid (Kabat number) of the heavy chain of human IgG1 antibody, is believed to not lose its antigen-binding ability due to protease cleavage.

[0181] Example 7 Evaluation of the migration activity of the anti-CXCL10 neutralizing antibody / CXCL10 complex with protease cleavage sequence introduced into the complex. The evaluation will assess whether the complex formed by the CXCL10 neutralizing antibody prepared in Example 5, which incorporates a protease cleavage sequence, and CXCL10 will release CXCL10 due to protease cleavage, and whether CXCL10 will exert cell migration activity. Cell migration activity of CXCL10 was evaluated using Ba / F3 transfected cells (hereinafter referred to as BaF3 / mCXCR3) that expressed mouse CXCR3 (mCXCR3). These cells were used with HTS Transwell™-96 Permeable Support with 5.0 μm Pore Polycarbonate Membrane (Cat. 3387, Corning). Five analytes were prepared: CXCL10+ protease, EEIVHC006a / EEIVL+CXCL10, EEIVHC006a / EEIVL+CXCL10+ protease, EEIVHC006a / EEIVL+ protease, and MabCXCL10+CXCL10+ protease. In a PROTEOSAVE SS 1.5 mL microtube (Cat. MS-4265M, Sumitomo Bakelite), add antibody (MabCXCL10 or EEIVHC006a / EEIVL) to a final concentration of 10 μg / mL, hCXCL10 (Cat. 300-12, Peprotech) to a final concentration of 100 ng / mL, or both antibody and hCXCL10. Incubate at room temperature for 30 minutes. For analytes containing proteases, after the above reaction, add mouse MT-SP1 (mMT-SP1, Cat. 4735-SE-010, R&D Systems) to bring the final concentration to 12.5 nM. 235 μL of each analyte was transferred to the lower chamber, and 75 μL of BaF3 / mCXCR3 cells were seeded into the upper chamber to achieve a cell density of 2.0 × 10⁵ cells / well. The reaction was carried out at 37°C with 5% carbon dioxide. After 6 hours of reaction, 100 μL of the solution in the lower chamber was transferred to a 96-well fluorescent plate (Cat. 3912, Corning), and 100 μL of CellTiter-Glo™ Luminescent Cell Viability Assay solution (Cat. G7571, Promega) was added. After reacting at room temperature for 10 minutes, the luminescence value was measured using a SpectraMax M3 Molecular Devices instrument to evaluate cell migration into the lower chamber. The results are shown in Figure 14. Compared to the CXCL10+ protease analyte, the luminescence intensity decreased upon addition of the EEIVHC006a / EEIVL+CXCL10 analyte. Since luminescence intensity reflects the amount of migrating cells, it indicates that EEIVHC006a / EEIVL forms a complex with CXCL10, neutralizing its effect. Conversely, the luminescence intensity recovered upon addition of the EEIVHC006a / EEIVL+CXCL10+ protease analyte, indicating that it induces cell migration similarly to the CXCL10+ protease analyte. No recovery of luminescence intensity was observed upon addition of the MabCXCL10+CXCL10+ protease analyte containing an antibody without the cleavage sequence. These results suggest that EEIVHC006a / EEIVL, along with antibody cleavage using the protease, reduces the neutralizing ability of CXCL10.

[0182] Example 8 Evaluation of the migration activity of the CXCL10 fusion protein with an anti-CXCL10 neutralizing antibody containing a protease cleavage sequence and associated protease cleavage. 8-1 Preparation and evaluation of anti-CXCL10 neutralizing antibody-CXCL10 fusion protein via protease cleavage Using the light chain of the neutralizing antibody against human CXCL10, namely MabCXCL10_G7 (heavy chain: G7H-G1T4 (sequence number: 368), light chain: G7L-LT0 (sequence number: 369)), a linker sequence composed of glycine-serine polymer was separated at the N-terminus of the light chain, and the human CXCL10 variant hCXCL10R75A (sequence number: 370) resistant to protease was linked to design the ligand fusion light chain hCXCL10R75A.G4SGGGG.G7L-LT0 (sequence number: 371). The ligand fusion light chain was combined with the G7H-G1T4 of the MabCXCL10_G7 heavy chain to form the fusion protein: G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 (heavy chain sequence number: 368, ligand fusion light chain sequence number: 371). The protein was expressed using a transient expression of Expi293 (Life Technologies) according to methods known to those skilled in the art, and purified using Protein A according to methods known to those skilled in the art. The CDR sequences of MabCXCL10_G7 are as follows: H-CDR1 (SFSIT, sequence number: 374), H-CDR2 (EITPMFGIANYAQKFQG, sequence number: 375), H-CDR3 (DGRFDVSDLLTDKPKVTINYNGMDV, sequence number: 376), L-CDR1 (SGSSSNIGSNTVN, sequence number: 377), L-CDR2 (NNDQRPS, sequence number: 378), and L-CDR3 (ASWDDSLNGRV, sequence number: 379). The fusion protein was examined to determine if it would be cleaved by a protease. Human urokinase (huPA) (R&D Systems, 1310-SE-010) was used as the protease. Reduction SDS-PAGE was used to evaluate the cleavage of the fusion protein using the protease. After incubating the fusion protein with 0.1 mg / ml of 30 nM huPA at 37°C for 1 hour, reduction SDS-PAGE was used to evaluate the cleavage of the fusion protein. The results showed that G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 was not cleaved by the protease (Figure 15).

[0183] 8-2 Evaluation of the migration activity of the anti-CXCL10 neutralizing antibody-CXCL10 fusion protein against its concomitant protease cleavage The study aimed to evaluate whether the anti-CXCL10 neutralizing antibody-CXCL10 fusion protein, obtained by fusing an anti-CXCL10 neutralizing antibody with a protease cleavage sequence with CXCL10, would cause CXCL10 to become free due to protease cleavage and induce cell migration. hCXCL10R75A-His (Sequence No.: 373), exhibiting only hCXCL10R75A activity, was prepared and purified using the following method as a comparative object for the activity of hCXCL10R75A isolated from the fusion protein. A histidine tag was added to the C-terminus of the human CXCL10 variant hCXCL10R75A (Sequence No.: 370), which is mutated into a protease-resistant variant, to prepare the histidine-tagged human CXCL10 variant hCXCL10R75A-His (Sequence No.: 373). hCXCL10R75A-His (Sequence No.: 373) was expressed using a transient expression with Expi293 (Life Technologies) according to methods known to those skilled in the art, and purified using a method known to those skilled in the art using Ni-sepharose. Cell migration activity was evaluated using Ba / F3 transfected cells (hereinafter referred to as BaF3 / mCXCR3) that expressed mouse CXCR3 (mCXCR3). These cells were used in conjunction with HTS Transwell™-96 Permeable Support with 5.0 μm Pore Polycarbonate Membrane (Cat. 3387, Corning). For the uPA(+) analyte, 0.15 μg / mL of hCXCL10R75A-His was added to a 2.0 mL 96-well deep-well plate (Cat. P-DW-20-CS, Axygen), and 1.5 μg / mL of recombinant huPA (Cat. 1310-SE, R&D systems) was added to bring the final concentration to 30 nM for the fusion protein G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 to 1.5 μg / mL. G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 1.5 μg / mL contains the equivalent of 0.15 μg / mL of hCXCL10R75A. The analyte for uPA(-) was hCXCL10R75A-His 0.15 μg / mL and the fusion protein G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 1.5 μg / mL, which does not have a protease cleavage sequence. 235 μL of each solution to be analyzed was transferred to the lower chamber, and 75 μL / well of BaF3 / mCXCR3 cells was seeded into the upper chamber to achieve a cell density of 2.0 × 10⁵ cells / well. The reaction was carried out for 6 hours at 37°C with 5% carbon dioxide. After 6 hours of reaction, 100 μL of the solution in the lower chamber was transferred to OptiPlate-96 (Cat. 6005299, PerkinElmer), and 100 μL of CellTiter-Glo™ Luminescent Cell Viability Assay solution (Cat. G7571, Promega) was added. After reacting at room temperature for 10 minutes, the luminescence value was measured using a SpectraMax M3 Molecular Devices instrument to evaluate cell migration into the lower chamber. The results are shown in Figure 16. Compared to the case with the addition of CXCL10R75A-His, no recovery of luminescence intensity was observed in the uncut sequence G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 even after protease treatment.

[0184] 8-3 Construction of the anti-CXCL10 neutralizing antibody-CXCL10 fusion protein with protease cleavage sequence introduced and evaluation of its migration activity accompanied by protease cleavage. The amino acid sequence was designed to contain a protease cleavage sequence near the boundary between the variable and invariant regions of the ligand fusion light chain hCXCL10R75A.G4SGGGG.G7L-LT0 (sequence number: 371) constructed in 8-1. The ligand fusion light chain with the introduced protease cleavage sequence was combined with the G7H-G1T4 of the MabCXCL10_G7 heavy chain. The fusion protein was represented using a transient representation with Expi293 (Life Technologies) according to methods known to those skilled in the art, and purified using methods known to those skilled in the art with Protein A. The fusion proteins were examined to determine whether they would be cleaved by a protease. Human urokinase (huPA) (R&D Systems, 1310-SE-010) was used as the protease. The cleavage of the fusion proteins using the protease was evaluated by reductive SDS-PAGE. After incubating 30 nM huPA at 37°C for 1 hour relative to 0.1 mg / ml of the fusion protein, the cleavage of the fusion protein was evaluated by reductive SDS-PAGE. This study aimed to evaluate whether the anti-CXCL10 neutralizing antibody-CXCL10 fusion protein, derived by fusing an anti-CXCL10 neutralizing antibody with a protease-cleaving sequence with CXCL10, would release CXCL10 due to protease cleavage and induce cell migration. Cell migration activity was assessed by creating Ba / F3 transfected cells (hereinafter referred to as BaF3 / mCXCR3) expressing mouse CXCR3 (mCXCR3), and evaluating these cells using HTS Transwell™-96 Permeable Support with 5.0 μm Pore Polycarbonate Membrane (Cat. 3387, Corning). For uPA(+) analytes, 0.15 μg / mL of hCXCL10R75A-His (prepared in 8-2) or 1.5 μg / mL of recombinant huPA (Cat. 1310-SE, R&D systems) was added to a 2.0 mL 96-well deep-well plate (Cat. P-DW-20-CS, Axygen) to a final concentration of 30 nM. The 1.5 μg / mL fusion protein with a protease cleavage sequence contained the equivalent of 0.15 μg / mL hCXCL10R75A. For uPA(-) analytes, 0.15 μg / mL of hCXCL10R75A-His or 1.5 μg / mL of the fusion protein with a protease cleavage sequence was used. 235 μL of each solution to be analyzed was transferred to the lower chamber, and 75 μL of BaF3 / mCXCR3 cells were seeded into the upper chamber to achieve a density of 2.0 × 10⁵ cells / well. The reaction was carried out for 6 hours at 37°C with 5% carbon dioxide. After 6 hours of reaction, 100 μL of the solution in the lower chamber was transferred to OptiPlate-96 (Cat. 6005299, PerkinElmer), and 100 μL of CellTiter-Glo™ Luminescent Cell Viability Assay solution (Cat. G7571, Promega) was added. After reacting at room temperature for 10 minutes, the luminescence value was measured using a SpectraMax M3 Molecular Devices instrument to evaluate cell migration into the lower chamber. The migration activity can be evaluated using luminescence intensity.

[0185] Example 9: Preparation of anti-IL-12 neutralizing antibody with protease cleavage sequence and mobile linker sequence and evaluation of IL-12 activation associated with protease cleavage. 9-1. Preparation of anti-IL-12 neutralizing antibody with protease cleavage sequence and mobile linker sequence inserted. IL-12 is one of the cytokines with immune-activating effects. IL-12 exerts its anti-tumor effect by activating immune cells, but it has been reported that systemic exposure can also cause serious side effects (Nat Immunol. 2012 Jul 19;13(8):722-8.). A sequence containing a mobile linker consisting of peptide sequence A (sequence number: 3) and glycine-serine polymer, reportedly cleaved by urokinase (uPA) and mastriptase (MT-SP1), was designed near the boundary between the variable and invariant regions of the heavy chain of an anti-IL12 antibody (UstkH-G1T4, heavy chain sequence number: 144) that has the same variable region as the human IL-12 neutralizing antibody Ustekinumab. An altered form of the Ustekinumab heavy chain, UstkH-G1T4CYTM1inP1 (sequence number: 146), was designed and combined with the light chain of Ustekinumab (UstkL-kT0, sequence number: 145). An expression vector encoding the altered form of Ustekinumab, UstkH-G1T4CYTM1inP1 / UstkL-kT0 (heavy chain sequence number: 146, light chain sequence number: 145), was prepared using methods known to those skilled in the art. The modified Ustekinumab, UstkH-G1T4CYTM1inP1 / UstkL-kT0, was expressed using a transient expression with FreeStyle 293 (Life Technologies) according to methods known to those skilled in the art, and purified using a method known to those skilled in the art with Protein A. The CDR sequences contained in the anti-IL12 antibody and its modified form in this embodiment are as follows: H-CDR1 (TYWLG, sequence number: 386), H-CDR2 (IMSPVDSDIRYSPSFQG, sequence number: 387), H-CDR3 (RRPGQGYFDF, sequence number: 388), L-CDR1 (RASQGISSWLA, sequence number: 389), L-CDR2 (AASSLQS, sequence number: 390), and L-CDR3 (QQYNIYPYT, sequence number: 391).

[0186] 9-2. Protease cleavage of anti-IL-12 neutralizing antibody with protease cleavage sequence and mobile linker sequence introduced. The antibody prepared in section 9-1 was examined to determine whether it would be cleaved by a protease. Recombinant human Matriptase / ST14 catalytic domain (human MT-SP1, hMT-SP1) (R&D Systems, 3946-SE-010), recombinant mouse Matriptase / ST14 catalytic domain (mouse MT-SP1, mMT-SP1) (R&D Systems, 4735-SE-010), and human urokinase (human uPA, huPA) (R&D Systems, 1310-SE-010) were used as proteases. The protease treatment involved adding hMT-SP1, mMT-SP1, or huPA to Ustekinumab (UstkH-G1T4 / UstkL-kT0) or its modified form UstkH-G1T4CYTM1inP1 / UstkL-kT0 to achieve final concentrations of 10.1, 16.9, and 9.17 μM, and then performing a one-night reaction at 37°C.

[0187] 9-3. Confirmation of cleavage of anti-IL-12 neutralizing antibodies against the introduced cleaved protease cleavage sequence and mobile linker sequence, and evaluation of IL-12 activation. The cleavage of antibodies treated with proteases was evaluated using reducing SDS-PAGE. The results showed that UstkH-G1T4 / UstkL-kT0 was not cleaved by any of the proteases. Conversely, UstkH-G1T4CYTM1inP1 / UstkL-kT0, which incorporated protease cleavage sequences and a mobile linker, exhibited new bands between 25 kDa and 50 kDa after protease treatment (Figure 17). Therefore, it was confirmed that the anti-IL-12 neutralizing antibody (UstkH-G1T4CYTM1inP1 / UstkL-kT0) with the inserted protease cleavage sequences and mobile linker sequences is cleaved by proteases. Then, the study evaluated whether IL-12 would be released from its antibody-antibody complex and exert its physiological activity when the antibody was cleaved using a protease. The physiological activity of IL-12 was evaluated based on the production of IFN-γ (also known as interferon gamma, IFN-g) in the NK92 human cell line. NK92 cells were seeded at 1 × 10⁵ cells / well in 96-well cell culture dishes. 10 ng / mL of IL-12 and protease-treated antibodies (UstkH-G1T4 / UstkL-kT0 or UstkH-G1T4CYTM1inP1 / UstkL-kT0, at concentrations of 20, 4, 0.8, 0.16, 0.032, 0.0054, and 0.0013 μg / mL) were added, and IFN-γ production was measured by ELISA after 48 hours. To evaluate the effect of the antibody on IL-12 activity, an experiment was also conducted with protease-treated IL-12 alone (No Ab) without antibody treatment. Figure 18 shows the results of measuring interferon gamma concentration. UstkH-G1T4 / UstkL-kT0 treated with various proteases (without protease cleavage sequences) inhibited the production of interferon gamma induced by IL-12 (a process known as neutralization), with the effect being the same at an antibody concentration of 0.8 μg / mL and without IL-12 (No IL-12). On the other hand, UstkH-G1T4CYTM1inP1 / UstkL-kT0 treated with various proteases (including protease cleavage sequences), at any antibody concentration, produced more interferon gamma compared to the case with UstkH-G1T4 / UstkL-kT0 without protease cleavage sequences. These results confirm that UstkH-G1T4CYTM1inP1 / UstkL-kT0, due to its accompanying cleavage, weakens its ability to neutralize IL-12, thus allowing IL-12 to act on cells.

[0188] Example 10: Evaluation of antibodies incorporating protease cleavage sequences into anti-human CXCL10 neutralizing antibodies. 10-1. Introduction of protease cleavage sequence for anti-human CXCL10 neutralizing antibody. The neutralizing antibodies MabCXCL10 (heavy chain: EEIVH (sequence number: 1), light chain: EEIVL (sequence number: 2)) and MabCXCL10_G7 (heavy chain: G7H-G1T4 (sequence number: 368), light chain: G7L-LT0 (sequence number: 369)) were prepared using methods known to those skilled in the art. The antibodies were expressed and purified using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) using methods known to those skilled in the art. Insert a cleavage sequence, indicated by sequence number 345, near the boundary between the variable and invariant regions of the heavy chain of MabCXCL10 or MabCXCL10_G7 to create the altered heavy chain EldHA0003-G1T4 (sequence number: 356) of MabCXCL10 and the altered heavy chain G7H.12aa-G1T4 (sequence number: 367) of MabCXCL10_G7.

[0189] The two modified heavy and light chains described above were combined, and the MabCXCL10 alteration EldHA0003 (heavy chain sequence number: 356, light chain sequence number: 2) and the MabCXCL10_G7 alteration G7H.12aa (heavy chain sequence number: 367, light chain sequence number: 369) were expressed transiently using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) according to methods known to those skilled in the art. The mixture was then purified using methods known to those skilled in the art that employed Protein A.

[0190] 10-2. Evaluation of protease-induced cleavage by anti-human CXCL10 neutralizing antibodies with protease-mediated sequence insertion into the heavy chain region. The study examined whether the antibody prepared by 10⁻¹ was cleaved by a protease. Recombinant human Matriptase / ST14 catalyst domain (human MT-SP1, hMT-SP1) (R&D Systems, 3946-SE-010) was used as the protease. The reaction was carried out at 10 nM protease, 50 μg / mL antibody, PBS, and 37°C for 20 hours, followed by SDS-PAGE reduction. The results are shown in Figures 19A and 19B. Both the MabCXCL10 altered variant EldHA0003 and the MabCXCL10_G7 altered variant G7H.12aa showed new bands around 37 kDa after hMT-SP1 treatment. This confirms that the protease cleavage sequence represented by sequence number 345 is cleaved by hMT-SP1. Similarly, it was confirmed that the protease cleavage sequence represented by sequence number 345 is also cleaved by human uPA and mouse uPA.

[0191] Example 11: Preparation and Evaluation of Peptides Incorporating Diverse Protease Cleavage Sequences 11-1 Production of multipeptides incorporating diverse protease sequences Expression vectors for the neutralizing antibody MRA against human IL6R (heavy chain: MRAH-G1T4 (sequence number: 147), light chain: MRAL-k0 (sequence number: 148)) were prepared using methods known to those skilled in the art. The CDR sequences of MRA are as follows: H-CDR1 (SDHAWS, sequence number: 398), H-CDR2 (YISYSGITTYNPSLKS, sequence number: 399), H-CDR3 (SLARTTAMDY, sequence number: 400), L-CDR1 (RASQDISSYLN, sequence number: 401), L-CDR2 (YTSRLHS, sequence number: 402), and L-CDR3 (QQGNTLPYT, sequence number: 403). Peptide sequences known to be cleaved by MMP-2, MMP-7, and MMP-9, and peptide sequences containing mobile linkers composed of glycine-serine polymers near these sequences, are shown in Table 4.

[0192] [Table 1]

[0193] The design involved inserting these sequences near the boundary between the variable and invariant regions of the heavy chain of MRA antibodies, resulting in the following altered heavy chains: MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4 (Sequence No.: 153), MEIVHG4SMP2.2G4S-MEIVHG4SMP2.2G4SG1T4 (Sequence No.: 154), MEIVHG4SMP2.4G4S-MEIVHG4SMP2.4G4SG1T4 (Sequence No.: 155), MEIVHG4SMP9G4S-MEIVHG4SMP9G4SG1T4 (Sequence No.: 156), MEIVHMP2.1-MEIVHMP2.1G1T4 (Sequence No.: 157), and MEIVHMP2.3-MEIVHMP2.3G1T4. (Sequence number: 158), MEIVHMP7.2-MEIVHMP7.2G1T4 (heavy chain sequence number: 159), the representation carrier encoding the altered heavy chains is made according to a method known to those skilled in the art. The modified heavy chain was combined with the MRA light chain, and the MRA modified organisms shown in Table 5 were expressed using transient expression of FreeStyle293 cells (Invitrogen) or Expi293 cells (Life technologies) in accordance with methods known to those skilled in the art, and purified using methods known to those skilled in the art using Protein A.

[0194] [Table 2] MRA alterations

[0195] 11-2. Evaluation of protease-mediated cleavage of multipeptides with diverse protease sequences. The presence of MRA altered variants prepared in step 11-1 was examined to determine whether they were cleaved by proteases. Recombinant human MMP-2 (R&D Systems, 902-MP-010), recombinant human MMP-7 (R&D Systems, 907-MP-010), and recombinant human MMP-9 (R&D Systems, 911-MP-010) were used as proteases. The proteases were mixed with 1 mM p-aminophenylmercuric acetate (APMA; abcam, ab112146) and activated at 37°C for 1 and 24 hours respectively before use. Proteases were prepared at 50 nM, 100 nM, or 500 nM, with 50 μg / mL antibody and assay buffer (using the MMP Activity Assay Kit (Fluorometric - Green) (ab112146), Component C: Assay Buffer). After reacting with 20 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, pH 7.2 (hereinafter referred to as Tris) at 37°C for 20 hours, the cleavage caused by the protease was evaluated by reducing SDS-PAGE. The results are shown in Figures 20A, 20B, and 21. MRA-altered antibodies reacted with the proteases shown in Table 5. In MMP-2, the following were observed: MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4 / MRAL-k0, MEIVHG4SMP2.2G4S-MEIVHG4SMP2.2G4SG1T4 / MRAL-k0, MEIVHG4SMP2.4G4S-MEIVHG4SMP2.4G4SG1T4 / MRAL-k0, MEIVHMP2.1-MEIVHMP2.1G1T4 / MRAL-k0. Cutting of MEIVHMP2.3-MEIVHMP2.3G1T4 / MRAL-k0 was observed in MMP-7. Cutting of MEIVHMP7.2-MEIVHMP7.2G1T4 / MRAL-k0 was observed in MMP-9. Cutting of MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4 / MRAL-k0 and MEIVHG4SMP9G4S-MEIVHG4SMP9G4SG1T4 / MRAL-k0 was observed in MMP-9.

[0196] Example 12 Evaluation of antibodies that introduce protease cleavage sequences at various positions on the heavy chain 12-1 Preparation of antibodies with protease cleavage sequences inserted at various positions on the heavy chain. Peptide sequence B (sequence number: 160), which is reported to be cleaved by urokinase (uPA) and mastriptase (MT-SP1), was inserted into different positions within the MRA heavy chain variable region (MRAH, sequence number: 161) to prepare altered versions of the MRA heavy chain variable region as shown in Table 6. These altered versions of the MRA heavy chain variable region were then linked to the MRA heavy chain invariant regions (G1T4, sequence number: 162) to create MRA heavy chain alterations, and expression vectors encoding the corresponding genes were prepared using methods known to those skilled in the art. Furthermore, peptide sequence B (sequence number: 160) was inserted into different positions within the MRA heavy chain invariant region (G1T4, sequence number: 162) to prepare altered versions of the MRA heavy chain invariant region as shown in Table 7. The altered MRA heavy chain invariant regions were linked to the variable regions of the MRA heavy chain (MRAH, sequence number: 161) to create MRA heavy chain alterations, and expression vectors encoding the corresponding genes were prepared according to methods known to those skilled in the art. The insertion sites of the protease cleavage sequences in the altered MRA heavy chain variable regions and the altered MRA heavy chain invariant regions are also shown in Tables 6 and 7. The insertion sites in Table 6 refer to the adjacent regions of the invariant regions at the recorded positions (Kabat numbers) in the antibody heavy chain variable regions, and the insertion sites in Table 7 refer to the adjacent regions of the variable regions at the recorded positions (EU numbers) in the antibody heavy chain invariant regions.

[0197] [Table 3] Alterations in the variable region of the MRA heavy chain and insertion sites of protease cleavage sequences

[0198] [Table 4] Alterations in the MRA heavy chain invariant region and insertion sites of protease cleavage sequences

[0199] The MRA heavy chain alteration prepared above was combined with the MRA light chain. The MRA alteration shown in Table 8 was expressed using a method known to those skilled in the art using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies), and then purified using a method known to those skilled in the art using Protein A.

[0200] [Table 5] MRA alterations

[0201] 12-2. Evaluation of the cleavage of anti-human IL6R neutralizing antibodies obtained by inserting protease cleavage sequences into antibody heavy chains. To examine whether the MRA altered mutants prepared in step 12-1 would be cleaved by proteases. Recombinant human Matriptase / ST14 catalytic domain (human MT-SP1, hMT-SP1) (R&D Systems, 3946-SE-010) was used as the protease. The reaction was carried out at 10 nM protease, 50 μg / mL antibody, PBS, and 37°C for 20 hours, followed by SDS-PAGE reduction. The results are shown in Figures 22A, 22B, 22C, 22D, 22E, 22F, 22G, 22H, 22I, 23A, 23B, and 23C. The protease-treated MRA altered mutant exhibited a heavy chain band at a smaller molecular weight compared to the untreated MRA altered mutant where the heavy chain was cleaved (the band appearing near 50 kDa in the MT-SP1(-) lane in the figure). This result confirms that the MRA alteration cells prepared in 12-1 were cleaved by hMT-SP1.

[0202] Example 13 Evaluation of antibodies that introduce protease cleavage sequences at various positions on the light chain 13-1 Preparation of antibodies with protease cleavage sequences inserted at various positions on the light chain. The peptide sequence B (sequence number: 160), which is reported to be cleaved by urokinase (uPA) and mastriptase (MT-SP1), was inserted into different positions within the MRA light chain variable region (MRAL, sequence number: 230) to create altered versions of the MRA light chain variable region as shown in Table 9. These altered versions of the MRA light chain variable region were then linked to the MRA light chain invariant region (k0, sequence number: 231) to create MRA light chain alterations, and expression vectors encoding the corresponding genes were prepared using methods known to those skilled in the art. Furthermore, peptide sequence B (sequence number: 160) was inserted into different positions within the MRA light chain invariant region (k0, sequence number: 231) to create altered versions of the MRA light chain invariant region as shown in Table 10. The modified MRA light chain invariant regions were linked to the variable regions of each MRA light chain (MRAL, sequence number: 230) to create MRA light chain modified regions, and expression vectors for the corresponding coding genes were prepared according to methods known to those skilled in the art. The insertion sites of the protease cleavage sequences in the modified MRA light chain variable regions and the modified MRA light chain invariant regions are also shown in Tables 9 and 10. The insertion sites in Table 9 are adjacent to the invariant regions of the antibody light chain variable regions containing the amino acids (Kabat number), and the insertion sites in Table 10 are adjacent to the variable regions of the antibody light chain invariant regions containing the amino acids (EU number).

[0203] [Table 6] Alterations in the variable region of the MRA light chain and insertion sites of protease cleavage sequences

[0204] [Table 7] Alterations in the variable region of the MRA light chain and insertion sites of protease cleavage sequences

[0205] The MRA light chain alteration prepared above was combined with the MRA heavy chain, and the MRA alteration shown in Table 11 was expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) in a transient manner according to methods known to those skilled in the art, and purified using Protein A in a manner known to those skilled in the art.

[0206] [Table 8] MRA alterations

[0207] 13-2. Evaluation of the use of anti-human IL6R neutralizing antibodies obtained by inserting protease cleavage sequences into the variable region of the antibody light chain. The presence of cleavage by protease was examined in the MRA alterations prepared in step 13-1. Recombinant human Matriptase / ST14 catalyst domain (MT-SP1) (R&D Systems, 3946-SE-010) was used as the protease. The reaction was carried out at 10 nM protease, 50 μg / mL antibody, PBS, and 37°C for 20 hours, followed by SDS-PAGE reduction. The results are shown in Figures 24A, 24B, 24C, 24D, 24E, 25A, and 25B. The cleaved MRA alterations after protease treatment exhibited light chain bands at smaller molecular weights compared to the untreated MRA alterations (the band appearing near 25 kDa in the MT-SP1(-) lane in the figure).

[0208] Example 14: Preparation of anti-human PD1 neutralizing antibody with protease cleavage sequence and evaluation of its binding to human PD1. 14-1. Introduction of protease cleavage sequence for anti-human PD1 neutralizing antibody The neutralizing antibody against human PD1 is 5C4H-G1T4 / 5C4L-KT0 (heavy chain 5C4H-G1T4, sequence number: 297; heavy chain variable region 5C4H, sequence number: 300; heavy chain invariant region G1T4, sequence number: 301; light chain 5C4L-KT0, sequence number: 298; light chain variable region 5C4L, sequence number: 302; light chain invariant region KT0, sequence number: 303;). Antibodies containing protease cleavage sequences were prepared by inserting either the heavy or light chain of H-CDR1 (NSGMH, sequence number: 392), H-CDR2 (VIWYDGSKRYYADSVKG, sequence number: 393), H-CDR3 (NDDY, sequence number: 394), L-CDR1 (RASQSVSSYLA, sequence number: 395), L-CDR2 (DASNRAT, sequence number: 396), and L-CDR3 (QQSSNWPRT, sequence number: 397) into the protease cleavage sequence. First, the peptide sequence (sequence number: 299) that is reported to be cleaved by the cancer-specific expression of Matriptase (MT-SP1) is inserted into the heavy chain 5C4H-G1T4 or the light chain 5C4L-KT0 of the aforementioned antibody to create the heavy chain variant shown in Table 12 and the light chain variant shown in Table 13, and then expressed using methods known to those skilled in the art.

[0209] [Table 9] Heavy chain alteration of anti-human PD1 neutralizing antibody Heavy chain alteration Serial Number Protease cleavage sequence insertion site 5C4HA12aa-G1T4 304 Between variable zone 113 (Kabat code) and invariant zone 118 (EU code) 5C4HA12aa-G1T4E 305 Between invariable zones 118 and 119 (EU number)

[0210] [Table 10] Light chain alterations of anti-human PD1 neutralizing antibodies

[0211] The heavy chain alteration variants of Table 12 combined with the light chain 5C4L-KT0, or the light chain alteration variants of Table 13 combined with the heavy chain 5C4H-G1T4, and the IgG1 antibodies containing protease cleavage sequences (Table 14) were expressed using a transient expression method known to those skilled in the art using Expi293 (Life Technologies), and purified using a method known to those skilled in the art using Protein A. Additionally, the 5C4H-G1T4 / 5C4L-KT0 (heavy chain sequence number: 297, light chain sequence number: 298), which does not contain the protease cleavage sequence, was expressed and purified as a control antibody.

[0212] [Table 11] Antibody with protease cleavage sequence introduced

[0213] 14-2. Evaluation of the binding of anti-human PD1 neutralizing antibodies with protease cleavage sequences to human PD1. 14-2-1 Protease Treatment Antibodies treated with protease were prepared by adding 10 μL of Recombinant Human Matriptase / ST14 Catalytic Domain (hMT-SP1, R&D systems 3946-SE-010) at a final concentration of 1.8 μg / mL using PBS to the antibody prepared in step 14-1 (final concentration 0.111 mg / mL). Antibodies not treated with protease were prepared by adding only 10 μL of PBS to the antibody prepared in step 14-1 (final concentration 0.111 mg / mL). The sample volume for the reaction was 90 μL, and the final concentration of protease was 0.2 μg / mL. All samples were incubated at 37°C for 12 hours.

[0214] 14-2-2 Preparation of Biotinylated Anti-Human PD1 Neutralizing Antibody A biotinylated anti-human PD1 neutralizing antibody with the same variable region sequence as 5C4H-G1T4 / 5C4L-KT0 was prepared. Specifically, a gene fragment of 5C4VH-G1dGSBAP (sequence number: 317), encoding the heavy chain invariant region of the heavy chain variable region 5C4H (sequence number: 300) with added antibody and biotin (AviTag sequence, sequence number: 316), was prepared and introduced into an animal cell expression vector using methods known to those skilled in the art. The constructed expression vector and the vector expressing the light chain 5C4L-KT0B (sequence number: 298) were introduced into FreeStyle293 cells (Invitrogen) using 293 Fectin (Invitrogen). At this time, the gene expressing EBNA1 (sequence number: 318) and the gene expressing biotin ligase (BirA, sequence number: 319) were simultaneously introduced, and biotin was added for biotin labeling purposes. Cells with the introduced gene were cultured at 37°C and 8% CO2 to allow the target biotinylated anti-human PD1 neutralizing antibody (5C4-bio) to be secreted into the culture supernatant. 5C4-bio was then purified from the culture supernatant using methods well-known to those skilled in the art.

[0215] 14-2-3 Evaluation of the binding of various antibodies to human PD1 before and after protease treatment 80 μL of either protease-treated or untreated antibody prepared in step 14-2-1 was added to a final concentration of 0.67 μM with human PD1, and the antibody was allowed to bind at room temperature for 30 minutes to prepare a binding evaluation sample. The binding of the antibody to PD1 was evaluated by assessing the amount of PD1 not bound to the antibody, in both protease-treated and untreated cases. Specifically, the amount of PD1 not bound to the antibody was evaluated using the biotinylated anti-human PD1 neutralizing antibody (5C4-bio) prepared in Example 14-2-2 by BioLayer Interferometry (BLI method). The binding evaluation sample, 5C4-bio, and PBS were injected into different wells of tiled bottom (TW384) microplates (ForteBio, 18-5076). The streptavidin biosensor (ForteBio, 18-0009) was hydrated with PBS and measured using an Octet RED 384 at 30°C. A 30-second baseline measurement was performed in the wells containing PBS, followed by 200 seconds of 5C4-bio binding to the streptavidin sensor. Another 30-second baseline measurement was performed in the wells containing PBS, followed by 180 seconds of binding measurement in the wells containing the binding evaluation sample, and then 180 seconds of dissociation measurement in the wells containing PBS. The real-time binding axis, showing the binding status, is shown in Figure 26. As shown in Figure 26, in the case of antibodies with introduced protease cleavage sequences, the amount of human PD1 bound to 5C4-bio was higher in the binding evaluation samples containing protease-treated antibodies compared to those containing untreated antibodies. That is, the antibodies with introduced protease cleavage sequences exhibit reduced PD1 binding activity due to protease treatment, resulting in free PD1 bound to 5C4-bio.

[0216] 14-2-4 Confirmation of antibody protease cleavage (SDS-PAGE) SDS-PAGE was used to confirm whether the antibodies used in 14-2-3 were cleaved by protease treatment. 10 μL of the protease-cleaved antibody prepared in 14-2-3 / untreated antibody was mixed with 3.3 μL of sample buffer and incubated at 95°C for 5 minutes. Electrophoresis was then performed using a Mini-PROTEAN TGX gel (4-20% 15well) (Bio-Rad #456-1096), and the proteins were stained with Sample Blue Safe Stain (novex, LC6065). The results are shown in Figure 27. As shown in Figure 27, the antibodies incorporating the protease-cleaving sequence have been cleaved by protease treatment.

[0217] 14-2-5 Evaluation of PD1 binding of antibodies before and after protease treatment The binding activity of each antibody with the protease cleavage sequence introduced was also determined by other methods before and after protease treatment for PD1. 10 μL of either protease-treated or untreated antibody prepared according to 14-2-1 was mixed with 70 μL of PBS to prepare the PD1 binding assay sample. PD1 binding was evaluated using BioLayer Interferometry (BLI). The protease-treated / untreated antibody prepared according to 14-2-1 and human PD1 (250 nM) were injected into different wells of Tilted Bottom (TW384) Microplates (ForteBio, 18-5076). The Protein G sensor (ForteBio, 18-0022) was hydrated with PBS, and the assay was performed using an Octet RED 384 at 30°C. A 30-second baseline measurement was performed in the well containing PBS, followed by antibody binding to the Protein G sensor for 200 seconds. Baseline measurements were performed again for 30 seconds in wells containing PBS, followed by 180-second binding measurements in wells containing human PD1, and then 180-second dissociation measurements in wells containing PBS. The instantaneous binding axis showing the binding status is shown in Figure 28. As shown in Figure 28, when using antibodies with protease cleavage sequences, the amount of human PD1 bound to protease-treated antibodies was reduced compared to untreated antibodies.

[0218] 14-3. The complex of an anti-human PD1 neutralizing antibody with a protease cleavage sequence and its ligand (human PD1) was evaluated using the ligand release mechanism induced by the protease. 14-3-1 Protease treatment in the presence of ligands 10 μL of human PD1 (prepared in PBS to a final concentration of 0.100 mg / mL) was added to the antibody prepared in step 14-1 to prepare an antibody-PD1 complex solution. For protease-treated samples, 10 μL of recombinant human Matriptase / ST14 Catalytic Domain (hMT-SP1, R&D systems 3946-SE-010) (prepared in PBS to a concentration of 5.28 μg / mL) was added to the antibody-PD1 complex solution. For untreated samples, only 10 μL of PBS was added. The final concentration of protease during the reaction was 0.528 μg / mL. All samples were incubated at 37°C for 12 hours.

[0219] 14-3-2 Evaluation of PD1 release after protease treatment The amount of PD1 that did not form a complex with the antibody was evaluated using the biotinylated anti-human PD1 neutralizing antibody (5C4-bio) prepared in Example 14-2-2 by BioLayer Interferometry (BLI method). Samples prepared in step 14-3-1, 5C4-bio, and PBS were injected into different wells of Tilted Bottom (TW384) Microplates (ForteBio, 18-5076). The streptavidin biosensor (ForteBio, 18-0009) was hydrated with PBS and measured using an Octet RED 384 at 30°C. A 30-second baseline measurement was performed in the wells containing PBS, followed by 200 seconds of 5C4-bio binding to the streptavidin sensor. Another 30-second baseline measurement was performed in the wells containing PBS, followed by 180 seconds of binding measurement in the wells containing either protease-treated or untreated samples, and then 180 seconds of dissociation measurement in the wells containing PBS. The real-time binding axis showing the binding status is shown in Figure 29. As shown in Figure 29, the amount of human PD1 bound to 5C4-bio increased in the protease-treated samples compared to the untreated samples, indicating the presence of antibodies with protease cleavage sequences. That is, because the protease treatment weakens the binding activity of each antibody to PD1, PD1 is released from the antibody-PD1 complex.

[0220] Example 15: Preparation and evaluation of a fusion protein (anti-PD1 neutralizing antibody-PD1 fusion protein) containing a protease cleavage sequence and human PD1 (anti-PD1 neutralizing antibody-PD1 fusion protein). 15-1. Preparation of the fusion protein between anti-human PD1 neutralizing antibody and human PD1 At the N-terminus of the heavy chain or heavy chain alteration of the antibody prepared in Example 14-1, a human PD1 sequence (Sequence Number: 320) is linked by a mobile linker composed of glycine-serine polymer (Sequence Number: 321) to produce a PD1 fusion heavy chain (Table 15).

[0221] [Table 12] PD1 Fusion Rechain

[0222] Furthermore, a human PD1 sequence (Sequence Number: 320) was connected to the N-terminus of the light chain or light chain alteration of the antibody prepared in Example 14-1 by means of a mobile linker composed of glycine-serine polymer (Sequence Number: 321) to form a PD1 fusion light chain (Table 16).

[0223] [Table 13] PD1 Fusion Light Chain

[0224] The following anti-PD1 neutralizing antibody-PD1 fusion proteins are obtained by combining PD1 fusion heavy chain and light chain 5C4L-KT0 from Table 15, or PD1 fusion light chain and heavy chain 5C4H-G1T4 from Table 16: hPD15C4HA12aa-G1T4 / 5C4L-KT0 (PD1 fusion heavy chain sequence number: 323, light chain sequence number: 298) hPD15C4HE12aa-G1T4E / 5C4L-KT0 (PD1 fusion heavy chain sequence number: 324, light chain sequence number: 298) 5C4H-G1T4 / hPD15C4LH12aa-KT0 (Heavy chain sequence number: 297, Light chain sequence number: 325) 5C4H-G1T4 / hPD15C4LI12aa-KT0 (Heavy chain sequence number: 297, PD1 fusion light chain sequence number: 326) 5C4H-G1T4 / hPD15C4LC12aa-KT0 (Heavy chain sequence number: 297, PD1 fusion light chain sequence number: 327) 5C4H-G1T4 / hPD15C4LD12aa-KT0 (Heavy chain sequence number: 297, PD1 fusion light chain sequence number: 328) 5C4H-G1T4 / hPD15C4LE12aa-KT0E (Heavy chain sequence number: 297, PD1 fusion light chain sequence number: 329) 5C4H-G1T4 / hPD15C4LB12aa-KT0B (Heavy chain sequence number: 297, PD1 fusion light chain sequence number: 330) 5C4H-G1T4 / hPD15C4LF12aa-KT0F (Heavy chain sequence number: 297, PD1 fusion light chain sequence number: 331) 5C4H-G1T4 / hPD15C4LG12aa-KT0G (Heavy chain sequence number: 297, PD1 fusion light chain sequence number: 332) 5C4H-G1T4 / hPD15C4LJ12aa-KT0J (Heavy chain sequence number: 297, PD1 fusion light chain sequence number: 333) 5C4H-G1T4 / hPD15C4LK12aa-KT0K (Heavy chain sequence number: 297, PD1 fusion light chain sequence number: 334) The antibody was expressed using a transient expression with Expi293 (Life Technologies) according to methods known to those skilled in the art, and purified using a method known to those skilled in the art with Protein A. Similarly, 5C4H-G1T4 / 5C4L-KT0 (heavy chain sequence number: 297, light chain sequence number: 298), a control antibody without the protease cleavage sequence, was expressed and purified.

[0225] 15-2. Evaluation of the use of protease cleavage by anti-PD1 neutralizing antibody-PD1 fusion protein 15-2-1 Protease Treatment The protease-treated fusion protein was prepared by adding 30 μg of the fusion protein (prepared in step 15-1) to 4.9 μL of Recombinant Human Matriptase / ST14 Catalytic Domain (hMT-SP1, R&D systems 3946-SE-010) prepared in PBS to a concentration of 16.7 μg / mL. The untreated fusion protein was prepared by adding only 4.9 μL of PBS to 30 μg of the fusion protein (prepared in step 15-1). Both protease-treated and untreated fusion proteins were incubated at 37°C for 12 hours.

[0226] 15-2-2 Evaluation of PD1 Free Cells Treated with Protease The biotinylated anti-human PD1 neutralizing antibody (5C4-bio) prepared in Example 14-2-2 was used to evaluate PD1 freeness induced by protease treatment using BioLayer Interferometry (BLI method). The protease-treated fusion protein, the untreated fusion protein, 5C4-bio, and PBS prepared in step 15-2-1 were injected into different wells of Tilted Bottom (TW384) Microplates (ForteBio, 18-5076). The streptavidin biosensor (ForteBio, 18-0009) was hydrated with PBS and measured using an Octet RED 384 at 30°C. A 30-second baseline measurement was performed in the wells containing PBS, followed by 200 seconds of binding to the streptavidin sensor with 5C4-bio. After another 30-second baseline measurement in the wells containing PBS, binding was measured for 180 seconds in wells containing either the protease-treated or untreated fusion protein, and dissociation was measured for 180 seconds in wells containing PBS. The real-time binding axis showing the binding status is shown in Figure 30. As shown in Figure 30, in the case of antibody-PD1 fusion protein containing an antibody with an introduced protease cleavage sequence, the amount of human PD1 bound to 5C4-bio increased in the protease-treated sample compared to the untreated sample. That is, because protease treatment weakens the binding activity of the antibody in the fusion protein to PD1, PD1 is released from the fusion protein.

[0227] 15-2-3 Confirmation of cleavage of anti-PD1 neutralizing antibody-PD1 fusion protein (SDS-PAGE) SDS-PAGE was used to confirm whether the protease-treated fusion protein prepared in step 15-2-1 was cleaved by the protease. 10 μL of either the protease-treated or untreated fusion protein prepared in step 15-2-1 was mixed with 3.3 μL of sample buffer and incubated at 95°C for 5 minutes. Electrophoresis was then performed using a Mini-PROTEAN TGX gel (4-20% 15-well) (Bio-Rad #456-1096), and the proteins were stained with Sample Blue Safe Stain (novex, LC6065). The results are shown in Figure 31. As shown in Figure 31, the fusion protein containing the antibody with the introduced protease-cleaving sequence has been cleaved by the protease.

[0228] The foregoing invention has been described in detail with reference to examples and illustrations to aid in clear understanding; however, the description and illustrations in this specification should not be construed as limiting the scope of the invention. All disclosures in the patent and scientific literature referenced in this specification are incorporated herein by reference in their entirety. [Industrial applicability]

[0229] The ligand-binding molecule of this invention is delivered into the body in a ligand-bound state. When the diseased tissue is cleaved, the binding to the ligand is weakened, allowing for the ligand to be released specifically to the diseased tissue. Therefore, the diseased tissue can be specifically exposed to the ligand. Furthermore, the ligand-binding molecule inhibits the biological activity of the ligand during delivery, thus reducing the possibility of systemic effects, which is extremely useful in disease treatment.

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Claims

1. A ligand-binding molecule, which is a molecule capable of binding to a ligand, the molecule being a polypeptide comprising: At least one cleavage site comprising a protease cleavage sequence, an antibody-invariant region, and an antigen-binding region or antibody fragment that binds to the ligand, and the binding to the ligand is weakened when the molecule is cleaved at at least one cleavage site, wherein the cleavage site is located between the antigen-binding region or antibody fragment and the antibody-invariant region, wherein the ligand is selected from: (a) intercytokines or chemokines; (b) Interleukins, interferons, hematopoietic factors, TNF superfamily, cell proliferation factors, TGF-β family, myokine, adipokine, or neurotrophic factors; or (c) CXCL10, IL-1, IL-2, IL-4, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-21, IFN-α, IFN-β, IFN-γ, TGF-β, TNF-α, MIG, I-TAC, RANTES, MCAF, MIP-1α, MIP-β, PD1, and IL-6R.

2. As in the ligand-binding molecule of claim 1, wherein, After the cleavage site is cleaved, the ligand is released from the ligand-binding molecule.

3. As in the ligand-binding molecule of claim 1, wherein, This protease is a tissue-specific protease.

4. A ligand-binding molecule as described in any of claims 1 to 3 of the patent application, wherein, The ligand-binding molecules include antibody VH and antibody VL.

5. As in claim 4, the ligand-binding molecule, wherein, The cleavage site or the protease cleavage sequence is located near the boundary between the antibody invariant region and the antibody VH, or / and near the boundary between the antibody invariant region and the antibody VL.

6. As in claim 4, the ligand-binding molecule, wherein, The antibody VL in the ligand-binding molecule assembles with the antibody VH, and the assembly is terminated by cleavage at the cleavage site or by cleavage by the protease via the protease cleavage sequence.

7. A ligand-binding molecule as described in any of claims 1 to 3 of the patent application, wherein, The ligand system contains biologically active molecules, and the ligand-binding molecule inhibits the biological activity of the ligand by binding to it.

8. A ligand-binding molecule as described in any of claims 1 to 3 of the patent application, wherein, The ligand regains its biological activity after at least one of the cleavage sites on the ligand-binding molecule is cleaved.

9. A ligand-binding molecule as described in any of claims 1 to 3 of the patent application, wherein, Compared to the ligand that is bound to the ligand-binding molecule before the cleavage site is cleaved, the ligand released from the ligand-binding molecule after the cleavage site is cleaved has a shorter half-reduction period.

10. A ligand-binding molecule as described in any of claims 1 to 3 of the patent application, wherein, (a) The protease is selected from: matriptase, urokinase (uPA), cathepsin, collagenase, furin, plasmagen, thrombin, serine protease, and metalloproteinase; or (b) the protease cleavage sequence contained in the ligand-binding molecule contains a sequence selected from the sequences listed in sequence numbers 3, 34, 66, 70, 71, 72, 73, 35, 75, 76, and 345.

11. A ligand-binding molecule as described in any of claims 1 to 3 of the patent application, wherein, The ligand binds to an IgG antibody.

12. The ligand-binding molecule in any of the claims 1 to 3 is bound to or fused with the ligand.

13. A pharmaceutical composition comprising a ligand-binding molecule as described in any one of claims 1 to 11, or a ligand-binding molecule as described in claim 12, wherein the ligand is bound to or fused to the ligand.

14. A method for manufacturing a ligand-binding molecule as claimed in any one of claims 1 to 12, comprising the step of culturing a host cell containing: a polynucleotide encoding a ligand-binding molecule as claimed in any one of claims 1 to 12 or a carrier containing the polynucleotide.

15. A method for manufacturing a fusion protein comprising a ligand-binding molecule as claimed in claim 12, comprising the step of fusing a ligand-binding molecule having the protease cleavage sequence with its ligand.

16. The ligand-binding molecule of claim 1, wherein (a) a first movable linker is attached to one end of the protease cleavage sequence, optionally wherein the first movable linker is composed of a glycine-serine polymer; or (b) a first movable linker is attached to one end of the protease cleavage sequence and a second movable linker is attached to the other end of the protease cleavage sequence, optionally wherein the second movable linker is composed of a glycine-serine polymer.

17. The ligand-binding molecule as described in claim 16, wherein, It includes the first movable linker and the second movable linker, wherein: (a) the protease cleavage sequence, the protease cleavage sequence and the first movable linker, or the protease cleavage sequence and the first movable linker and the second movable linker are located within the antibody invariant region; (b) the protease cleavage sequence, the protease cleavage sequence and the first movable linker, or the protease cleavage sequence and the first movable linker and the second movable linker are inserted at any position in the sequence from amino acid 118 (EU number) to amino acid 140 (EU number) in the antibody heavy chain invariant region; (c) The protease cleavage sequence, the protease cleavage sequence and the first movable linker, or the protease cleavage sequence and the first movable linker and the second movable linker are inserted at any position in the sequence from amino acid 108 (EU number) (Kabat number 108) to amino acid 131 (EU number) (Kabat number 131) in the antibody light chain invariant region; (d) The protease cleavage sequence, the protease cleavage sequence and the first movable linker, or the protease cleavage sequence and the first movable linker and the second movable linker are located within the antibody VH or the antibody VL; (e) The protease cleavage sequence, the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence and the first mobile linker and the second mobile linker are inserted at any position in the sequence of the group consisting of antibody VH7 amino acids (Kabat number) to 16 amino acids (Kabat number), 40 amino acids (Kabat number) to 47 amino acids (Kabat number), 55 amino acids (Kabat number) to 69 amino acids (Kabat number), 73 amino acids (Kabat number) to 79 amino acids (Kabat number), 83 amino acids (Kabat number) to 89 amino acids (Kabat number), 95 amino acids (Kabat number) to 99 amino acids (Kabat number), and 101 amino acids (Kabat number) to 113 amino acids (Kabat number); (f) The protease cleavage sequence, the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence and the first mobile linker and the second mobile linker are inserted at any position in the sequence of the group consisting of antibody VL7 amino acids (Kabat number) to 19 amino acids (Kabat number), 39 amino acids (Kabat number) to 46 amino acids (Kabat number), 49 amino acids (Kabat number) to 62 amino acids (Kabat number), and 96 amino acids (Kabat number) to 107 amino acids (Kabat number);(g) The protease cleavage sequence, the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence and the first mobile linker and the second mobile linker are located near the boundary between the antibody invariant region and the antibody VH, or / and near the boundary between the antibody invariant region and the antibody VL. (h) The protease cleavage sequence, or the protease cleavage sequence and the first movable linker, or the protease cleavage sequence and the first movable linker and the second movable linker, is inserted at any position in the sequence from amino acid (Kabat number) of antibody VH109 to amino acid (EU number) of antibody heavy chain invariant region 122; or (i) The protease cleavage sequence, the protease cleavage sequence and the first movable linker, or the protease cleavage sequence and the first movable linker and the second movable linker, is inserted at any position in the sequence from amino acid (Kabat number) of antibody VL104 to amino acid (EU number) of antibody light chain invariant region 113 (Kabat number position 113).

18. The ligand-binding molecule as described in claim 16, wherein, The ligand binds to a molecular mediator linker and fuses with the ligand, optionally wherein the linker is composed of a glycine-serine polymer, and optionally wherein the linker does not contain the protease cleavage sequence.

19. The ligand-binding molecule as described in claim 18, wherein, The ligand-binding molecule contains an antibody light chain and an antibody heavy chain, wherein the antibody light chain or the antibody heavy chain is fused to the ligand, and optionally the cleavage site is contained in the antibody light chain or the antibody heavy chain.

20. The ligand-binding molecule as described in claim 1, wherein, The ligand is IL12.

21. As in claim 20, the ligand-binding molecule, wherein, The protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence and the first mobile linker and the second mobile linker, is inserted at any position in the sequence from amino acid (Kabat number) of antibody VH109 to amino acid (EU number) of antibody heavy chain invariant region 122.

22. As in the ligand-binding molecule of claim 1, wherein, The ligand is IL12 and the at least one cleavage site includes the protease cleavage sequence, wherein a first movable linker is attached to one end of the protease cleavage sequence and a second movable linker is attached to the other end of the protease cleavage sequence, and wherein the protease cleavage sequence, or the protease cleavage sequence and the first movable linker, or the protease cleavage sequence and the first movable linker and the second movable linker, is inserted at any position in the sequence from amino acid (Kabat number) of antibody VH109 to amino acid (EU number) of antibody heavy chain invariant region 122, optionally wherein the second movable linker is composed of a glycine-serine polymer.

23. The ligand-binding molecule as described in claim 22, wherein, The protease is a mastriptase, and the protease cleavage sequence in the ligand-binding molecule contains the sequence described in sequence number 345.

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