Polypeptide including transport section, its use, pharmaceutical composition comprising it and method for production of polypeptide

Abstract

The present invention relates to a polypeptide comprising an antigen-binding domain and a carrier portion having an inhibitory domain that inhibits the antigen-binding activity of the antigen-binding domain, and having a longer half-life than that of the antigen-binding domain existing alone; methods for producing and analyzing the polypeptide; a pharmaceutical composition comprising the polypeptide; methods for producing and analyzing the single-domain antibody whose antigen-binding activity is inhibited by association with particular vl, vh or vhh; and a fusion polypeptide library including a single-domain antibody whose antigen-binding activity is inhibited by association with particular vl, vh or vhh.

Description

POLYPEPTIDE INCLUDING TRANSPORT SECTION, ITS USE, PHARMACEUTICAL COMPOSITION COMPRISING IT AND METHOD FOR PRODUCING THE POLYPEPTIDE TECHNICAL FIELD

[001] The present invention relates to a polypeptide comprising an antigen-binding domain and a carrier portion having an inhibitory domain that inhibits the antigen-binding activity of the antigen-binding domain, and having a longer half-life than the half-life of the antigen-binding domain existing alone, methods for producing and analyzing the polypeptide, a pharmaceutical composition comprising the polypeptide, methods for producing and analyzing the single-domain antibody whose antigen-binding activity can be inhibited by association with particular VL, VH or VHH, and a fusion polypeptide library, in which a single-domain antibody whose antigen-binding activity can be inhibited by association with particular VL, VH or VHH is included. PREVIOUS TECHNIQUE

[002] Antibodies have received attention as drugs because they are highly stable in plasma and cause few adverse reactions. Among them, many IgG-type antibody drugs have been launched, and a large number of antibody drugs are currently under development (Non-Patent Literature 1 and 2).

[003] Rituxan against CD20, cetuximab against EGFR, Herceptin against HER2, and similar drugs have been approved so far as therapeutic drugs for cancer using antibody drugs (Non-Patent Literature 3). These antibody molecules bind to their antigens expressed on cancer cells and thus exert cytotoxic activity against cancer cells through ADCC activity, etc. Such cytotoxic activity based on ADCC activity, etc. is known to depend on the number of antigens expressed in Petition 870260070097, dated 07 / 15 / 2026, page 8 / 455 2 / 202 target cells of therapeutic antibodies (Non-Patent Literature 4). Therefore, high levels of expression of targeted antigens are preferred from the point of view of the effects of therapeutic antibodies. However, if an antigen, although having a high expression level, is expressed in normal tissues, cytotoxic activity based on ADCC activity, etc., is exerted against normal cells. Therefore, adverse reactions become a serious problem. Therefore, it is preferable that antigens targeted by therapeutic antibodies as therapeutic drugs for cancer should be specifically expressed in cancer cells. For example, an antibody molecule against EpCAM, known as a cancer antigen, has been considered promising as a therapeutic drug for cancer. However, EpCAM is known to be expressed in the pancreas.In fact, it has been reported in clinical trials that administration of an anti-EpCAM antibody causes pancreatitis as an adverse reaction due to cytotoxic activity against the pancreas (Non-Patent Literature 5).

[004] Following the success of antibody drugs exerting cytotoxic activity based on ADCC activity, enhanced, second-generation antibody molecules exerting strong cytotoxic activity have been reported as a result of, for example, enhancing ADCC activity by removing fucose from the N-linked oligosaccharide of a natural human IgG1 Fc region (Non-Patent Literature 6) or enhancing ADCC activity by enhancing FcyRIIIa binding through amino acid substitution of a natural human IgG1 Fc region (Non-Patent Literature 7). Enhanced antibody molecules exerting stronger cytotoxic activity, such as an antibody-drug conjugate (ADC) containing an antibody conjugated to a drug having strong cytotoxic activity (Non-Patent Literature 8), and a low molecular weight antibody that exerts Petition 870260070097, dated 07 / 15 / 2026, p. 9 / 455 3 / 202 cytotoxic activity against cancer cells through the recruitment of T cells to cancer cells (Non-Patent Literature 9) have been reported as antibody drugs exerting cytotoxic activity against cancer cells under a mechanism different from the NK cell-mediated ADCC activity as mentioned above.

[005] Such antibody molecules exerting stronger cytotoxic activity can exert cytotoxic activity even against cancer cells expressing an antigen at a level that is not elevated, but also exert cytotoxic activity against normal tissues expressing the antigen at a low level, similarly to cancer cells. In fact, EGFR-BiTE, a bispecific antibody against CD3 and EGFR, can exert strong cytotoxic activity against cancer cells and exert an antitumor effect by recruiting T cells to cancer cells, as compared to cetuximab, a natural human IgG1 against EGFR. On the other hand, it has also been found that serious adverse reactions appear when EGFR-BiTE is administered to cynomolgus monkeys, because EGFR is also expressed in normal tissues (Non-Patent Literature 10).Furthermore, ADC bivatuzumab mertansine containing mertansine conjugated with an antibody against CD44v6 highly expressed in cancer cells has been clinically found to cause various dermal toxicities and hepatotoxicity, because CD44v6 is also expressed in normal tissues (Non-Patent Literature 11).

[006] As mentioned above, the use of an antibody that can exert strong cytotoxic activity even against cancer cells expressing an antigen at low levels requires that the target antigen be expressed in a manner that is extremely cancer-specific. However, considering that a Herceptin HER2 target antigen or a cetuximab EGFR target antigen is also expressed in tissues Petition 870260070097, dated 07 / 15 / 2026, page 10 / 455 4 / 202 normal, only a limited number of cancer antigens can be expressed in a highly cancer-specific manner. Therefore, adverse reactions attributable to a cytotoxic effect on normal tissues may become a problem, although cytotoxic activity against cancer may be increased.

[007] Recently, ipilimumab, which enhances tumor immunity by inhibiting CTLA4 that contributes to immunosuppression in cancer, has been shown to prolong overall survival in metastatic melanoma (Non-Patent Literature 12). However, ipilimumab systemically inhibits CTLA4 and therefore causes adverse reactions similar to autoimmune disease due to systemic activation of immunity, although it enhances tumor immunity (Non-Patent Literature 13).

[008] However, antibody drugs exerting a therapeutic effect through the inhibition of inflammatory cytokines in inflammatory or autoimmune diseases are known as antibody drugs against diseases other than cancer (Non-Patent Literature 14). It is known that, for example, Remicade or Humira targeting TNF, and Actenra targeting IL-6R exert a high therapeutic effect in rheumatoid arthritis, while infectious disease is seen as an adverse reaction due to the systemic neutralization of these cytokines (Non-Patent Literature 15).

[009] Several techniques have been developed applicable to second-generation antibody drugs. For example, techniques for improving effector functions, antigen binding capacity, pharmacokinetics, or stability or reducing the risk of immunogenicity have been reported (Non-Patent Literature 16). However, there are still some reports on techniques that allow antibody drugs to act specifically on a target tissue in order to resolve adverse reactions, as described above. The reported techniques include a method that involves: attaching an antibody to a peptide Petition 870260070097, dated 07 / 15 / 2026, page 11 / 455 5 / 202 masking by means of a ligand that is cleaved by protease expressed at a site of injury such as cancerous tissue or inflammatory tissue, thereby masking the antibody's antigen-binding site with the masking peptide and inhibiting the antibody's antigen-binding activity; and dissociating the masking peptide from the antibody by protease cleavage of this ligand so that the antibody restores its antigen-binding activity and becomes capable of binding to the antigen in a target pathological tissue (Non-Patent Literature 17 and 18 and Patent Literature 1). [Citation List] [Patent Literature] [Patent Literature 1] International Publication No. WO2010 / 081173 [Non-Patent Literature]

[010] [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

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

[012] [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

[013] [Non-Patent Literature 4] Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies. Lewis GD, Figari I, Fendly B, Wong WL, Carter P, Gorman C, Shepard HM, Cancer Immunol. Immunotherapy (1993) 37, 255-263

[014] [Non-Patent Literature 5] ING-1, a monoclonal antibody targeting Ep-CAM in patients with advanced adenocarcinomas. de Bono JS, Tolcher AW, Forero A, Vanhove GF, Takimoto C, Bauer RJ, Hammond LA, Patnaik A, White ML, Shen S, Khazaeli MB, Rowinsky Petition 870260070097, dated 07 / 15 / 2026, p. 12 / 455 6 / 202 EK, LoBuglio AF, Clin. Cancer Res. (2004) 10 (22), 7555-7565

[015] [Non-Patent Literature 6] Non-fucosylated therapeutic antibodies as next-generation therapeutic antibodies. Satoh M, Iida S, Shitara K., Expert Opin. Biol. Ther. (2006) 6 (11), 1161-1173

[016] [Non-Patent Literature 7] Optimizing engagement of the immune system by anti-tumor antibodies: an engineer's perspective. Desjarlais JR, Lazar GA, Zhukovsky EA, Chu SY., Drug Discov. Today (2007) 12 (21-22), 898-910

[017] [Non-Patent Literature 8] Antibody-drug conjugates: Delivery of targeted cancer drugs. Alley SC, Okeley NM, Senter PD., Curr. Opin. Chem. Biol. (2010) 14 (4), 529-537

[018] [Non-Patent Literature 9] BiTE: Teaching antibodies to engage T cells for cancer therapy. Baeuerle PA, Kufer P, Bargou R., Curr. Opin. Mol. Ther. (2009) 11 (1), 22-30

[019] [Non-Patent Literature 10] EGFR-specific T-cell-involving BiTE antibodies potentially eliminate KRAS- and BRAF colorectal cancer cells. Lutterbuese R, Raum T, Kischel R, Hoffmann P, Mangold S, Rattel B, Friedrich M, Thomas O, Lorenczewski G, Rau D, Schaller E, Herrmann I, Wolf A, Urbig T, Baeuerle PA, Kufer P., Proc. Natl. Acad. Sci. USA (2010) 107 (28), 12605-12610

[020] [Non-Patent Literature 11] Phase I trial with the CD44v6targeting immunoconjugate bivatuzumab mertansine in head and neck squamous cell carcinoma. Riechelmann H, Sauter A, Golze W, Hanft G, Schroen C, Hoermann K, Erhardt T, Gronau S., Oral Oncol. (2008) 44 (9), 823-829

[021] [Non-Patent Literature 12] Ipilimumab in the treatment of melanoma. Trinh VA, Hwu WJ., Expert Opin. Biol. Ther., April 14 (2012) (doi: 10.1517 / 14712598.2012.675325)

[022] [Non-Patent Literature 13] IPILIMUMAB - A NOVEL IMMUNOMODULATING THERAPY CAUSING AUTOIMMUNE Petição 870260070097, de 15 / 07 / 2026, pág. 13 / 455 7 / 202 HYPOPHYSITIS: A CASE REPORT AND REVIEW. Juszczak A, Gupta A, Karavitaki N, Middleton MR, Grossman A., Eur. J. Endocrinol. 10 de Abril (2012) (doi: 10.1530 / EJE-12-0167)

[023] [Literatura Não Patentária 14] The Japanese experience com biologic therapies for rheumatoid arthritis. Takeuchi T, Kameda H., Nat. Rev. Rheumatol. (2010) 6 (11), 644-652

[024] [Literatura Não Patentária 15] Current evidence for the management of rheumatoid arthritis with biological disease-modifying antirheumatic drugs: a systematic literature review informing a EULAR recommendations for the management of RA. Nam JL, Winthrop KL, van Vollenhoven RF, Pavelka K, Valesini G, Hensor EM, Worthy G, Landewe R, Smolen JS, Emery P, Buch MH., Ann. Rheum. Dis. (2010) 69 (6), 976986

[025] [Non-Patent Literature 16] Antibody engineering for the development of therapeutic antibodies. Kim SJ, Park Y, Hong HJ., Mol. Cells. (2005) 20 (1), 17-29

[026] [Non-Patent Literature 17] Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index. Desnoyers LR, Vasiljeva O, Richardson JH, Yang A, Menendez EE, Liang TW, Wong C, Bessette PH, Kamath K, Moore SJ, Sagert JG, Hostetter DR, Han F, Gee J, Flandez J, Markham K, Nguyen M, Krimm M, Wong KR, Liu S, Daugherty PS, West JW, Lowman HB. Sci Transl Med. October 16, 2013; 5(207): 207ra144.

[027] [Non-Patent Literature 18] Body therapy to target antibodies to diseased tissue. Polu KR, Lowman HB. Expert Opin Biol Ther. August 2014; 14(8): 1049-53. SUMMARY OF THE INVENTION Technical Problem to be Solved

[028] The present inventors thought that the dissociation techniques, by protease cleavage, a masking peptide Petition 870260070097, dated 07 / 15 / 2026, page 14 / 455 8 / 202 inhibiting the antigen-binding activity of an antibody, so that the antibody restores its antigen-binding activity, as described above, could cause adverse reactions because the antibody cleaved at a site of injury can distribute to normal tissues via the bloodstream, since protease cleavage is irreversible.

[029] The present invention was made based on such an idea. One objective of the present invention is to provide a pharmaceutical composition useful in the treatment of disease with a reduced adverse reaction, and an active ingredient thereof. Another objective of the present invention is to provide methods for analysis and production of the pharmaceutical composition and the active ingredient. Solution to the Problem

[030] The present inventors have conducted diligent studies and consequently developed a polypeptide comprising an antigen-binding domain and a carrier portion having an inhibitory domain that inhibits the binding activity of the antigen-binding domain, and having a longer half-life than the half-life of the antigen-binding domain existing alone. It is considered that the use of the polypeptide may allow the antigen-binding domain to restore its antigen-binding activity in a diseased tissue and exert antigen-binding activity in the diseased tissue. Furthermore, the systemic distribution of an activated form of the antigen-binding domain may be suppressed due to the difference in half-life between the polypeptide comprising the antigen-binding domain whose antigen-binding activity is inhibited and a polypeptide comprising the antigen-binding domain whose antigen-binding activity is restored.Furthermore, the present inventors have discovered that the polypeptide or a pharmaceutical composition comprising the polypeptide is useful in the treatment of Petition 870260070097, dated 07 / 15 / 2026, page 15 / 455 9 / 202 disease and also discovered that: the polypeptide or pharmaceutical composition is useful in the treatment of disease involving administration of the polypeptide; and the polypeptide is useful in the production of a drug for the treatment of disease. The present inventors have also developed methods for analysis and production of the polypeptide, methods for production and analysis of the single-domain antibody whose antigen-binding activity can be inhibited by association with particular VL, VH or VHH, and a library including a single-domain antibody whose antigen-binding activity can be inhibited by association with particular VL, VH or VHH, completing the present invention.

[031] The present invention is based on these findings and especially encompasses the exemplary embodiments described below.

[032] (1) A polypeptide comprising an antigen-binding domain and a carrier portion, the carrier portion having an inhibitory domain that inhibits the antigen-binding activity of the antigen-binding domain, and the antigen-binding domain having a shorter half-life in blood than that of the carrier portion.

[033] (2) The polypeptide according to (1), wherein the molecular weight of the antigen-binding domain is less than that of the carrier portion.

[034] (3) The polypeptide according to (1) or (2), wherein the molecular weight of the antigen-binding domain is 60 kDa or less.

[035] (4) The polypeptide according to any one of (1) to (3), wherein the carrier portion has FcRn binding activity, and the antigen-binding domain has no FcRn binding activity or has weaker FcRn binding activity than that of the carrier portion.

[036] (5) The polypeptide according to any one of (1) to (4), Petition 870260070097, dated 07 / 15 / 2026, page 16 / 455 10 / 202 in which the antigen-binding domain is able to be released from the polypeptide, and the antigen-binding domain released from the polypeptide has greater antigen-binding activity than before release.

[037] (6) The polypeptide according to any one of (1) to (5), wherein the inhibitory domain of the carrier portion associates with the antigen-binding domain and thereby inhibits the antigen-binding activity of the antigen-binding domain.

[038] (7) The polypeptide according to (5), wherein the polypeptide comprises a cleavage site, wherein the cleavage site is cleaved in such a way that the antigen-binding domain becomes capable of being released from the polypeptide.

[039] (8) The polypeptide according to (6), wherein the polypeptide comprises a cleavage site, wherein the cleavage site is cleaved in such a way that the association of the inhibitory domain of the carrier portion with the antigen-binding domain is cancelled.

[040] (9) The polypeptide according to (7) or (8), wherein the cleavage site comprises a protease cleavage sequence.

[041] (10) The polypeptide according to (9), wherein the protease is a target tissue-specific protease.

[042] (11) The polypeptide according to (10), wherein the target tissue is a cancerous tissue or an inflammatory tissue.

[043] (12) The polypeptide according to (9), wherein the protease is at least one protease selected from matriptase, urokinase (uPA), and metalloproteinase.

[044] (13) The polypeptide according to (12), wherein the protease is at least one protease selected from MT-SP1, uPA, MMP2, MMP9, ADAMTS5, MMP7 and MMP13.

[045] (14) The polypeptide according to (9), wherein a protease cleavage sequence comprises a sequence Petition 870260070097, dated 07 / 15 / 2026, page 17 / 455 11 / 202 selected from SEQ ID Nos: 12, 25, 34, 35, 70 to 73, 75, 76, 91, 178, and 193 to 195.

[046] (15) The polypeptide according to any one of (9) to (14), wherein a first flexible linker is also attached to one end of a protease cleavage sequence.

[047] (16) The polypeptide according to (15), in which a second flexible linker is also attached to the other end of a protease cleavage sequence.

[048] (17) The polypeptide according to (15), wherein the first flexible linker is a flexible linker consisting of a glycine-serine polymer.

[049] (18) The polypeptide according to (16), wherein the second flexible linker is a flexible linker consisting of a glycine-serine polymer.

[050] (19) The polypeptide according to any one of (1) to (18), wherein the antigen-binding domain comprises a single-domain antibody or is a single-domain antibody wherein the inhibitory domain of the carrier portion inhibits the antigen-binding activity of the single-domain antibody.

[051] (20) The polypeptide according to (19), wherein the single domain antibody is VHH, VH having antigen-binding activity on its own, or VL having antigen-binding activity on its own.

[052] (21) The polypeptide according to any one of (1) to (20), wherein the antigen-binding domain comprises a single-domain antibody, and the carrier portion inhibition domain is VHH, VH antibody, or VL antibody, wherein the antigen-binding activity of the single-domain antibody is inhibited by VHH, VH antibody, or VL antibody.

[053] (22) The polypeptide according to any one of (1) to (21), wherein the antigen-binding domain comprises an antibody of Petition 870260070097, dated 07 / 15 / 2026, page 18 / 455 12 / 202 single domain, and the inhibitory domain of the carrier portion is VHH, antibody VH, or antibody VL, wherein the antigen-binding activity of the single domain antibody is inhibited by association with VHH, antibody VH, or antibody VL.

[054] (23) The polypeptide according to any one of (19) to (22), wherein the single domain antibody is VHH or VH having antigen-binding activity by itself, and the inhibiting domain of the carrier portion is VL of antibody, wherein the antigen-binding activity of VHH or VH having antigen-binding activity by itself is inhibited by association with VL of antibody.

[055] (24) The polypeptide according to any one of (19) to (23), wherein the single domain antibody is VHH, wherein VHH has an amino acid substitution at at least one position selected from amino acid positions 37, 44, 45, and 47 (all according to Kabat numbering).

[056] (25) The polypeptide according to any one of (19) to (23), wherein the single domain antibody is VHH, wherein VHH contains at least one amino acid selected from amino acids 37V, 44G, 45L and 47W (all according to Kabat numbering).

[057] (26) The polypeptide according to any one of (19) to (23), wherein the single domain antibody is VHH, wherein VHH contains at least one amino acid substitution selected from amino acid substitutions F37V, Y37V, E44G, Q44G, R45L, H45L, G47W, F47W, L47W, T47W and S47W (all according to Kabat numbering).

[058] (27) The polypeptide according to any one of (19) to (23), wherein the single domain antibody is VHH, wherein VHH has amino acid substitutions at least one selected group of positions 37 / 44, positions 37 / 45, positions 37 / 47, positions 44 / 45, positions 44 / 47, positions 45 / 47, positions 37 / 44 / 45, Petition 870260070097, dated 07 / 15 / 2026, p. 19 / 455 13 / 202 positions 37 / 44 / 47, positions 37 / 45 / 47, positions 44 / 45 / 47, and positions 37 / 44 / 45 / 47 (all according to Kabat numbering).

[059] (28) The polypeptide according to any one of (19) to (23), wherein the single-domain antibody is VHH, wherein the VHH contains at least one amino acid group selected from 37V / 44G, 37V / 45L, 37V / 47W, 44G / 45L, 44G / 47W, 45L / 47W, 37V / 44G / 45L, 37V / 44G / 47W, 37V / 45L / 47W, 44G / 45L / 47W and 37V / 44G / 45L / 47W (all according to Kabat numbering).

[060] (29) The polypeptide according to any one of (19) to (23), wherein the single-domain antibody is VHH, wherein the VHH contains at least one amino acid substitution group selected from F37V / R45L, F37V / G47W, R45L / G47W and F37V / R45L / G47W (all according to Kabat numbering).

[061] (30) The polypeptide according to any one of (19) to (22), wherein the single domain antibody is VL having antigen-binding activity by itself, and the inhibiting domain of the carrier portion is VH antibody, wherein the antigen-binding activity of VL having antigen-binding activity by itself is inhibited by association with VH antibody.

[062] (31) The polypeptide according to any one of (1) to (30), wherein the carrier portion has an FcRn binding region.

[063] (32) The polypeptide according to any one of (1) to (31), wherein the carrier portion comprises a constant region of the antibody.

[064] (33) The polypeptide according to (32), in which the constant region of the antibody carrier portion and the antigen-binding domain are fused by means of a linker or without a linker.

[065] (34) The polypeptide according to (32), wherein the carrier portion comprises an antibody heavy chain constant region, wherein the antibody heavy chain constant region and Petition 870260070097, dated 07 / 15 / 2026, page 20 / 455 14 / 202 The antigen-binding domains are fused with or without a ligand.

[066] (35) The polypeptide according to (32), wherein the carrier portion comprises an antibody light chain constant region, wherein the antibody light chain constant region and the antigen-binding domain are fused by means of a linker or without a linker.

[067] (36) The polypeptide according to (34), wherein in the polypeptide, the N-terminal of the antibody heavy chain constant region of the carrier portion and the C-terminal of the antigen-binding domain are fused by means of a linker or without a linker, and the polypeptide also has a protease cleavage sequence, wherein a protease cleavage sequence is linked within the antigen-binding domain sequence, or next to the antigen-binding domain compared to amino acid position 122 (EU numbering) of the antibody heavy chain constant region.

[068] (37) The polypeptide according to (35), wherein in the polypeptide, the N-terminal of the antibody light chain constant region of the carrier portion and the C-terminal of the antigen-binding domain are fused by means of a linker or without a linker, and the polypeptide also has a protease cleavage sequence, wherein a protease cleavage sequence is linked within the antigen-binding domain sequence, or next to the antigen-binding domain compared to amino acid position 113 (EU numbering) (Kabat numbering position 113) of the antibody light chain constant region.

[069] (38) The polypeptide according to any one of (33) to (35), wherein in the polypeptide, the N-terminal of the antibody constant region of the carrier portion and the C-terminal of the antigen-binding domain are fused by means of a ligand or without a ligand, the Petition 870260070097, dated 07 / 15 / 2026, page 21 / 455 15 / 202 antigen-binding domain is a single-domain antibody prepared from VH, or VHH, and the polypeptide also has a protease cleavage sequence, wherein a protease cleavage sequence is located within the antibody constant region sequence, or next to the antibody constant region compared to amino acid position 109 (Kabat numbering) of the single-domain antibody antigen-binding domain.

[070] (39) The polypeptide according to (33), wherein in the polypeptide, the N-terminal of the constant region of the antibody carrier portion and the C-terminal of the antigen-binding domain are fused by means of a linker or without a linker, and the polypeptide also has a protease cleavage sequence, wherein a protease cleavage sequence is located near the boundary between the antigen-binding domain and the constant region of the antibody.

[071] (40) The polypeptide according to (34), wherein in the polypeptide, the N-terminal of the antibody heavy chain constant region of the carrier portion and the C-terminal of the antigen-binding domain are fused by means of a linker or without a linker, and the polypeptide also has a protease cleavage sequence, wherein a protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody heavy chain constant region.

[072] (41) The polypeptide according to (35), wherein in the polypeptide, the N-terminal of the antibody light chain constant region of the carrier portion and the C-terminal of the antigen-binding domain are fused by means of a linker or without a linker, and the polypeptide also has a protease cleavage sequence, wherein a protease cleavage sequence is located near the boundary between the antigen-binding domain and the constant region of Petition 870260070097, dated 07 / 15 / 2026, page 22 / 455 16 / 202 antibody light chain.

[073] (42) The polypeptide according to (40), wherein the antigen-binding domain is a single-domain antibody prepared from VH, or VHH, and a protease cleavage sequence is located at any position between amino acid position 109 (Kabat numbering) of the single-domain antibody antigen-binding domain and amino acid position 122 (EU numbering) of the antibody heavy chain constant region.

[074] (43) The polypeptide according to (41), wherein the antigen-binding domain is a single-domain antibody prepared from VH, or VHH, and a protease cleavage sequence is located at any position between amino acid position 109 (Kabat numbering) of the single-domain antibody antigen-binding domain and amino acid position 113 (EU numbering) (Kabat numbering position 113) of the antibody light chain constant region.

[075] (44) The polypeptide according to (40), wherein the antigen-binding domain is a single-domain antibody prepared from VL, and a protease cleavage sequence is located at any position between amino acid position 104 (Kabat numbering) of the single-domain antibody antigen-binding domain and amino acid position 122 (EU numbering) of the antibody heavy chain constant region.

[076] (45) The polypeptide according to (41), wherein the antigen-binding domain is a single-domain antibody prepared from VL, and a protease cleavage sequence is located at any position between amino acid position 109 (Kabat numbering) of the single-domain antibody antigen-binding domain and amino acid position 113 (EU numbering) (Kabat numbering position 113) of the antibody light chain constant region. Petition 870260070097, dated 07 / 15 / 2026, page 23 / 455 17 / 202

[077] (46) The polypeptide according to any one of (32) to (45), wherein the antibody constant region of the polypeptide is an IgG antibody constant region.

[078] (47) The polypeptide according to any one of (1) to (46), wherein the polypeptide is a molecule similar to the IgG antibody.

[079] (48) The polypeptide according to any one of (1) to (47), wherein when the antigen-binding domain is assayed in a non-released state by the use of BLI (biolayer interferometry) (Octet), the binding of the antigen-binding domain to the antigen is not seen.

[080] (49) The polypeptide according to any one of (1) to (48), wherein a second antigen-binding domain is also linked to the antigen-binding domain.

[081] (50) The polypeptide according to (49), in which the second antigen-binding domain has different antigen-binding specificity than that of the antigen-binding domain.

[082] (51) The polypeptide according to (49) or (50), wherein the second antigen-binding domain comprises a second single-domain antibody.

[083] (52) The polypeptide according to (51), wherein the antigen-binding domain is a single-domain antibody, the second antigen-binding domain is a second single-domain antibody, and the antigen-binding domain and the second antigen-binding domain are capable of being released from the polypeptide, wherein the single-domain antibody and the second single-domain antibody form a bispecific antigen-binding molecule in states released from the antigen-binding domain and the second antigen-binding domain.

[084] (53) The polypeptide according to any one of (49) to (52), in which the second antigen-binding domain is Petition 870260070097, dated 07 / 15 / 2026, page 24 / 455 18 / 202 targeting HER2 or GPC3 as a target antigen.

[085] (54) The polypeptide according to any one of (1) to (53), wherein the polypeptide also has an additional antigen-binding domain other than the antigen-binding domain, wherein the antigen-binding activity of the additional antigen-binding domain is also inhibited by binding to the carrier portion of the polypeptide.

[086] (55) The polypeptide according to (54), in which the additional antigen-binding domain and the antigen-binding domain differ in antigen-binding specificity.

[087] (56) The polypeptide according to any one of (1) to (55), wherein the antigen-binding domain is an antigen-binding domain directed to plexin A1, IL6R or CD3 as a target antigen.

[088] (57) A pharmaceutical composition comprising the polypeptide of any one of (1) to (56).

[089] (58) A method for producing the polypeptide of any of (1) to (56).

[090] (59) The production method according to (58), comprising the following steps:

[091] (a) obtaining a single-domain antibody that binds to a target antigen;

[092] (b) linking the single-domain antibody obtained in step (a) to a carrier moiety such that the antigen-binding activity of the single-domain antibody is inhibited by an inhibitory domain of the carrier moiety, to form a polypeptide precursor; and

[093] (c) introduction of a protease cleavage sequence into the polypeptide precursor.

[094] (60) The production method according to (58), comprising the following steps: Petition 870260070097, dated 07 / 15 / 2026, page 25 / 455 19 / 202

[095] (a) obtaining a single-domain antibody that binds to a target antigen;

[096] (b) linking the single-domain antibody obtained in step (a) to a carrier moiety such that the antigen-binding activity of the single-domain antibody is inhibited by an inhibitory domain of the carrier moiety, to form a polypeptide precursor; and

[097] (c) introduction of a protease cleavage sequence near the boundary between the single-domain antibody and the carrier portion.

[098] (61) The production method according to (58), comprising the following steps:

[099] (a) obtaining a single-domain antibody that binds to a target antigen; and

[0100] (b) linking the single-domain antibody obtained in step (a) to a carrier moiety via a protease cleavage sequence such that the antigen-binding activity of the single-domain antibody is inhibited by an inhibitory domain of the carrier moiety, to form a polypeptide.

[0101] (62) The production method according to any one of (59) to (61), also comprising the following steps:

[0102] (d) confirmation that the binding activity of the single-domain antibody incorporated into the polypeptide or polypeptide precursor against the target antigen is weakened or lost.

[0103] (63) The production method according to any one of (59) to (62), also comprising the following steps:

[0104] (e) release of single-domain antibody by protease cleavage of a protease cleavage sequence and confirmation that the released single-domain antibody binds to the antigen. Petition 870260070097, dated 07 / 15 / 2026, page 26 / 455 20 / 202

[0105] (64) The production method according to (58), in which the polypeptide is a molecule similar to the IgG antibody.

[0106] (65) The production method according to (64), comprising the following steps:

[0107] (a) obtaining a single-domain antibody that binds to a target antigen;

[0108] (b) association of the single-domain antibody obtained in step (a) as a VH substitute for an IgG antibody with VL, or association of the single-domain antibody as a VL substitute for an IgG antibody with VH such that the antigen-binding activity of the single-domain antibody is inhibited, to form a molecule similar to the IgG precursor antibody hosting the single-domain antibody; and

[0109] (c) introduction of a protease cleavage sequence into the precursor molecule similar to the IgG antibody housing the single-domain antibody.

[0110] (66) The production method according to (64), comprising the following steps:

[0111] (a) obtaining a single-domain antibody that binds to a target antigen;

[0112] (b) association of the single-domain antibody obtained in step (a) as a VH substitute for an IgG antibody with VL, or association of the single-domain antibody as a VL substitute for an IgG antibody with VH such that the antigen-binding activity of the single-domain antibody is inhibited, to form a molecule similar to the IgG precursor antibody hosting the single-domain antibody; and

[0113] (c) introduction of a protease cleavage sequence near the boundary between another single-domain antibody and a single-domain antibody and a constant region of the antibody in Petition 870260070097, dated 07 / 15 / 2026, page 27 / 455 21 / 202 precursor molecule similar to IgG antibody.

[0114] (67) The production method according to (64), comprising the following steps:

[0115] (a) obtaining a single-domain antibody that binds to a target antigen; and

[0116] (b) linking the single-domain antibody obtained in step (a) as a surrogate for IgG VH or VL antibody to a constant light chain region or constant heavy chain region of IgG antibody by means of a protease cleavage sequence such that the antigen-binding activity of the single-domain antibody is inhibited, to form a molecule similar to the IgG antibody harboring the single-domain antibody.

[0117] (68) The production method according to any one of (65) to (67), also comprising the following steps:

[0118] (d) confirmation that the single-domain antibody binding activity lodged in the IgG antibody-like molecule or in the IgG antibody-like molecule precursor against the target antigen is weakened or lost.

[0119] (69) The production method according to any one of (65) to (68), also comprising the following steps:

[0120] (e) release of single-domain antibody by protease cleavage of a protease cleavage sequence and confirmation that the released single-domain antibody binds to the target antigen.

[0121] (70) The production method according to (64), comprising the following steps:

[0122] (a) substitution of an amino acid residue in a single-domain antibody that involves the association of the single-domain antibody without the single-domain antibody with VH of antibody, or substitution of an amino acid residue in a Petition 870260070097, dated 07 / 15 / 2026, page 28 / 455 22 / 202 single-domain antibody that involves the association of the single-domain antibody without the single-domain antibody with VL antibody, to prepare a single-domain antibody variant while maintaining the binding activity of the single-domain antibody against the target antigen;

[0123] (b) association of the single-domain antibody variant prepared in step (a) with antibody VL, or association of the single-domain antibody variant with antibody VH such that the antigen-binding activity of the single-domain antibody variant is inhibited, to form a molecule similar to the IgG precursor antibody harboring the single-domain antibody variant; and

[0124] (c) introduction of a protease cleavage sequence into the precursor molecule similar to the IgG antibody harboring the single-domain antibody variant.

[0125] (71) The production method according to (64), comprising the following steps:

[0126] (a) replacement of an amino acid residue in a single-domain antibody that engages in association with antibody VH, or replacement of an amino acid residue in a single-domain antibody that engages in association with antibody VL, to prepare a single-domain antibody variant while maintaining the binding activity of the single-domain antibody against the target antigen;

[0127] (b) association of the single-domain antibody variant prepared in step (a) with antibody VL, or association of the single-domain antibody variant with antibody VH such that the antigen-binding activity of the single-domain antibody variant is inhibited, to form a molecule similar to the IgG precursor antibody harboring the single-domain antibody variant; Petition 870260070097, dated 07 / 15 / 2026, page 29 / 455 23 / 202 and

[0128] (c) introduction of a protease cleavage sequence near the boundary between the single-domain antibody variant and a constant region in the IgG antibody-like precursor molecule.

[0129] (72) The production method according to (64), comprising the following steps:

[0130] (a) substitution of an amino acid residue in a single-domain antibody that engages in association with antibody VH, or substitution of an amino acid residue in a single-domain antibody that engages in association with antibody VL, to prepare a single-domain antibody variant while maintaining the binding activity of the single-domain antibody against the target antigen; and

[0131] (b) linking the single-domain antibody variant prepared in step (a) to a constant region of IgG antibody heavy chain by means of a protease cleavage sequence, or linking the single-domain antibody variant to a constant region of IgG antibody light chain by means of a protease cleavage sequence such that the antigen-binding activity of the single-domain antibody variant is inhibited, to form an IgG antibody-like molecule harboring the single-domain antibody variant.

[0132] (73) The production method according to any one of (70) to (72), also comprising the following steps:

[0133] (d) confirmation that the binding activity of the single-domain antibody variant hosted in the IgG antibody-like molecule or the binding activity of the single-domain antibody variant hosted in the IgG antibody-like molecule precursor against the target antigen is weakened or lost. Petition 870260070097, dated 07 / 15 / 2026, page 30 / 455 24 / 202

[0134] (74) The production method according to any one of (70) to (73), also comprising the following steps:

[0135] (e) release of the single-domain antibody variant by cleavage of a protease cleavage sequence with the protease and confirmation that the released single-domain antibody variant binds to the target antigen.

[0136] (75) A polynucleotide encoding the polypeptide according to any one of (1) to (56).

[0137] (76) A vector comprising the polynucleotide according to (75).

[0138] (77) A host cell comprising the polynucleotide according to (75) or the vector according to (76).

[0139] (78) A method for the production of the polypeptide according to any one of (1) to (56), comprising the step of culturing the host cell according to (77).

[0140] (79) A method for the analysis of a single domain antibody whose antigen-binding activity can be inhibited by association with a particular VL, association with a particular VH, or association with a particular VHH.

[0141] (80) The method of analysis according to (79), wherein the method is a method for the analysis of a single domain antibody whose antigen-binding activity can be inhibited by association with a particular VL.

[0142] (81) The method of analysis according to (80), comprising the following steps:

[0143] (a) obtaining a single-domain antibody having target antigen binding activity;

[0144] (b) association of the single-domain antibody obtained in step (a) with the particular VL; and

[0145] (c) confirmation that the antibody binding activity Petition 870260070097, dated 07 / 15 / 2026, page 31 / 455 25 / 202 single domain associated with the particular VL in step (b) against the antigen is weakened or lost as compared to that before association.

[0146] (82) The method of analysis according to (80), comprising the following steps:

[0147] (a) association of a single-domain antibody with a particular VL;

[0148] (b) selection of a VL and single-domain antibody combination based on that of the single-domain antibody associated with the particular VL in step (a) which has no binding activity or binding activity of a predetermined value or lower against the antigen; and

[0149] (c) confirmation that the single-domain antibody in the associate selected in step (b) has stronger binding activity against the antigen in a state not associated with the particular VL than that in a state associated with the same.

[0150] (83) The method of analysis according to (79), wherein the method is a method for the analysis of a single domain antibody whose antigen-binding activity can be inhibited by association with a particular HV.

[0151] (84) The method of analysis according to (83), comprising the following steps:

[0152] (a) obtaining a single-domain antibody having target antigen binding activity;

[0153] (b) association of the single-domain antibody obtained in step (a) with the particular HV; and

[0154] (c) confirmation that the binding activity of the single-domain antibody associated with the particular HV in step (b) against the antigen is weakened or lost as compared to that before association. Petition 870260070097, dated 07 / 15 / 2026, page 32 / 455 26 / 202

[0155] (85) The method of analysis according to (83), comprising the following steps:

[0156] (a) association of a single-domain antibody with a particular HV;

[0157] (b) selection of an HV-single-domain antibody association based on the fact that the single-domain antibody associated with the particular HV in step (a) has no binding activity or binding activity of a predetermined value or lower against the antigen; and

[0158] (c) confirmation that the single-domain antibody in the associate selected in step (b) has stronger binding activity against the antigen in a state not associated with the particular HV than that in a state associated with the same.

[0159] (86) The method of analysis according to (79), wherein the method is a method for the analysis of a single domain antibody whose antigen-binding activity can be inhibited by association with a particular VHH.

[0160] (87) The method of analysis according to (86), comprising the following steps:

[0161] (a) obtaining a single-domain antibody having target antigen binding activity;

[0162] (b) association of the single-domain antibody obtained in step (a) with a particular VHH; and

[0163] (c) confirmation that the binding activity of the single-domain antibody associated with the particular HHV in step (b) against the antigen is weakened or lost as compared to that before association.

[0164] (88) The method of analysis according to (86), comprising the following steps:

[0165] (a) association of a single-domain antibody with a Petition 870260070097, dated 07 / 15 / 2026, page 33 / 455 27 / 202 VHH particular;

[0166] (b) selection of a VHH and single-domain antibody combination based on that of the single-domain antibody associated with the particular VHH in step (a) which has no binding activity or binding activity of a predetermined value or lower against the antigen; and

[0167] (c) confirmation that the single-domain antibody in the associate selected in step (b) has stronger binding activity against the antigen in a state not associated with the particular HHV than that in a state associated with the same.

[0168] (89) A method for producing a single-domain antibody whose antigen-binding activity can be inhibited by association with a particular VL, association with a particular VH, or association with a particular VHH.

[0169] (90) The production method according to (89), wherein the method is a method for the production of a single domain antibody whose antigen-binding activity can be inhibited by association with a particular VL.

[0170] (91) The production method according to (90), comprising the following steps:

[0171] (a) substitution of an amino acid residue in a single-domain antibody that involves association with antibody VL, to prepare a single-domain antibody variant while maintaining the binding activity of the single-domain antibody against the target antigen.

[0172] (92) The production method according to (91), also comprising the following steps:

[0173] (b) association of the single-domain antibody variant prepared in step (a) with the VL; and

[0174] (c) confirmation that the antigen-binding activity of Petition 870260070097, dated 07 / 15 / 2026, page 34 / 455 The 28 / 202 single-domain antibody variant associated with VL is weakened or lost compared to that before association.

[0175] (93) The production method according to (89), wherein the method is a method for the production of a single domain antibody whose antigen-binding activity can be inhibited by association with a particular HV.

[0176] (94) The production method according to (93), comprising the following steps:

[0177] (a) substitution of an amino acid residue in a single-domain antibody that engages in association with a molecule similar to the IgG VH antibody, to prepare a single-domain antibody variant while maintaining the binding activity of the single-domain antibody against the target antigen.

[0178] (95) The production method according to (94), also comprising the following steps:

[0179] (b) association of the single-domain antibody variant prepared in step (a) with VH; and

[0180] (c) confirmation that the antigen-binding activity of the HV-associated single-domain antibody variant is weakened or lost as compared to that before association.

[0181] (96) The production method according to (89), wherein the method is a method for the production of a single domain antibody whose antigen-binding activity can be inhibited by association with a particular VHH.

[0182] (97) The production method according to (96), comprising the following steps:

[0183] (a) substitution of an amino acid residue in a single-domain antibody that is involved in association with VHH, Petition 870260070097, dated 07 / 15 / 2026, page 35 / 455 29 / 202 to prepare a single-domain antibody variant while maintaining the binding activity of the single-domain antibody against the target antigen.

[0184] (98) The production method according to (97), also comprising the following steps:

[0185] (b) association of the single-domain antibody variant prepared in step (a) with VHH; and

[0186] (c) confirmation that the antigen-binding activity of the HHV-associated single-domain antibody variant is weakened or lost compared to that before association.

[0187] (99) A library comprising a plurality of single-domain antibody fusion polypeptides each linked to a first supporting domain by association, wherein the single-domain antibodies include a single-domain antibody whose antigen-binding activity may be inhibited or lost by association with a particular VL, a single-domain antibody whose antigen-binding activity may be inhibited or lost by association with a particular VH, or a single-domain antibody whose antigen-binding activity may be inhibited or lost by association with a particular VHH.

[0188] (100) The library according to (99), wherein the single-domain antibody portions of the fusion polypeptides in the library include a single-domain antibody obtained from an animal of the Camelidae family or a transgenic animal harboring a gene capable of increasing single-domain antibody, or a humanized antibody thereof, a single-domain antibody obtained by immunization of an animal of the Camelidae family or a transgenic animal harboring a gene capable of increasing single-domain antibody, or a humanized antibody thereof, or a single-domain antibody Petition 870260070097, dated 07 / 15 / 2026, page 36 / 455 30 / 202 artificially prepared that originates from human VH or VL antibody.

[0189] (101) The library according to (99) or (100) is a library comprising a plurality of single-domain antibody fusion polypeptides each linked to a first supporting domain by association, wherein the single-domain antibodies include a single-domain antibody whose antigen-binding activity may be inhibited or lost by association with a particular VL.

[0190] (102) The library according to (99) or (100) is a library comprising a plurality of single-domain antibody fusion polypeptides each linked to a first supporting domain by association, wherein the single-domain antibodies include a single-domain antibody whose antigen-binding activity may be inhibited or lost by association with particular HV.

[0191] (103) The library according to (99) or (100) is a library comprising a plurality of single-domain antibody fusion polypeptides each linked to a first supporting domain by association, wherein the single-domain antibodies include a single-domain antibody whose antigen-binding activity may be inhibited or lost by association with particular VHH.

[0192] (104) A method for analyzing a library according to (99) or (100) for a fusion polypeptide comprising a single-domain antibody whose antigen-binding activity may be inhibited or could be lost by association with a particular VL, a single-domain antibody whose antigen-binding activity may be inhibited or lost by association with a particular VH, or a single-domain antibody whose antigen-binding activity may Petition 870260070097, dated 07 / 15 / 2026, page 37 / 455 31 / 202 may be inhibited or lost due to association with a particular VHH.

[0193] (105) A method for analyzing a library according to (101) for a fusion polypeptide comprising a single domain antibody whose antigen-binding activity may be inhibited or lost by association with a particular VL.

[0194] (106) The analysis method according to (105), comprising the following steps:

[0195] (a) in vitro display of library fusion polypeptides;

[0196] (b) providing a joint venture partner for a second domain of joint venture with a particular VL;

[0197] (c) association of the fusion polypeptides exhibited in step (a) with the association partner provided in step (b) and selection of a fusion polypeptide that does not bind to the antigen or has antigen-binding activity of a predetermined value or lower in a state where the single-domain antibody associates with the VL; and

[0198] (d) selection of the fusion polypeptides thus selected in step (c) a fusion polypeptide that binds to the antigen or has antigen-binding activity of a predetermined value or higher in a state where the single-domain antibody contained therein does not associate with the VL.

[0199] (107) The analysis method according to (106), wherein the association partner provided in step (b) also comprises a protease cleavage sequence, and step (d) comprises cleaving the association partner by protease treatment so that the association of the single-domain antibody without the single-domain antibody with the VL is cancelled.

[0200] (108) The analysis method according to (107), wherein a protease cleavage sequence of the association partner provided in step (b) is located close to the boundary between the particular VL and the Petition 870260070097, dated 07 / 15 / 2026, page 38 / 455 32 / 202 second domain of support by association.

[0201] (109) The analysis method according to (106), wherein the library fusion polypeptides also comprise a protease cleavage sequence, and step (d) comprises cleaving the fusion polypeptides by protease treatment so that the association of the single-domain antibody without the single-domain antibody with the VL is cancelled.

[0202] (110) The analysis method according to (109), in which a protease cleavage sequence contained in each fusion polypeptide is located close to the boundary between the single-domain antibody and the first association support domain.

[0203] (111) The method of analysis according to (106), wherein step (d) comprises the in vitro display of the complete complements of the fusion polypeptides selected in step (c) or their portions comprising the single domain antibodies.

[0204] (112) The analysis method according to (106), wherein step (d) comprises displaying in vitro the complete complements of the fusion polypeptides selected in step (c) and selecting a fusion polypeptide that binds to the antigen or has antigen-binding activity of a predetermined value or higher in a state associated only with the second association support domain.

[0205] (113) A method for analyzing a library according to (102) for a fusion polypeptide comprising a single domain antibody whose antigen-binding activity may be inhibited or lost by association with particular HV.

[0206] (114) The analysis method according to (113), comprising the following steps:

[0207] (a) in vitro display of library fusion polypeptides;

[0208] (b) provision of a membership partner of a Petition 870260070097, dated 07 / 15 / 2026, page 39 / 455 33 / 202 second support domain by association fused with the particular VH;

[0209] (c) association of the fusion polypeptides exhibited in step (a) with the association partner provided in step (b) and selection of a fusion polypeptide that does not bind to the antigen or has antigen-binding activity of a predetermined value or lower in a state where the single-domain antibody associates with HV; and

[0210] (d) selection of the fusion polypeptides thus selected in step (c) a fusion polypeptide that binds to the antigen or has antigen-binding activity of a predetermined value or higher in a state where the single-domain antibody contained therein does not associate with HV.

[0211] (115) The analysis method according to (114), wherein the association partner provided in step (b) also comprises a protease cleavage sequence, and step (d) comprises cleaving the association partner by protease treatment so that the association of the single-domain antibody without the single-domain antibody with VH is cancelled.

[0212] (116) The analysis method according to (115), wherein a protease cleavage sequence of the association partner provided in step (b) is located close to the boundary between the particular VH and the second association support domain.

[0213] (117) The analysis method according to (114), wherein the library fusion polypeptides also comprise a protease cleavage sequence, and step (d) comprises cleaving the fusion polypeptides by protease treatment so that the association of the single-domain antibody without the single-domain antibody with VH is cancelled.

[0214] (118) The analysis method according to (117), in which a protease cleavage sequence contained in each polypeptide of Petition 870260070097, dated 07 / 15 / 2026, page 40 / 455 34 / 202 fusion is located near the boundary between the single-domain antibody and the first supporting domain by association.

[0215] (119) The analytical method according to (114), wherein step (d) comprises the in vitro display of the complete complements of the fusion polypeptides selected in step (c) or their portions comprising the single domain antibodies.

[0216] (120) The analysis method according to (114), wherein step (d) comprises displaying in vitro the complete complements of the fusion polypeptides selected in step (c) and selecting a fusion polypeptide that binds to the antigen or has antigen-binding activity of a predetermined value or higher in a state associated only with the second association support domain.

[0217] (121) A method for analyzing a library according to (103) for a fusion polypeptide comprising a single domain antibody whose antigen-binding activity may be inhibited or could be lost by association with particular VHH.

[0218] (122) The method of analysis according to (121), comprising the following steps:

[0219] (a) in vitro display of library fusion polypeptides;

[0220] (b) provision of a joint venture partner for a second joint venture support domain with the particular VHH;

[0221] (c) association of the fusion polypeptides shown in step (a) with the association partner provided in step (b) and selection of a fusion polypeptide that does not bind to the antigen or has antigen-binding activity of a predetermined value or lower in a state where the single-domain antibody associates with the particular HHV; and

[0222] (d) selection of fusion polypeptides, in this way Petition 870260070097, dated 07 / 15 / 2026, page 41 / 455 35 / 202 selected in step (c), a fusion polypeptide that binds to the antigen or has antigen-binding activity of a predetermined value or higher in a state where the single-domain antibody contained therein does not associate with HHV.

[0223] (123) The method of analysis according to (122), wherein the association partner provided in step (b) also comprises a protease cleavage sequence, and step (d) comprises cleavage of the association partner by protease treatment so that the association of the single-domain antibody without the single-domain antibody with VHH is cancelled.

[0224] (124) The analysis method according to (123), wherein a protease cleavage sequence of the association partner provided in step (b) is located close to the boundary between the particular VHH and the second association support domain.

[0225] (125) The analysis method according to (122), wherein the library fusion polypeptides also comprise a protease cleavage sequence, and step (d) comprises cleaving the fusion polypeptides by protease treatment so that the association of the single-domain antibody without the single-domain antibody with VHH is cancelled.

[0226] (126) The analysis method according to (125), in which a protease cleavage sequence contained in each fusion polypeptide is located close to the boundary between the single-domain antibody and the first association support domain.

[0227] (127) The method of analysis according to (122), wherein step (d) comprises the in vitro display of the complete complements of the fusion polypeptides selected in step (c) or their portions comprising the single domain antibodies.

[0228] (128) The method of analysis according to (122), wherein step (d) comprises the in vitro display of complete complements Petition 870260070097, dated 07 / 15 / 2026, page 42 / 455 36 / 202 of the fusion polypeptides selected in step (c) and selection of a fusion polypeptide that binds to the antigen or has antigen-binding activity of a predetermined value or higher in a state associated only with the second association support domain.

[0229] (129) The method of analysis according to any one of (106) to (112), (114) to (120), and (122) to (128), wherein the step of providing an association partner in step (b) is the step of displaying the association partner and the fusion polypeptides together.

[0230] (130) The library according to any one of (99) to (103), wherein the first association support domain comprises an IgG antibody CH1 domain or an antibody light chain constant region.

[0231] (131) The method of analysis according to any one of (106) to (112), (114) to (120), and (122) to (128), wherein the first association support domain comprises an IgG antibody CH1 domain, and the second association support domain comprises an antibody light chain constant region.

[0232] (132) The method of analysis according to any one of (106) to (112), (114) to (120), and (122) to (128), wherein the first association support domain comprises a constant region of antibody light chain, and the second association support domain comprises a CH1 domain of IgG antibody.

[0233] (133) The method of analysis according to (105), comprising the following steps:

[0234] (a) in vitro display of library fusion polypeptides;

[0235] (b) providing a joint venture partner for a second joint venture support domain with a particular VL; Petition 870260070097, dated 07 / 15 / 2026, page 43 / 455 37 / 202

[0236] (c) selection of a fusion polypeptide comprising a single-domain antibody that binds to the antigen or has antigen-binding activity of a predetermined or higher value; and

[0237] (d) association of the fusion polypeptides thus selected in step (c) with the association partner provided in step (b) and selection of a fusion polypeptide that does not bind to the antigen or has antigen-binding activity of a predetermined or lower value in a state where the single-domain antibody associates with the VL.

[0238] (134) The method of analysis according to (129), wherein step (d) comprises in vitro display against fusion polypeptides selected in step (c).

[0239] (135) The analysis method according to (133), wherein step (c) comprises the association of the fusion polypeptide only with the second support domain by association or confirmation of the binding to the antigen of the single domain antibody contained in the fusion polypeptide associated only with the second support domain by association.

[0240] (136) The method of analysis according to (113), comprising the following steps:

[0241] (a) in vitro display of library fusion polypeptides;

[0242] (b) provision of a joint venture partner for a second joint venture support domain with the particular VH;

[0243] (c) selection of a fusion polypeptide comprising a single-domain antibody that binds to the antigen or has antigen-binding activity of a predetermined or higher value; and

[0244] (d) association of the fusion polypeptides thus selected in step (c) with the association partner provided in step (b) and selection of a fusion polypeptide that does not bind to Petition 870260070097, dated 07 / 15 / 2026, page 44 / 455 38 / 202 antigen or has antigen-binding activity of a predetermined value or lower in a state where the single-domain antibody associates with HV.

[0245] (137) The method of analysis according to (136), wherein step (d) comprises in vitro display against fusion polypeptides selected in step (c).

[0246] (138) The analysis method according to (136), wherein step (c) comprises the association of the fusion polypeptide only with the second support domain by association or confirmation of antigen binding of the single domain antibody contained in the fusion polypeptide associated only with the second support domain by association.

[0247] (139) The method of analysis according to (121), comprising the following steps:

[0248] (a) in vitro display of library fusion polypeptides;

[0249] (b) provision of a joint venture partner for a second joint venture support domain with the particular VHH;

[0250] (c) selection of a fusion polypeptide comprising a single-domain antibody that binds to the antigen or has antigen-binding activity of a predetermined or higher value; and

[0251] (d) association of the fusion polypeptides thus selected in step (c) with the association partner provided in step (b) and selection of a fusion polypeptide that does not bind to the antigen or has antigen-binding activity of a predetermined or lower value in a state where the single-domain antibody associates with VHH.

[0252] (140) The method of analysis according to (139), wherein step (d) comprises in vitro display against fusion polypeptides selected in step (c). Petition 870260070097, dated 07 / 15 / 2026, page 45 / 455 39 / 202

[0253] (141) The analysis method according to (139), wherein step (c) comprises the association of the fusion polypeptide only with the second support domain by association or confirmation of the binding to the antigen of the single domain antibody contained in the fusion polypeptide associated only with the second support domain by association.

[0254] (142) The method of analysis according to any one of (133) to (141), wherein the step of association of the fusion polypeptides with the association partner in step (d) is the step of displaying the association partner and the fusion polypeptides together.

[0255] (143) The method of analysis according to any one of (133) to (142), wherein the first association support domain comprises an IgG antibody CH1 domain, and the second association support domain comprises an antibody light chain constant region.

[0256] (144) The method of analysis according to any one of (133) to (142), wherein the first association support domain comprises a constant region of antibody light chain, and the second association support domain comprises a CH1 domain of IgG antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0257] Figure 1 is a diagram showing the Probody technology concept. Probody is an antibody molecule whose antigen-binding activity is inhibited by attaching an antibody to a peptide masking the antibody's antigen-binding site by means of a ligand that is cleaved by protease expressed at a site of injury.

[0258] Figure 2 is a diagram showing a cause of adverse reactions that may be exhibited by Probody. Activated Probody accumulated in the blood may exhibit adverse reactions by binding to a Petition 870260070097, dated 07 / 15 / 2026, page 46 / 455 40 / 202 antigen expressed in normal tissue.

[0259] Figure 3 is a diagram showing a cause of adverse reactions that can be exhibited by Probody. Probody is in equilibrium between a state where the masking peptide bound via the ligand is bound to the antigen-binding site and a state where the masking peptide is dissociated. A molecule in the dissociated state can bind to the antigen.

[0260] Figure 4 is a diagram showing a cause of adverse reactions that may be exhibited by Probody. An antidrug antibody against the masking peptide (antimasking peptide antibody) may bind to the Probody masking peptide before activation and thus activate Probody without protease cleavage.

[0261] Figure 5 is a diagram showing the concept of a polypeptide comprising an antigen-binding domain and a carrier portion. (A) The polypeptide with the antigen-binding domain attached to the carrier portion has a long half-life and does not bind to the antigen. (B) The antigen-binding domain is released by, for example, cleavage at a cleavage site to bind to the antigen, and the antigen-binding domain thus released has a short half-life.

[0262] Figure 6 is a diagram showing one embodiment of a method for producing the polypeptide of the present invention. In the present embodiment, the polypeptide of interest is a molecule similar to an IgG antibody. (A) A single-domain antibody that binds to the target antigen is obtained. (B) The single-domain antibody is associated as a VH surrogate of an IgG antibody with VL so that the antigen-binding activity of the single-domain antibody is inhibited. (C) A protease cleavage sequence is introduced into a molecule similar to the IgG precursor antibody housing the single-domain antibody. Petition 870260070097, dated 07 / 15 / 2026, p. 47 / 455 41 / 202

[0263] Figure 7 is a diagram showing one embodiment of the polypeptide of the present invention. In the present embodiment, the polypeptide is a molecule similar to the IgG antibody, and antigen-binding domains are respectively established in portions that correspond to the two variable regions of the IgG antibody. The two antigen-binding domains may have the same antigen-binding specificity or may differ in antigen-binding specificity.

[0264] Figure 8 is a diagram showing an embodiment in which a second antigen-binding domain is also linked to the antigen-binding domain of the present invention. In this embodiment, the antigen-binding domain and the second antigen-binding domain form a bispecific antigen-binding molecule after release. Figure 8(A) is a diagram showing the polypeptide in an unreleased state. The antigen-binding activity of the antigen-binding domain is inhibited. Figure 8(B) is a diagram showing the release of the specific antigen-binding molecule formed by the antigen-binding domain and the second antigen-binding domain. Figure 8(C) is a diagram showing a bispecific antigen-binding molecule against, for example, a T-cell surface antigen and a cancer cell surface antigen, as an example of the bispecific antigen-binding molecule after release.

[0265] Figure 9A is a diagram showing an example of a method for the analysis of a fusion polypeptide comprising a single-domain antibody whose antigen-binding activity may be inhibited or could be lost by association with a particular inhibitory domain, from a library comprising a plurality of single-domain antibody fusion polypeptides each linked to a first supporting domain by association. Petition 870260070097, dated 07 / 15 / 2026, page 48 / 455 42 / 202 Figure 9A (1) is a diagram showing the library comprising a plurality of single-domain antibody fusion polypeptides each linked to a first association support domain. Figure 9A (2) is a diagram showing that the antigen-binding activity of each single-domain antibody is confirmed in a state where the fusion polypeptide associates with an association partner. A fusion polypeptide comprising a single-domain antibody that does not bind to the target antigen or has antigen-binding activity of a predetermined value or lower in this association state is selected. Figure 9A (3) is a diagram showing that the association of the single-domain antibody in the fusion polypeptide selected in (2) with the inhibition domain in the association partner is canceled, and the antigen-binding activity of the single-domain antibody is confirmed.A fusion polypeptide comprising a single-domain antibody that binds to the target antigen or has antigen-binding activity of a predetermined value or higher in this non-association state is selected. Figure 9A (2') is a diagram showing that the antigen-binding activity of the single-domain antibody in each fusion polypeptide is confirmed. A fusion polypeptide comprising a single-domain antibody that binds to the target antigen or has antigen-binding activity of a predetermined value or higher in this state of the fusion polypeptide existing alone is selected. Figure 9A (3') is a diagram showing that the antigen-binding activity of the single-domain antibody is confirmed in a state where the fusion polypeptide selected in (2') associates with an association partner.A fusion polypeptide comprising a single-domain antibody that does not bind to the target antigen or has antigen-binding activity of a predetermined value or lower in this state. Petition 870260070097, dated 07 / 15 / 2026, page 49 / 455 Association 43 / 202 is selected.

[0266] Figure 9B is a diagram showing a more specific example of the method for analyzing a fusion polypeptide comprising a single-domain antibody whose antigen-binding activity may be inhibited or could be lost by association with a particular inhibition domain, from a library comprising a plurality of single-domain antibody fusion polypeptides each linked to a first holding domain by association.(1) Fusion polypeptides, each comprising a single-domain antibody and a first association support domain and an association partner housing a protease cleavage sequence between an inhibition domain and a second association support domain, are displayed together to form a Fab-like structure; (2) among the Fab-like structures thus displayed, a structure that does not bind to the antigen or has antigen-binding activity of a predetermined value or lower is selected; and (3) the association partner is protease-cleaved, and a fragment comprising a single-domain antibody that binds to the antigen or has antigen-binding activity of a predetermined value or higher is selected.

[0267] Figure 9C is a diagram showing another more specific example of the method for analysis for a fusion polypeptide comprising a single-domain antibody whose antigen-binding activity may be inhibited or could be lost by association with a particular inhibition domain, from a library comprising a plurality of single-domain antibody fusion polypeptides each linked to a first support domain by association. (1) The fusion polypeptides, each harboring a protease cleavage sequence between a Petition 870260070097, dated 07 / 15 / 2026, page 50 / 455 44 / 202 a single-domain antibody and a first binding support domain, and a binding partner of an inhibitory domain linked to a second binding support domain are displayed together to form a Fab-like structure; (2) among the Fab-like structures thus displayed, a structure that does not bind to the antigen or has antigen-binding activity of a predetermined value or lower is selected; and (3) the fusion polypeptide is cleaved by protease, and a fragment comprising a single-domain antibody that binds to the antigen or has antigen-binding activity of a predetermined value or higher is selected.

[0268] Figure 9D is a diagram showing an alternative example of the method for analysis for a fusion polypeptide comprising a single-domain antibody whose antigen-binding activity may be inhibited or could be lost by association with a particular inhibitory domain, from a library comprising a plurality of single-domain antibody fusion polypeptides each linked to a first supporting domain by association.(1) Fusion polypeptides, each comprising a single-domain antibody and a first binding support domain and a binding partner of an inhibitory domain linked to a second binding support domain, are displayed together to form a Fab-like structure, and among the Fab-like structures thus displayed, a structure that does not bind to the antigen or has antigen-binding activity of a predetermined value or lower is selected; and (2) portions comprising the single-domain antibodies in the Fab-like structures thus selected in (1) are displayed again so as not to express the inhibitory domain at the same time as the same, and a fragment that binds to the same. Petition 870260070097, dated 07 / 15 / 2026, page 51 / 455 45 / 202 antigen or has antigen-binding activity of a predetermined value or higher is selected. Each of Figures 9D(2') and 9D(2'') is a diagram showing an alternative embodiment, in which the portions comprising the single-domain antibodies in (2) are displayed again so as not to express the inhibitory domain along with them. The order of (1) and (2), (2') or (2'') may be (2), (2') or (2'') preceding (1). Specifically, the portions comprising the single-domain antibodies are displayed so as not to express the inhibitory domain along with them, and a fragment having antigen-binding activity of a predetermined value or higher is selected.Next, fusion polypeptides, each comprising a single-domain antibody comprising the fragment having predetermined or higher binding and a first binding support domain, and a binding partner of an inhibitory domain linked to a second binding support domain, are displayed together to form a Fab-like structure, and among the Fab-like structures thus displayed, a structure that does not bind to the antigen or has antigen-binding activity of a predetermined or lower value is selected.

[0269] Figure 10 is a diagram showing results of IL6R binding assessment of antibody-like molecules prepared by associating multiple light chains with IL6R90-G1m containing VHH of anti-human IL6R (IL6R90) fused with a constant region of human IgG1 (CH1-hinge-CH2-CH3). The onset time of action of antibody-like molecules on antigen-immobilized sensors is a starting point on the abscissa.

[0270] Figure 11(A) is a diagram showing a model of an antibody-like molecule prepared by inserting a protease cleavage sequence near the boundary between VHH and the region Petition 870260070097, dated 07 / 15 / 2026, p. 52 / 455 46 / 202 constant in IL6R90-G1m. Figure 11(B) is a diagram showing the name of each prepared antibody heavy chain, the amino acid sequence insertion site, and the inserted amino acid sequence. The insertion site is indicated by [insertion].

[0271] Figure 12-1 is a diagram showing the results of the cleavage degree assessment by SDS-PAGE reduction after protease (MT-SP1) treatment of IL6R90-G1m or antibody-like molecules prepared by inserting a protease cleavage sequence near the boundary between the VHH and the constant region in IL6R90-G1m. Of the two bands resulting from protease treatment, the band appearing at 25 kDa or less is a VHH-derived band, and the band appearing at a position of 25 to 50 kDa is a constant-region-derived band.

[0272] Figure 12-2 is a continuation diagram of Figure 12-1.

[0273] Figure 13 is a diagram showing the results of evaluating the binding of human IL6R to IL6R90-G1m or antibody-like molecules prepared by inserting a protease cleavage sequence near the boundary between the VHH and the constant region in IL6R90-G1m, or these samples after protease treatment (MT-SP1). Protease- describes sensorgrams evaluating the binding of unprotease-treated antibody-like molecules to the antigen, and Protease+ describes sensorgrams evaluating the binding of protease-treated antibody-like molecules to the antigen. 30 seconds before the onset of action of antibody-like molecules on antigen-immobilized sensors is a starting point on the abscissa.

[0274] Figure 14 is a diagram showing results of IL6R binding assessment of antibody-like molecules prepared by associating multiple light chains with 20A11-G1m containing VHH of anti-human IL6R (20A11) fused with a region Petition 870260070097, dated 07 / 15 / 2026, p. 53 / 455 47 / 202 constant of human IgG1 (CH1-hinge-CH2-CH3). 30 seconds before the onset time of action of antibody-like molecules on antigen-immobilized sensors is a starting point on the abscissa.

[0275] Figure 15 is a diagram showing the results of evaluating the binding of human IL6R to 20A11-G1m or antibody-like molecules prepared by introducing mutations in the amino acids present at the interface between 20A11 and VL and associating various light chains with 20A11hu-G1m containing the 20A11hu thus prepared fused with a constant region of human IgG1 (CH1-hinge-CH2-CH3). 60 seconds before the onset time of action of the antibody-like molecules on antigen-immobilized sensors is a starting point on the abscissa.

[0276] Figure 16 is a diagram showing the results of the cleavage degree assessment by SDS-PAGE reduction after protease (MT-SP1) treatment of 20A11-G1m or 4 types of antibody-like molecules prepared by inserting a protease cleavage sequence near the boundary between 20A11hu and the constant region in 20A11hu-G1m. Of the two bands resulting from protease treatment, the band appearing at 25 kDa or less is a VHH-derived band, and the band appearing at a position of 25 to 50 kDa is a constant-region-derived band.

[0277] Figure 17 is a diagram showing the results of evaluating the binding of human IL6R to 20A11-G1m or antibody-like molecules prepared by inserting a protease cleavage sequence near the boundary between VHH and the constant region in 20A11hu-G1m, or these samples after protease treatment (MT-SP1). Protease- describes sensorgrams evaluating the binding of unprotease-treated antibody-like molecules to the antigen, and Protease+ describes sensorgrams evaluating the Petition 870260070097, dated 07 / 15 / 2026, page 54 / 455 48 / 202 binding of protease-treated antibody-like molecules to the antigen. 60 seconds before the onset of antibody action on antigen-immobilized sensors is a starting point on the abscissa. The term "not tested" indicates that the sample was not assayed.

[0278] Figure 18 is a diagram showing the results of the cleavage degree assessment by migration in SDS-PAGE reduction and CBB detection after protease treatment (MT-SP1) of antibody-like molecules that had anti-human CD3 VHH in their variable heavy chain regions and were prepared by inserting a protease cleavage sequence near the boundary between the VHH and the constant heavy chain region. Of the two bands resulting from protease treatment, the band appearing around 10 to 15 kDa is a VHH-derived band, and the band appearing around 37 kDa is a band derived from the constant heavy chain region.

[0279] Figure 19 is a diagram showing the results of the evaluation of human CD3ed-Fc binding of samples after protease treatment (MT-SP1) of antibody-like molecules that had anti-human CD3 VHH in their variable heavy chain regions and were prepared by inserting a protease cleavage sequence near the boundary between the VHH and the constant heavy chain region. Protease- describes sensorgrams evaluating the binding of unprotease-treated antibody-like molecules to the antigen, and Protease+ describes sensorgrams evaluating the binding of protease-treated antibody-like molecules to the antigen. 30 seconds before the onset of action of the antibody-like molecules on antigen-immobilized sensors is a starting point on the abscissa. Binding is shown when a response before antigen binding was defined as 0 Petition 870260070097, dated 07 / 15 / 2026, page 55 / 455 49 / 202 and a response time before antibody action was defined as 100. The time starting 30 seconds before antibody action is shown.

[0280] Figure 20 is a diagram showing the results of the assessment of the degree of cleavage by migration in SDS-PAGE reduction and detection with CBB after protease treatment (MT-SP1) of a molecule having IL6R90-G1m as a heavy chain and Vk1-39-k0MT as a light chain, or antibody-like molecules prepared by inserting a protease cleavage sequence near the boundary between the variable region of the light chain and the constant region of the light chain of the molecule having IL6R90-G1m as a heavy chain and Vk1-39-k0MT as a light chain. Two bands derived from the light chain resulting from protease treatment, and the light chain was cleaved by protease.

[0281] Figure 21 is a diagram showing the results of evaluating the binding of human IL6R to samples after protease treatment (MT-SP1) of a molecule having IL6R90-G1m as a heavy chain and Vk1-39-k0MT as a light chain, or antibody-like molecules prepared by inserting a protease cleavage sequence near the boundary between the variable light chain region and the constant light chain region of the molecule having IL6R90G1m as a heavy chain and Vk1-39-k0MT as a light chain. Protease- describes sensorgrams evaluating the binding of unprotease-treated antibody-like molecules to the antigen, and Protease+ describes sensorgrams evaluating the binding of protease-treated antibody-like molecules to the antigen. An antibody (MRA) confirmed to bind to IL6R was used as a positive control. The onset time of action of antibody-like molecules on antigen-immobilized sensors is a starting point on the abscissa. Petition 870260070097, dated 07 / 15 / 2026, page 56 / 455 50 / 202

[0282] Figure 22 is a diagram showing SDS-PAGE results of protease cleavage evaluation of IgG antibody-like molecules with VHH binding incorporated into human plexin A1. The protease(+) band describes samples treated with protease cleavage, and the protease(-) band describes negative control samples without protease cleavage treatment.

[0283] Figure 23 is a diagram showing Octet sensorgrams evaluating the binding to human plexin A1 of VHH released by protease cleavage of IgG antibody-like molecules with VHH binding incorporated into human plexin A1. Protease+ describes samples treated by protease cleavage, and protease- describes samples without protease cleavage treatment. The concentrations of the IgG antibody-like molecules used are described on the left side of the diagram.

[0284] Figure 24 is a diagram showing the SDSPAGE results of the evaluation of a bispecific VHH-VHH-containing polypeptide protease cleavage.

[0285] Figure 25 is a diagram showing luciferase activity before and after protease cleavage. The broken line depicts samples without protease treatment, and the solid line depicts samples with protease treatment.

[0286] Figure 26 is a diagram showing luciferase activity before and after protease cleavage. The broken line depicts samples without protease treatment, and the solid line depicts samples with protease treatment.

[0287] Figure 27 is a diagram showing the SDSPAGE evaluation of a protease cleavage of an IgG antibody-like molecule containing VHH of anti-human IL6R.

[0288] Figure 28 is a diagram showing the evaluation of a protease cleavage of IgG antibody-like molecules. Petition 870260070097, dated 07 / 15 / 2026, page 57 / 455 51 / 202 housing a protease cleavage sequence in its light chains.

[0289] Figure 29 is a diagram showing the assessment of the degree of activation based on the presence or absence of a protease treatment of IgG-like antibody molecules harboring a protease cleavage sequence in their light chains.

[0290] Figure 30A is a diagram showing the evaluation of a protease cleavage of IgG antibody-like molecules harboring a protease cleavage sequence in their heavy chains.

[0291] Figure 30B is a diagram showing the evaluation of a protease cleavage of IgG antibody-like molecules harboring a protease cleavage sequence in their heavy chains. Protease cleavage was performed using an assay buffer (MMP Activity Assay Kit (Fluorometric - Green) (ab112146), Component C: Assay Buffer). DESCRIPTION OF THE MODALITIES

[0292] The polypeptide according to the present invention generally refers to a peptide having a length on the order of 4 amino acids or longer, and a protein. Furthermore, the polypeptide according to the present invention is generally a polypeptide consisting of an artificially designed sequence, but is not limited to the same. For example, an organism-derived polypeptide may be used. Alternatively, the polypeptide according to the present invention may be any of a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, and the like. In addition, fragments of these polypeptides are also included in the polypeptide of the present invention.

[0293] In the present application, each amino acid is indicated by a one-letter code or a three-letter code, or both, as represented, Petition 870260070097, dated 07 / 15 / 2026, page 58 / 455 52 / 202, for example, by Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, or Val / V. For the expression of an amino acid located at a particular position, an expression using a number representing the particular position in combination with the one-letter code or the three-letter code of the amino acid may be appropriately used. For example, an amino acid 37V, which is an amino acid contained in a single-domain antibody, represents Val located at position 37 as defined by Kabat numbering.

[0294] For the alteration of an amino acid in the amino acid sequence of a polypeptide, a method known in the art such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) or overlap extension PCR may be appropriately adopted. A plurality of methods known in the art may also be adopted as alteration methods for the substitution of an amino acid by an amino acid other than a natural amino acid (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system containing tRNA (Clover Direct (Protein Express)) having an unnatural amino acid linked with amber suppressor tRNA complementary to the UAG codon (amber codon), which is a stop codon, is preferably also used. In the present application, examples of the alteration include, but are not limited to, substitution.

[0295] In the present application, the term and / or used to represent amino acid modification sites is intended to include each combination appropriately represented by ee or. Specifically, for example, the phrase amino acids at positions 37, 45, and / or 47 are substituted includes the following amino acid modification variations:

[0296] (a) position 37, (b) position 45, (c) position 47, (d) positions 37 and Petition 870260070097, dated 07 / 15 / 2026, p. 59 / 455 53 / 202 45, (e) positions 37 and 47, (f) positions 45 and 47, and (g) positions 37, 45 and 47.

[0297] In the present application, the expression in which one-letter or three-letter codes of amino acids before and after the change are used before and after a number representing a particular position may be appropriately used for representing an amino acid change. For example, a change of F37V or Phe37Val used for the substitution of an amino acid contained in a variable region of an antibody or a single-domain antibody represents the substitution of Phe at position 37 defined by Kabat numbering by Val. Specifically, the number represents an amino acid position defined by Kabat numbering; the one-letter or three-letter code of the amino acid preceding the number represents the amino acid before the substitution; and the one-letter or three-letter code of the amino acid following the number represents the amino acid after the substitution.Similarly, a P238A or Pro238Ala alteration used for amino acid substitution in an Fc region contained within a constant region of an antibody represents the substitution of Pro at position 238 defined by EU numbering by Ala. Specifically, the number represents an amino acid position defined by EU numbering; the one-letter or three-letter code of the amino acid preceding the number represents the amino acid before the substitution; and the one-letter or three-letter code of the amino acid following the number represents the amino acid after the substitution.

[0298] In the present application, the term antibody is used in the broadest sense and encompasses various antibody structures including, but not limited to, a monoclonal antibody, a polyclonal antibody, a multispecific antibody (e.g., a bispecific antibody), a single-domain antibody, and an antibody fragment provided Petition 870260070097, dated 07 / 15 / 2026, p. 60 / 455 54 / 202 that the antibody exhibits the desired antigen-binding activity.

[0299] An antibody fragment refers to a molecule, other than a complete antibody, containing a portion of the complete antibody and binding to an antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diacorpo, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0300] The terms natural-size antibody, complete antibody, and whole antibody are used interchangeably in the present application and refer to an antibody having a structure substantially similar to a natural antibody structure, or having heavy chains containing an Fc region defined in the present application.

[0301] The term variable region or variable domain refers to a region or domain of an antibody heavy chain or light chain involved in the binding of the antibody to its antigen. Generally, antibody heavy chain and light chain variable domains (VH and VL, respectively) are structurally similar and each contains 4 conserved structural regions (FRs) and 3 complementary determinant regions (CDRs) (see, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0302] The term region determining complementarity or The CDR used in this application is hypervariable in sequence, and / or forms a structurally determined loop (hypervariable loop), and / or refers to antigen contact residues (antigen contacts) or each region of a compound's variable domain. Generally, an antibody contains 6 CDRs: three in VH (H1, H2, and H3), and three in VL (L1, L2, L3). Petition 870260070097, dated 07 / 15 / 2026, p. 61 / 455 55 / 202 L2 and L3). In this application, exemplary CDRs include the following:

[0303] (a) hypervariable loops formed at amino acid residues 26 to 32 (L1), 50 to 52 (L2), 91 to 96 (L3), 26 to 32 (H1), 53 to 55 (H2), and 96 to 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987));

[0304] (b) CDRs formed at amino acid residues 24 to 34 (L1), to 56 (L2), 89 to 97 (L3), 31 to 35b (H1), 50 to 65 (H2), and 95 to 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0305] (c) antigen contacts formed at amino acid residues 27c to 36 (L1), 46 to 55 (L2), 89 to 96 (L3), 30 to 35b (H1), 47 to 58 (H2), and 93 to 101 (H3) (MacCallum et al., J. Mol. Biol. 262: 732-745 (1996)); and

[0306] (d) a combination of (a), (b), and / or (c) containing HVR residues of amino acids 46 to 56 (L2), 47 to 56 (L2), 48 to 56 (L2), 49 to 56 (L2), 26 to 35 (H1), 26 to 35b (H1), 49 to 65 (H2), 93 to 102 (H3), and 94 to 102 (H3).

[0307] In the present application, CDR residues and other residues (e.g., FR residues) in a variable domain are numbered according to Kabat et al. (above), unless otherwise specified.

[0308] The term structure or FR refers to variable domain residues other than complementarity-determining region (CDR) residues. FRs in a variable domain consist of 4 FR domains: FR1, FR2, FR3, and FR4. Consequently, CDR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0309] In the present application, the term constant region or constant domain refers to a region or domain other than the regions Petition 870260070097, dated 07 / 15 / 2026, page 62 / 455 56 / 202 variables in an antibody. For example, an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 Da consisting of two identical light chains and two identical heavy chains connected by disulfide bonds. Each heavy chain has a variable region (VH), also called a variable heavy chain domain or variable heavy chain domain, followed by a heavy chain region (CH) containing a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain, from the N-terminal to the C-terminal. Similarly, each light chain has a variable region (VL), also called a variable light chain domain or variable light chain domain, followed by a constant light chain domain (CL), from the N-terminal to the C-terminal. The light chains of natural antibodies can be assigned to one of two types called kappa (κ) and lambda (λ) based on the amino acid sequence of their constant domains.

[0310] In the present application, the term Fc region is used to define the C-terminal region of immunoglobulin heavy chains, including at least a portion of constant regions. This term includes an Fc region having a natural sequence and a mutant Fc region. In one embodiment, the human IgGq heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycinalysine (Gly446-Lys447) of the Fc region may be present or absent. In the present application, amino acid residues in an Fc region or a constant region are numbered according to the EU numbering system (also called 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, unless otherwise specified.

[0311] The class of an antibody refers to the type of a domain Petition 870260070097, dated 07 / 15 / 2026, page 63 / 455 57 / 202 constant or a constant region carried by the antibody heavy chain. Antibodies have 5 main classes: IgA, IgD, IgE, IgG, and IgM. Some of these classes can also be divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Constant heavy chain domains corresponding to immunoglobulins of different classes are called α, δ, ε, γ, and μ, respectively.

[0312] In the present application, the antigen-binding domain is limited only by what binds to the antigen of interest. The antigen-binding domain may be a domain having any structure, provided that the domain used binds to the antigen of interest. Examples of such a domain include, but are not limited to, a variable region of antibody heavy chain (VH), a variable region of antibody light chain (VL), a single-domain antibody (sdAb), a module called a domain of approximately 35 amino acids containing an avimer of cell membrane protein in vivo (International Publications Nos. WO2004 / 044011 and WO2005 / 040229), adnectin containing a 10Fn3 domain that serves as a protein-binding domain derived from a glycoprotein fibronectin expressed in cell membranes (International Publication No.WO2002 / 032925), Affibody containing an IgG-binding domain scaffold constituting a three-helix bundle composed of 58 amino acids of protein A (International Publication No. WO1995 / 001937), DARPins (ankyrin repeat proteins) which are molecular surface-exposed regions of ankyrin (AR) repeats each having a 33-amino acid residue structure folded into a one-turn subunit, two antiparallel helices, and a loop (International Publication No. WO2002 / 020565), anticalin having four loop regions connecting eight antiparallel filaments folded towards the central axis at one end of a highly conserved barrel structure. Petition 870260070097, dated 07 / 15 / 2026, page 64 / 455 58 / 202 lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (International Publication No. WO2003 / 029462), and a depressed region in the inner parallel sheet structure of a horseshoe-shaped fold composed of leucine-rich repeat (LRR) modules of a free-form immunoglobulin (VLR) lymphocyte receptor structure as observed in the acquired immune systems of jawless vertebrates such as lamprey or myxini (International Publication No. WO2008 / 016854).

[0313] Preferred examples of the antigen-binding domain of the present invention include an antigen-binding domain that can perform an antigen-binding function by a molecule consisting only of the antigen-binding domain, and an antigen-binding domain that can perform an antigen-binding function on its own after being released from an additional peptide bound to it. Examples of such an antigen-binding domain include, but are not limited to, a single-domain antibody, scFv, Fv, Fab, Fab', and F(ab')2.

[0314] A preferred example of the antigen-binding domain of the present invention includes an antigen-binding domain having a molecular weight of 60 kDa or less. Examples of such an antigen-binding domain include, but are not limited to, single-domain antibodies, scFv, Fab, and Fab'. The antigen-binding domain having a molecular weight of 60 kDa or less is generally likely to cause renal clearance when existing as a monomer in the blood (see, J Biol Chem. October 15, 1988; 263 (29): 15064-70).

[0315] From another point of view, a preferred example of the antigen-binding domain of the present invention includes an antigen-binding domain having a half-life in the blood of 12 hours or less. Examples of such an antigen-binding domain include, but are not limited to, single-domain antibodies, scFv, Fab, and Fab'. Petition 870260070097, dated 07 / 15 / 2026, page 65 / 455 59 / 202

[0316] A preferred example of the antigen-binding domain of the present invention includes a single-domain antibody (sdAb).

[0317] In the present application, the term single-domain antibody is not limited by its structure as long as the domain can exert antigen-binding activity on its own. It is known that a general antibody, for example, an IgG antibody, exhibits its antigen-binding activity in a state where a variable region is formed by the pairing of VH and VL, whereas the single-domain antibody structure itself can exert antigen-binding activity on its own without pairing with another domain. Generally, the single-domain antibody has a relatively low molecular weight and exists in the form of a monomer.

[0318] Examples of single-domain antibodies include, but are not limited to, congenitally antigen-binding molecules lacking a light chain, such as the VHH of an animal in the Camelidae family and shark Vnar, and antibody fragments containing all or a portion of a VH antibody domain or all or a portion of a VL antibody domain. Examples of single-domain antibodies that are antibody fragments containing all or a portion of a VH or VL antibody domain include, but are not limited to, artificially prepared single-domain antibodies originating from a human VH antibody or human VL antibody as described in U.S. Patent No. 6,248,516 B1, etc. In some embodiments of the present invention, a single-domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0319] Single-domain antibodies can be obtained from an animal capable of producing single-domain antibodies or by immunizing an animal capable of producing single-domain antibodies. Examples of animals capable of producing single-domain antibodies include, but are not limited to, animals of the Camelidae family, Petition 870260070097, dated 07 / 15 / 2026, page 66 / 455 60 / 202 and transgenic animals harboring a gene capable of increasing single-domain antibody. Animals of the Camelidae family include camels, llamas, alpacas, dromedaries, and guanacos, etc. Examples of transgenic animals harboring a gene capable of increasing single-domain antibody include, but are not limited to, transgenic animals described in International Publication No. WO2015 / 143414 and U.S. Patent Publication No. US2011 / 0123527 A1. The structural sequences of the single-domain antibody obtained from the animal can be converted into human germline sequences or sequences similar to these to obtain a humanized single-domain antibody. The humanized single-domain antibody (e.g., humanized VHH) is also an embodiment of the single-domain antibody of the present invention.

[0320] Alternatively, the single-domain antibody can be obtained by ELISA, panning, or similar methods from a polypeptide library containing single-domain antibodies. Examples of polypeptide libraries containing single-domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78); and Biochimica et Biophysica Acta Proteins and Proteomics 2006 1764: 8 (1307-1319)), antibody libraries obtained by immunization of various animals (e.g., Journal of Applied Microbiology 2014 117: 2 (528-536)), and antibody libraries prepared from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21: 1 (35-43); Journal of Biological Chemistry 2016 291:24 (12641-12657); and AIDS 2016 30: 11). (1691-1701)).

[0321] In the present application, the antigen is limited only by containing an epitope to which the antigen-binding domain binds. Petition 870260070097, dated 07 / 15 / 2026, page 67 / 455 61 / 202 Preferred examples of antigens include, but are not limited to, animal- or human-derived peptides, polypeptides, and proteins. Preferred examples of antigens for use in treating a disease caused by a target tissue include, but are not limited to, molecules expressed on the surface of target cells (e.g., cancer cells and inflammatory cells), molecules expressed on the surface of other cells in tissues containing target cells, molecules expressed on the surface of cells having an immunological role against target cells and tissues containing target cells, and large molecules present in the stroma of tissues containing target cells.

[0322] Examples of antigen may include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, adenosine receptor A1, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, adressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK,bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), antigens, Petition 870260070097, dated 07 / 15 / 2026, page 68 / 455 62 / 202 associados com câncer, catepsina A, catepsina B, catepsina C / DPPI, catepsina D, catepsina E, catepsina H, catepsina L, catepsina O, catepsina S, catepsina V, catepsina X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, toxina botulínica, toxina Clostridium perfringens, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF,CTLA-4, PD1, PDL1, LAG3, TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigens, DAN, DCC, DcR3, DC-SIGN, decline-accelerating factor, des(1-3)IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, Eselectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activating protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, Petition 870260070097, dated 07 / 15 / 2026, page 69 / 455 63 / 202 FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha 1, GFR-alpha 2, GFR-alpha 3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone-releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV envelope glycoprotein gB, HCMV envelope glycoprotein gH, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 loop V3, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human heart myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM,I309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, receptor de IgA, IgE, IGF, proteína de ligação ao IGF, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon (INF)alfa, INF-beta, INF-gama, inhibin, iNOS, cadeia de insulina A, cadeia de insulina B, fator de crescimento similar à insulina 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gama, IP-10, I-TAC, JE, calicreína 2, calicreína 5, calicreína 6, calicreína 11, calicreína 12, calicreína 14, calicreína 15, calicreína L1, calicreína L2, calicreína L3, calicreína L4, KC, KDR, queratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, laminin TGF-1 bp1, LBP, LDGF,LECT2, lefty, antígeno de, Petition 870260070097, 15 / 07 / 2026, pág. 70 / 455 64 / 202 Lewis-Y, antigen related to Lewis-Y, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metaloproteinases, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibiting substance, Mug, MuSK, NAIP, NAP, NCAD, NC adherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin chain A, relaxin chain B, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP,STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptor (e.g., T-cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, Pan-specific TGF-beta, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGFbeta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, thymus CK-1, thyroid-stimulating hormone, TIE, TIMP, Petition 870260070097, dated 07 / 15 / 2026, page 71 / 455 65 / 202 TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNFalpha / beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICKB), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFFR), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY CROWN, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60) TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3). Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL ligand Apo-2, TL2), TNFSF11 (TRANCE / RANK ODF ligand, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF LEAD, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (AITR ligand of GITR ligand, TL6), TNFSF1A (TNF-a connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33); TNFSF4 (gp34 of OX40 ligand, TXGP1), TNFSF5 (CD154 of CD40 ligand, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Apo-1 ligand of Fas ligand, APT1 ligand), TNFSF7 (CD70 of CD27 ligand), TNFSF8 (CD153 of CD30 ligand), TNFSF9 (ligand 4-1BB ligand CD137), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (twist-like receptor) 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, Petition 870260070097, of 15 / 07 / 2026, p. 72 / 455 66 / 202 TLR9, TLR10, TSG, TSLP, CA125 tumor-associated antigen, Lewis-Y carbohydrate-expressing tumor-associated antigen, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, PCSK9, prekallikrein, RON, TMEM16F, SOD1, chromogranin A, chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA, S1P, and receptors for growth hormones or growth factors.

[0323] Although the antigen examples listed above also include receptors, these receptors, even existing in a soluble form in a body fluid, can be used as the antigen to which the antigen-binding domain of the present invention binds. A non-limiting example of the soluble form of such a receptor may include the protein represented by SEQ ID NO: 35, which is soluble IL-6R as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968). Petition 870260070097, dated 07 / 15 / 2026, page 73 / 455 67 / 202

[0324] The antigen examples listed above include membrane molecules expressed on cell membranes, and soluble molecules secreted to the exterior of cells. When the antigen-binding domain of the present invention binds to a soluble molecule secreted from cells, the antigen-binding domain preferably has neutralizing activity.

[0325] The solution containing the soluble molecule is not limited, and this soluble molecule may exist in a body fluid, that is, any vascular fluid or any liquid load between tissues or cells in living bodies. In a non-limiting aspect, the soluble molecule to which the antigen-binding domain of the present invention binds may exist in an extracellular fluid. Extracellular fluid refers to a generic name for plasma, intercellular fluid, lymph, tight connective tissues, cerebrospinal fluid, spinal fluid, aspirates, synovial fluid, or such components in bone and cartilage, alveolar fluid (bronchoalveolar lavage), ascitic fluid, pleural effusion, cardiac effusion, cyst fluid, aqueous humor (hydratoid), or such transcellular fluids (various fluids in glandular cavities resulting from the active transport or secretory activity of cells, and fluids in the lumen of the intestine and other body cavities) in vertebrates.

[0326] The epitope, meaning an antigenic determinant, present on the antigen means a site on the antigen to which the antigen-binding domain described in this application binds. Consequently, for example, the epitope can be defined by its structure. Alternatively, the epitope can be defined by the antigen-binding activity of the antigen-binding domain that recognizes the epitope. When the antigen is a peptide or a polypeptide, the epitope can be identified by amino acid residues constituting the epitope. When the epitope is a sugar chain, the epitope can be identified by a chain structure. Petition 870260070097, dated 07 / 15 / 2026, page 74 / 455 68 / 202 private.

[0327] A linear epitope refers to an epitope comprising an epitope that is recognized by its primary amino acid sequence. The linear epitope typically contains at least 3 and more commonly at least 5, for example, approximately 8 to approximately 10 or 6 to 20 amino acids, in its single sequence.

[0328] Unlike the linear epitope, a conformational epitope refers to an epitope that is contained in a primary amino acid sequence containing a component different from the single defined component of the epitope to be recognized (for example, an epitope whose primary amino acid sequence may not be recognized by an antibody that determines the epitope). The conformational epitope may contain an increased number of amino acids, as compared to the linear epitope. As for the recognition of the conformational epitope, the antigen-binding domain recognizes the three-dimensional structure of the peptide or protein.For example, when a protein molecule is folded to form a three-dimensional structure, certain amino acids and / or polypeptide structure constituting the conformational epitope are arranged in parallel to allow the antibody to recognize the epitope. Examples of methods for determining epitope conformation include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and spectral labeling by centrifugation and electron paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris ed.

[0329] The structure of the antigen-binding domain binding to the epitope is called a paratope. The paratope stably binds to the epitope through a hydrogen bond, electrostatic force, van der Waals forces, a hydrophobic bond, or similar forces acting between the Petition 870260070097, dated 07 / 15 / 2026, page 75 / 455 69 / 202 epitope and paratope. This binding strength between the epitope and the paratope is called affinity. The total binding strength when a plurality of antigen-binding domains binds to a plurality of antigens is called activity. Affinity works synergistically when, for example, an antibody comprising a plurality of antigen-binding domains (i.e., a polyvalent antibody) binds to a plurality of epitopes. Therefore, avidity is greater than affinity.

[0330] In a particular embodiment, the antigen-binding domain provided in the present application has a dissociation constant (Kd) of <1 pM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM or <0.001 nM (e.g., 10-8M or less, e.g., 10-8M to 10-13M, e.g., 10-9M to 10-13M).

[0331] Hereafter, an exemplary method for confirming the binding of an antigen-binding domain directed to IL-6R, or a polypeptide comprising the antigen-binding domain to the epitope, will be shown. However, a method for confirming the binding of an antigen-binding domain directed to an antigen other than IL-6R, or a polypeptide comprising the antigen-binding domain to the epitope, can also be appropriately carried out according to the example given below.

[0332] For example, whether the antigen-binding domain directed to IL-6R recognizes a linear epitope present in the IL-6R molecule can be confirmed, for example, as follows: a linear peptide comprising an amino acid sequence constituting the extracellular domain of IL-6R is synthesized for the purpose described above. The peptide can be chemically synthesized.Alternatively, the peptide is obtained by a genetic engineering method using a region encoding an amino acid sequence corresponding to the extracellular domain in IL-6R cDNA. Next, the... Petition 870260070097, dated 07 / 15 / 2026, page 76 / 455 The 70 / 202 antigen-binding domain directed at IL-6R is evaluated for its binding capacity against the linear peptide comprising an amino acid sequence constituting the extracellular domain. For example, the binding activity of the antigen-binding domain against the peptide can be evaluated by ELISA using an immobilized linear peptide as an antigen. Alternatively, the binding activity against the linear peptide can be determined based on the level at which the linear peptide inhibits the binding of the antigen-binding domain to IL-6R-expressing cells. These tests can determine the binding activity of the antigen-binding domain against the linear peptide.

[0333] Furthermore, whether the IL-6R-directed antigen-binding domain recognizes the conformational epitope can be confirmed as follows: IL-6R-expressing cells are prepared for the purpose described above. Recognition of the conformational epitope by the IL-6R-directed antigen-binding domain is confirmed, for example, when the IL-6R-directed antigen-binding domain strongly binds to IL-6R-expressing cells on cell contact, while the antigen-binding domain does not substantially bind to an immobilized linear peptide comprising an amino acid sequence constituting the extracellular domain of IL-6R or a denatured linear peptide (using a general denaturant such as guanidine) comprising an amino acid sequence constituting the extracellular domain of IL-6R.In this context, the term "not substantially bound" means that the binding activity is 80% or less, generally 50% or less, preferably 30% or less, and particularly preferably 15% or less of binding activity against cells expressing human IL-6R.

[0334] The method for confirming the binding activity to Petition 870260070097, dated 07 / 15 / 2026, p. 77 / 455 71 / 202 antigen-binding domain also includes a method for measuring a Kd value, for example, by radiolabeled antigen-binding assay (RIA). In one embodiment, RIA is performed using the antigen-binding domain of interest and its antigen. For example, the binding affinity in a solution of the antigen-binding domain to the antigen is measured by equilibrizing the antigen-binding domain with the lowest concentration of a (125I)-labeled antigen in the presence of a titration series of an unlabeled antigen, and subsequently capturing the bound antigen by a plate coated with the antigen-binding domain (see, for example, Chen et al., J. Mol. Biol. 293: 865-881(1999)).

[0335] According to an alternative embodiment, Kd is measured by a surface plasmon resonance method using BIACORE(R). For example, the assay using BIACORE(R)-2000 or BIACORE(R)-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C using a CM5 chip with approximately 10 response units (RU) of the antigen immobilized on it. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIAcore, Inc.) is activated using N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 pg / mL (approximately 0.2 pM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 pg / min in order to achieve protein binding in approximately 10 response units (RU). After antigen injection, 1 M ethanolamine is injected to block unreacted groups.For kinetic measurement, 2-fold dilutions (0.78 nM to 500 nM) of the antigen-binding domain in PBS containing 0.05% Polysorbate 20 (TWEEN-20™) as a surfactant (PBST) are injected at a flow rate of approximately 25 µl / min at 25°C. An association rate (kon) e. Petition 870260070097, dated 07 / 15 / 2026, page 78 / 455 72 / 202 A dissociation rate (koff) is calculated by fitting the association and dissociation sensorgrams simultaneously using a simple 1:1 Langmuir linkage model (BIACORE® evaluation software version 3.2). An equilibrium dissociation constant (Kd) is calculated as a koff / kon ratio. Additionally, an apparent dissociation constant (Kd) can be determined using equilibrium analysis. For these procedures, see the protocol linked to BIACORE®. See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999) and Methods Enzymol. 2000; 323: 325-40. In the surface plasmon resonance assay, the amount of immobilized protein, the amount of protein used in the reaction, temperature, and solution composition can be variably changed by those skilled in the art.When the on-rate in the surface plasmon resonance assay described above exceeds 106 M-1s-1, an on-rate can be determined by using a fluorescence quenching technique using a spectrometer (e.g., a stopped-flow spectrometer (Aviv Instruments, Inc.) or SLMAMINCO(TM) 8000 series spectrophotometer (Thermo. Spectronic / Thermo Fisher Scientific Inc.) using a stirring cuvette) to measure the increase or decrease in fluorescence intensity (excitation = 295 nm; emission = 340 nm, band size: 16 nm) at 25°C for 20 nM antigen-binding domain in PBS (pH 7.2) in the presence of gradually increasing antigen concentrations.

[0336] In addition, the antigen-binding activity of the antigen-binding domain can also be measured by a known molecule-molecule interaction measurement method such as electrogenerated chemiluminescence.

[0337] Examples of the method for measuring the binding activity of the antigen-binding domain directed to IL-6R against cells Petition 870260070097, dated 07 / 15 / 2026, page 79 / 455 73 / 202 expressing IL-6R include methods described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, binding activity can be assessed based on the principles of ELISA or FACS (fluorescence-activated cell sorting) using cells expressing IL-6R as an antigen.

[0338] In the ELISA format, the binding activity of the antigen-binding domain directed to IL-6R against IL-6R-expressing cells is quantitatively assessed by comparing the signal levels generated through enzymatic reaction. Specifically, a test polypeptide associate is added to an ELISA plate with IL-6R-expressing cells immobilized on it. Then, the test antigen-binding domain bound to the cells is detected using an antibody labeled with an enzyme recognizing the test antigen-binding domain. Alternatively, in FACS, a dilution series of a test antigen-binding domain is prepared, and the antibody binding titer to IL-6R-expressing cells can be determined to compare the binding activity of the test antigen-binding domain against IL-6R-expressing cells.

[0339] The binding of the test antigen-binding domain to the antigen expressed on the surface of cells suspended in a buffer solution or similar can be detected using a flow cytometer. For example, the following devices are known as flow cytometers:

[0340] FACSCanto(TM) II

[0341] FACSAria(TM)

[0342] FACSArray(TM)

[0343] FACSVantage(TM) SE

[0344] FACSCalibur(TM) (all are trade names of BD) (Biosciences) Petition 870260070097, dated 07 / 15 / 2026, page 80 / 455 74 / 202

[0345] EPICS ALTRA HyPerSort

[0346] Cytomics FC 500

[0347] EPICS XL-MCL ADC EPICS XL ADC

[0348] Cell Lab Quanta / Cell Lab Quanta SC (all are trade names of Beckman Coulter, Inc.)

[0349] A preferred example of the method for measuring the antigen-binding activity of the IL-6R-targeted antigen-binding domain includes the following method: first, IL-6R-expressing cells reacted with a test antigen-binding domain are stained with a FITC-labeled secondary antibody recognizing the test antigen-binding domain. The test antigen-binding domain is appropriately diluted with a suitable buffer solution to prepare the antigen-binding domain at the desired concentration for use. The antigen-binding domain can be used, for example, at any concentration from 10 µg / mL to 10 ng / mL. Next, the fluorescence intensity and cell number are measured using FACSCalibur (Becton, Dickinson and Company).The amount of antigen-binding domain bound to cells is reflected in the fluorescence intensity obtained by analysis using the CELL QUEST software (Becton, Dickinson and Company), that is, a geometric mean value. In summary, the binding activity of the test antigen-binding domain, indicated by the amount of the bound test antigen-binding domain, can be determined by obtaining the geometric mean value.

[0350] If the antigen-binding domain targeting IL-6R shares an epitope with a certain antigen-binding domain, this can be confirmed by competition between these antigen-binding domains for the same epitope. Competition between antigen-binding domains is detected by cross-blocking assays or similar methods. The cross-blocking assay is preferably, for example, Petition 870260070097, dated 07 / 15 / 2026, page 81 / 455 75 / 202 competitive ELISA assay.

[0351] Specifically, in the cross-block assay, IL-6R protein-coated cells from a microtiter plate are pre-incubated in the presence or absence of a candidate competitor antigen-binding domain. Then, a test antigen-binding domain is added to this. The amount of test antigen-binding domain bound to the IL-6R protein in the wells indirectly correlates with the binding capacity of the competing competitor antigen-binding domain for binding to the same epitope. In summary, higher affinity of the competitor antigen-binding domain for the same epitope means lower binding activity of the test antigen-binding domain against IL-6R protein-coated wells.

[0352] The amount of antigen-binding domain linked to the test pits via the IL-6R protein can be easily measured by labeling the antigen-binding domain beforehand. For example, a biotin-labeled antigen-binding domain is assayed using an avidin peroxidase conjugate and an appropriate substrate. In particular, the cross-blocking assay using enzyme markers such as peroxidase is called a competitive ELISA assay. The antigen-binding domain can be labeled with an alternative detectable or measurable labeling material. Specifically, radiolabels, fluorescent markers, and the like are known in the art.

[0353] Provided that the competing antigen-binding domain can block the binding of the targeted antigen-binding domain to IL-6R by at least 20%, preferably at least 20 to 50%, more preferably at least 50% as compared to binding activity obtained in a control test performed on Petition 870260070097, dated 07 / 15 / 2026, page 82 / 455 76 / 202 absence of the candidate competing antigen-binding domain associate, the test antigen-binding domain is determined to be an antigen-binding domain that substantially binds to the same epitope as that for the competing antigen-binding domain, or that competes with respect to binding to the same epitope.

[0354] When the epitope to which the IL-6R-targeted antigen-binding domain binds has an identified structure, whether a test antigen-binding domain and a control antigen-binding domain share an epitope can be assessed by comparing the binding activity of these antigen-binding domains against a peptide or polypeptide prepared by introducing an amino acid mutation to a peptide constituting the epitope.

[0355] In such a method for measuring binding activity, for example, the binding activity of a test antigen-binding domain and a control antigen-binding domain against a linear peptide containing an introduced mutation can be compared in the ELISA format described above. In a different ELISA method, the binding activity against the mutated peptide bound to a column can be measured by flowing the test antigen-binding domain and the control antigen-binding domain into the column, and then quantifying the eluted antigen-binding domain in the eluate. A method for adsorbing a mutated peptide, for example, as a GST fusion peptide, to a column is known in the art.

[0356] When the identified epitope is a conformational epitope, whether a test antigen-binding domain and a control antigen-binding domain share an epitope can be assessed by the following method: first, cells expressing IL-6R and cells expressing IL-6R with an introduced mutation in the epitope are prepared. The test antigen-binding domain and the control antigen-binding domain are added to the cell suspensions. Petition 870260070097, dated 07 / 15 / 2026, page 83 / 455 77 / 202 containing these cells suspended in an appropriate buffer solution such as PBS. Subsequently, the cell suspensions are appropriately washed with a buffer solution, and a FITC-labeled antibody capable of recognizing the test antigen-binding domain and the control antigen-binding domain is then added to it. The fluorescence intensity and the number of cells stained with the labeled antibody are measured using FACSCalibur (Becton, Dickinson and Company). The test antigen-binding domain and the control antigen-binding domain are appropriately diluted with a suitable buffer solution and used at concentrations thus adjusted to those desired. These antigen-binding domains are used, for example, at any concentration from 10 µg / mL to 10 ng / mL.The amount of labeled antibody bound to cells is reflected in the fluorescence intensity obtained by analysis using CELL QUEST software (Becton, Dickinson and Company), that is, a geometric mean value. In summary, the binding activity of the test antigen-binding domain and the control antigen-binding domain, indicated by the amount of labeled antibody bound, can be determined by obtaining the geometric mean value.

[0357] Competition of the antigen-binding domain with another antigen-binding domain for the same epitope can also be confirmed by using radiolabeled antigen-binding assay (RIA), BIACORE(R) surface plasmon resonance assay, electrogenerated chemiluminescence, or similar assays, in addition to ELISA or FACS described above.

[0358] In the present method, whether or not substantially binding to cells expressing mutated IL-6R can be determined, for example, by the following method: first, a test antigen-binding domain and a control antigen-binding domain are linked with the Petition 870260070097, dated 07 / 15 / 2026, page 84 / 455 78 / 202 cells expressing mutated IL-6R are stained with a labeled antibody. Subsequently, the fluorescence intensity of the cells is detected. In the case of using FACSCalibur for fluorescence detection by flow cytometry, the obtained fluorescence intensity can be analyzed using CELL QUEST software. From the geometric mean values ​​obtained in the presence and absence of the polypeptide associate, its comparison value (AGeo-Mean) can be calculated according to expression 1 provided below to determine the rate of increase in fluorescence intensity caused by the binding of the antigen-binding domain.

[0359] (Expression 1)

[0360] Average Geometric Comparison Value = Average Geometric Comparison Value (in the presence of the polypeptide associate) / Average Geometric Comparison Value (absence of the polypeptide associate)

[0361] The average geometric comparison value (average Geometric Comparison Value for the mutated IL-6R molecule), thus obtained by analysis, which reflects the amount of the test antigen-binding domain bound to cells expressing mutated IL-6R, is compared with the average Geometric Comparison Value which reflects the amount of the test antigen-binding domain bound to cells expressing IL-6R. In this case, the concentrations of the test antigen-binding domain used to determine the average Geometric Comparison Values ​​for cells expressing mutated IL-6R and cells expressing IL-6R are particularly adjusted preferably to equal or substantially equal concentrations. An antigen-binding domain already confirmed to recognize an epitope on IL-6R is used as the control antigen-binding domain.

[0362] Provided that the mean AGeo comparison value of the test antigen-binding domain for cells expressing mutated IL6R is less than at least 80%, preferably 50%, Petition 870260070097, dated 07 / 15 / 2026, page 85 / 455 79 / 202 more preferably 30%, particularly preferably 15% of the average geometric (GEO) comparison value of the test antigen-binding domain for cells expressing IL-6R, the test antigen-binding domain does not substantially bind to cells expressing mutated IL-6R. The calculation expression for determining the average geometric value is described in the CELL QUEST Software User Guide (BD biosciences). The epitope for the test antigen-binding domain and the control antigen-binding domain can be evaluated as being equal when their comparison values ​​can be considered to be substantially equivalent as a result of comparison.

[0363] In the present application, the term carrier portion refers to a portion other than an antigen-binding domain in a polypeptide. The carrier portion of the present invention is generally a peptide or a polypeptide consisting of amino acids. In a specific embodiment, the carrier portion in the polypeptide is linked to the antigen-binding domain by means of a cleavage site. The carrier portion of the present invention may be a series of peptides or polypeptides connected via an amide linkage, or it may be a complex formed from a plurality of peptides or polypeptides through a covalent linkage such as a disulfide bond or a non-covalent linkage such as a hydrogen bond or hydrophobic interaction.

[0364] The carrier portion of the present invention has an inhibition domain that inhibits the antigen-binding activity of the antigen-binding domain. In the present application, the term inhibition domain is limited only to the inhibition of the antigen-binding activity of the antigen-binding domain. The inhibition domain may be a domain having any structure, provided that the domain used can inhibit the antigen-binding activity of the antigen-binding domain. Petition 870260070097, dated 07 / 15 / 2026, page 86 / 455 80 / 202 antigen. Examples of such an inhibitory domain include, but are not limited to, an antibody heavy chain variable region (HV), an antibody light chain variable region (LV), pre-B cell receptors, and single-domain antibodies. The inhibitory domain may constitute the entire carrier portion or may constitute a portion of the carrier portion.

[0365] In some embodiments of the present invention, the antigen-binding domain released from the polypeptide has higher antigen-binding activity than before release. In other words, the antigen-binding activity of the antigen-binding domain is inhibited by the inhibition domain in a state where the antigen-binding domain is not released from the polypeptide. Whether the antigen-binding activity of the antigen-binding domain is inhibited by the inhibition domain is confirmed by a method such as FACS (fluorescence-activated cell sorting), ELISA (enzyme-linked immunosorbent assay), ECL (electrogenerated chemiluminescence), a SPR (surface plasmon resonance) method (Biacore), BLI (biolayer interferometry) (Octet).In some embodiments of the present invention, the antigen-binding activity of the antigen-binding domain released from the polypeptide is equal to or greater than 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, 2000 times, or 3000 times the binding activity of the antigen-binding domain not released from the polypeptide. In some more specific embodiments of the present invention, the binding of the antigen-binding domain prior to release to the antigen is not observed when the antigen-binding activity of the antigen-binding domain is measured by a method. Petition 870260070097, dated 07 / 15 / 2026, page 87 / 455 81 / 202 selected from among the methods described above.

[0366] In some aspects of the present invention, the cleavage site is cleaved in such a way that the antigen-binding domain becomes capable of being released from the polypeptide. In such aspects, therefore, the antigen-binding activity can be compared between before and after cleavage of the polypeptide. Specifically, the antigen-binding activity measured using the cleaved polypeptide is a value equal to or greater than 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, 2000 times, or 3000 times the antigen-binding activity measured using the uncleaved polypeptide.In some more specific embodiments, the binding of the antigen-binding domain of the uncleaved polypeptide to the antigen is not seen when antigen-binding activity is measured by a method selected from among the methods described above.

[0367] In some aspects of the present invention, the cleavage site is cleaved by protease. In such aspects, therefore, the antigen-binding activity can be compared before and after protease treatment of the polypeptide. Specifically, the antigen-binding activity measured using the polypeptide after protease treatment is a value equal to or greater than 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, 2000 times, or 3000 times the antigen-binding activity measured using the polypeptide without protease treatment. In some more specific embodiments, the binding of the antigen-binding domain to Petition 870260070097, dated 07 / 15 / 2026, page 88 / 455 82 / 202 antigen of the untreated protease polypeptide is not seen when antigen-binding activity is measured by a method selected from those described above.

[0368] In the present invention, the polypeptide comprising an antigen-binding domain and a carrier portion has a longer half-life in the blood than that of the antigen-binding domain existing alone. In some embodiments of the present invention, for the longer half-life of the polypeptide, the carrier portion is designed to have a longer half-life in the blood. In such embodiments, examples of the method of prolonging the half-life in the blood of the carrier portion include, but are not limited to, a higher molecular weight of the carrier portion, FcRn binding activity possessed by the carrier portion, albumin binding activity possessed by the carrier portion, and PEGylation of the carrier portion.In some embodiments of the present invention, the carrier portion has a longer half-life in the blood than that of the antigen-binding domain (in other words, the antigen-binding domain has a shorter half-life in the blood than that of the carrier portion).

[0369] In the present invention, the half-lives of the antigen-binding domain alone and the polypeptide, or the blood half-lives of the antigen-binding domain and the carrier portion are preferably compared in terms of their blood half-lives in humans. If blood half-lives are difficult to measure in humans, human blood half-lives can be predicted based on their blood half-lives in mice (e.g., normal mice, transgenic mice expressing a human antigen, and transgenic mice expressing human FcRn) or monkeys (e.g., cynomolgus monkeys). Petition 870260070097, dated 07 / 15 / 2026, page 89 / 455 83 / 202

[0370] In one embodiment, the method of extending the blood half-life of the carrier portion involves a large molecular weight of the carrier portion. In another embodiment, the method of making the blood half-life of the carrier portion longer than that of the antigen-binding domain involves a higher molecular weight of the carrier portion than that of the antigen-binding domain.

[0371] In one embodiment, the method of extending the half-life in the blood of the carrier portion includes FcRn binding activity possessed by the carrier portion. The carrier portion can generally possess FcRn binding activity by a method of establishing an FcRn binding region in the carrier portion. The FcRn binding region refers to a region having binding activity against FcRn and can have any structure, as long as the region used has binding activity against FcRn.

[0372] The carrier portion containing an FcRn-binding region is capable of being captured in cells and then brought back into the plasma via the salvage function of FcRn. For example, an IgG molecule has a relatively long circulation time in the plasma (slow disappearance) because of FcRn, known as a salvage receptor for IgG molecules. An IgG molecule captured in the endosome via pinocytosis binds to the FcRn expressed in the endosome under acidic intraendosomal conditions. An IgG molecule that does not bind to FcRn is moved to the lysosome and degraded there, while the FcRn-bound IgG molecule is transferred to the cell surface, then dissociated from FcRn under neutral conditions in the plasma, and thus brought back into the plasma.

[0373] The FcRn binding region is preferably a region that binds directly to FcRn. Preferred examples of the FcRn binding region may include antibody Fc regions. However, Petition 870260070097, dated 07 / 15 / 2026, p. 90 / 455 84 / 202 a region capable of binding to a polypeptide, such as albumin or IgG, which has FcRn binding capacity is capable of indirectly binding to FcRn via albumin, IgG or similar substances. Therefore, the FcRn binding region according to the present invention can be a region that binds to such a polypeptide having FcRn binding activity.

[0374] The binding activity of the FcRn binding region according to the present invention against FcRn, particularly human FcRn, can be measured by a method known to those skilled in the art, as mentioned in the section above on binding activity. The conditions for this can be appropriately determined by those skilled in the art. The binding activity against human FcRn can be evaluated as KD (dissociation constant), apparent KD (apparent dissociation constant), kd (dissociation rate), or apparent kd (apparent dissociation rate), etc. These values ​​can be measured by methods known to those skilled in the art. For example, Biacore (GE Healthcare Japan Corp.), Scatchard plot, a flow cytometer, and the like can be used.

[0375] The conditions for measuring the binding activity of the FcRn binding region against FcRn are not particularly limited and can be appropriately selected by those skilled in the art. Binding activity can be measured under conditions involving, for example, a MES buffer and 37°C, as described in WO2009 / 125825. Furthermore, the binding activity of the FcRn binding region of the present invention against FcRn can be measured by a method known to those skilled in the art and can be measured using, for example, Biacore (GE Healthcare Japan Corp.). In measuring the binding activity of the FcRn binding region against FcRn, FcRn and the FcRn binding region or the carrier portion Petition 870260070097, dated 07 / 15 / 2026, page 91 / 455 85 / 202 containing the FcRn binding region can be injected as analytes into chips where the FcRn binding region or the carrier portion containing the FcRn binding region and FcRn, respectively, are immobilized, followed by evaluation.

[0376] Regarding the pH for use in the measurement conditions, the binding affinity of the FcRn-to-FcRn binding region can be evaluated at any pH from 4.0 to 6.5. Preferably, a pH of 5.8 to 6.0, which is close to the pH in the initial endosome in vivo, is used for determining the binding affinity of the FcRn-to-human FcRn binding region. Regarding the temperature for use in the measurement conditions, the binding affinity of the FcRn-to-FcRn binding region can be evaluated at any temperature from 10°C to 50°C. Preferably, a temperature of 15°C to 40°C is used for determining the binding affinity of the FcRn-to-human FcRn binding region. More preferably, any temperature from 20°C to 35°C, for example, any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is also used for determining the binding affinity of the FcRn binding region to FcRn.A temperature of 25°C is a non-limiting example of the temperature range of the present invention.

[0377] An example of an FcRn binding region includes, but is not limited to, an IgG Fc region antibody. In the case of using an IgG Fc region antibody, its type is not limited and, for example, the Fc region of IgG1, IgG2, IgG3, or IgG4 can be used. For example, an Fc region containing a selected sequence of the amino acid sequences represented by SEQ ID NOs: 21, 22, 23, and 24 can be used.

[0378] An Fc region of a natural IgG antibody, as well as a variant of an Fc region having one or more amino acid substitutions, may be used, provided that the Fc region has activity. Petition 870260070097, dated 07 / 15 / 2026, page 92 / 455 86 / 202 connection to FcRn.

[0379] For example, an Fc region variant containing an amino acid sequence derived from an IgG Fc region antibody by replacing at least one amino acid selected from EU numbering positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 can be replaced by another amino acid.

[0380] More specifically, an Fc region variant containing at least one amino acid substitution selected from

[0381] an amino acid substitution to replace Gly at position 237 with Met,

[0382] an amino acid substitution to replace Pro at position 238 with Ala,

[0383] an amino acid substitution to replace Ser at position 239 with Lys,

[0384] an amino acid substitution to replace Lys at position 248 with Ile,

[0385] an amino acid substitution to replace Thr at position 250 with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr,

[0386] an amino acid substitution to replace Met at position 252 with Phe, Trp, or Tyr,

[0387] an amino acid substitution to replace Ser at position 254 with Thr,

[0388] an amino acid substitution to replace Arg at position 255 with Glu,

[0389] an amino acid substitution to replace Thr at position 256 with Asp, Glu, or Gln,

[0390] an amino acid replacement to replace Pro in Petition 870260070097, dated 07 / 15 / 2026, p. 93 / 455 87 / 202 position 257 by Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val,

[0391] an amino acid substitution to replace Glu at position 258 with His,

[0392] an amino acid substitution to replace Asp at position 265 with Ala,

[0393] an amino acid substitution to replace Asp at position 270 with Phe,

[0394] an amino acid substitution to replace Asn at position 286 with Ala or Glu,

[0395] an amino acid substitution to replace Thr at position 289 with His,

[0396] an amino acid substitution to replace Asn at position 297 with Ala,

[0397] an amino acid substitution to replace Ser at position 298 with Gly,

[0398] an amino acid substitution to replace Val at position 303 with Ala,

[0399] an amino acid substitution to replace Val at position 305 with Ala,

[0400] an amino acid substitution to replace Thr at position 307 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr,

[0401] an amino acid substitution to replace Val at position 308 with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr,

[0402] an amino acid substitution to replace Leu or Val at position 309 with Ala, Asp, Glu, Pro, or Arg,

[0403] an amino acid substitution to replace Gln at position 311 with Ala, His, or Ile,

[0404] an amino acid substitution to replace Asp at position 312 with Ala or His, Petition 870260070097, dated 07 / 15 / 2026, p. 94 / 455 88 / 202

[0405] an amino acid substitution to replace Leu at position 314 with Lys or Arg,

[0406] an amino acid substitution at position 315 with Ala or His to replace Asn at

[0407] an amino acid substitution at position 317 with Ala to replace Lys at

[0408] an amino acid substitution at position 325 with Gly to replace Asn at

[0409] an amino acid substitution at position 332 with Val to replace Ile at

[0410] an amino acid substitution at position 334 with Leu to replace Lys at

[0411] an amino acid substitution at position 360 with His to replace Lys at

[0412] an amino acid substitution at position 376 with Ala to replace Asp at

[0413] an amino acid substitution at position 380 with Ala to replace Glu at

[0414] a substitution at position 382 with Ala, amino acid to replace Glu at

[0415] an amino acid substitution to replace Asn or Ser at position 384 with Ala,

[0416] a substitution at position 385 with Asp or His,amino acid to replace Gly at

[0417] a substitution at position 386 for Pro, amino acid to replace Gln at

[0418] a substitution at position 387 for Glu, amino acid to replace Pro at

[0419] a substitution at position 389 for Ala or Ser, amino acid to replace Asn at, Petition 870260070097, dated 07 / 15 / 2026, p. 95 / 455 89 / 202

[0420] an amino acid substitution to replace Ser at position 424 with Ala,

[0421] an amino acid substitution to replace Met at position 428 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr,

[0422] an amino acid substitution to replace His at position 433 with Lys,

[0423] an amino acid substitution to replace Asn at position 434 with Ala, Phe, His, Ser, Trp, or Tyr, and

[0424] an amino acid substitution to replace Tyr or Phe at position 436 with His

[0425] (all according to EU numbering)

[0426] in an Fc region antibody of IgG can be used.

[0427] From another point of view, an Fc region containing at least one amino acid selected from

[0428] Met as the amino acid at position 237,

[0429] Ala as the amino acid at position 238,

[0430] Lys as the amino acid at position 239,

[0431] Ile as the amino acid at position 248,

[0432] Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr as the amino acid at position 250,

[0433] Phe, Trp, or Tyr as the amino acid at position 252,

[0434] Thr as the amino acid at position 254,

[0435] Glu as the amino acid at position 255,

[0436] Asp, Glu, or Gln as the amino acid at position 256,

[0437] Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val as the amino acid at position 257,

[0438] His as the amino acid at position 258,

[0439] Ala as the amino acid at position 265,

[0440] Phe as the amino acid at position 270, Petition 870260070097, dated 07 / 15 / 2026, page 96 / 455 90 / 202

[0441] Ala or Glu as the amino acid at position 286,

[0442] His as the amino acid at position 289,

[0443] Ala as the amino acid at position 297,

[0444] Gly as the amino acid at position 298,

[0445] Ala as the amino acid at position 303,

[0446] Ala as the amino acid at position 305,

[0447] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr as the amino acid at position 307,

[0448] Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr as the amino acid at position 308,

[0449] Ala, Asp, Glu, Pro, or Arg as the amino acid in position 309,

[0450] Ala, His, or Ile as the amino acid at position 311,

[0451] Ala or His as the amino acid at position 312,

[0452] Lys or Arg as the amino acid at position 314,

[0453] Ala or His as the amino acid at position 315,

[0454] Ala as the amino acid at position 317,

[0455] Gly as the amino acid at position 325,

[0456] Val as the amino acid at position 332,

[0457] Read as the amino acid at position 334,

[0458] His as the amino acid at position 360,

[0459] Ala as the amino acid at position 376,

[0460] Ala as the amino acid at position 380,

[0461] Ala as the amino acid at position 382,

[0462] Ala as the amino acid at position 384,

[0463] Asp or His as the amino acid at position 385,

[0464] Pro as the amino acid at position 386,

[0465] Glu as the amino acid at position 387,

[0466] Ala or Ser as the amino acid at position 389,

[0467] Ala as the amino acid at position 424, Petition 870260070097, dated 07 / 15 / 2026, page 97 / 455 91 / 202

[0468] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr as the amino acid at position 428,

[0469] Lys as the amino acid at position 433,

[0470] Ala, Phe, His, Ser, Trp, or Tyr as the amino acid in position 434, and

[0471] His as the amino acid at position 436

[0472] (all according to EU numbering)

[0473] in an Fc region antibody of IgG can be used.

[0474] The FcRn binding activity possessed by the carrier portion does not mean that the antigen-binding domain has no FcRn binding activity. In embodiments where the carrier portion has a longer half-life in blood than that of the antigen-binding domain, the antigen-binding domain may routinely have no FcRn binding activity, or the antigen-binding domain may have FcRn binding activity provided that the FcRn binding activity is weaker than that of the carrier portion.

[0475] In one embodiment, the method for prolonging the blood half-life of the carrier portion involves binding the carrier portion to albumin. Since albumin is not excreted renally and has FcRn binding activity, its blood half-life is as long as 17 to 19 days (J Clin Invest. 1953 Aug; 32 (8): 746-768). Therefore, it has been reported that a protein bound to albumin becomes bulky and able to indirectly bind to FcRn and thus has an increased blood half-life (Antibodies 2015, 4 (3), 141-156).

[0476] In one embodiment, the alternative method for prolonging the blood half-life of the carrier portion involves PEGylation of the carrier portion. PEGylation of a protein is considered to make the protein bulky and also suppress its degradation by protease in the blood, thereby prolonging the blood half-life of the protein. Petition 870260070097, dated 07 / 15 / 2026, p. 98 / 455 92 / 202 (J Pharm Sci. October 2008; 97 (10): 4167-83).

[0477] In some embodiments of the present invention, the carrier portion contains an antibody Fc region. In one specific embodiment, the carrier portion contains a CH2 domain and a CH3 domain of a human IgG antibody. In another specific embodiment, the carrier portion contains a portion extending from the human IgG1 antibody heavy chain Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may be present or absent.

[0478] In some embodiments of the present invention, the carrier portion contains a constant region of the antibody. In a more preferred embodiment, the carrier portion contains a constant region of IgG antibody. In a more preferred embodiment, the carrier portion contains the human constant region of IgG antibody.

[0479] In some embodiments of the present invention, the carrier portion contains: a region substantially similar in structure to a constant region of antibody heavy chain; and a region substantially similar in structure to an antibody light chain, connected to the region by means of a covalent bond such as a disulfide bond or a non-covalent bond such as a hydrogen bond or hydrophobic interaction.

[0480] In the present application, the polypeptide comprising an antigen-binding domain and a carrier portion is generally a series of polypeptides connected via an amide linkage, or a protein containing a plurality of polypeptides connected via an amide linkage.

[0481] In some embodiments of the present invention, the antigen-binding domain is capable of being released from the polypeptide, and the Petition 870260070097, dated 07 / 15 / 2026, page 99 / 455 93 / 202 The antigen-binding domain released from the polypeptide has higher antigen-binding activity. In the present application, the term release refers to the mutual separation of two portions of the polypeptide. The release of the antigen-binding domain from the polypeptide can be attributed to the cancellation of the interaction between the antigen-binding domain and the carrier portion. The antigen-binding activity of the antigen-binding domain incorporated into the polypeptide is inhibited. Therefore, the antigen-binding domain released from the polypeptide can be confirmed by measuring the antigen-binding activity of an individual and comparing it with the antigen-binding activity of the antigen-binding domain incorporated into the polypeptide.

[0482] In some embodiments, the polypeptide comprises a cleavage site, and the cleavage site is cleaved so that the antigen-binding domain is released from the polypeptide. The cleavage site may be cleaved, for example, by an enzyme, may be reduced with a reducing agent, or may be photodegraded. The cleavage site may be placed in any position on the polypeptide provided that the antigen-binding domain can be released and does not lose its antigen-binding activity after release. The polypeptide may also contain an additional cleavage site different from the cleavage site for the release of the antigen-binding domain. In one embodiment of the present invention, the cleavage site comprises a protease cleavage sequence and may be cleaved by protease.

[0483] In the present application, the term cleaved refers to a state where the antigen-binding domain and the carrier portion are separated from each other after alteration of the cleavage site by protease, reduction of a cysteine-cysteine ​​disulfide bond at the cleavage site, and / or photoactivation. In the present application, the term uncleaved refers to a state where the antigen-binding domain is Petition 870260070097, dated 07 / 15 / 2026, page 100 / 455 94 / 202 bound to the carrier portion in the absence of protease cleavage at the cleavage site, in the absence of reduction of a cysteine-cysteine ​​disulfide bond at the cleavage site, and / or in the absence of light.

[0484] Cleavage at the cleavage site can be detected by subjecting a solution containing the cleavage-containing polypeptide to SDS-PAGE (polyacrylamide gel electrophoresis) and measuring the molecular weights of the fragments or detecting a change in molecular weight between before and after cleavage.

[0485] The cleavage site can be specifically modified (cleaved, reduced, or photodegraded) by an agent (i.e., protease, a reducing agent, or light) at a rate of approximately 0.001 to 1500 x 104M-1S-1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 x 104M-1S-1.

[0486] Specific protease cleavage is performed by contact between the protease and the cleavage site or a molecule containing the cleavage site. The cleavage site can be cleaved in the presence of sufficient enzyme activity. Sufficient enzyme activity may refer to the enzyme's ability to perform cleavage in contact with the cleavage site.

[0487] In the present application, the term protease refers to an enzyme such as endopeptidase or exopeptidase that hydrolyzes a peptide bond, typically endopeptidase. The protease used in the present invention is limited only by being able to cleave a protease cleavage sequence and is not particularly limited by its type. In some embodiments, target tissue-specific protease is used. Target tissue-specific protease may refer to, for example, any of the

[0488] (1) protease that is expressed at a higher level in the target tissue than in normal tissues, Petition 870260070097, dated 07 / 15 / 2026, p. 101 / 455 95 / 202

[0489] (2) protease that has greater activity in target tissue than in normal tissues,

[0490] (3) protease that is expressed at a higher level in target cells than in normal cells, and

[0491] (4) protease that has greater activity in target cells than in normal cells.

[0492] In a more specific embodiment, a cancerous tissue-specific protease or an inflammatory tissue-specific protease is used.

[0493] In the present application, the term target tissue means a tissue containing at least one target cell. In some embodiments of the present invention, the target tissue is a cancerous tissue. In some embodiments of the present invention, the target tissue is an inflammatory tissue.

[0494] The term cancerous tissue means a tissue containing at least one cancerous cell. Thus, considering that, for example, cancerous tissue contains cancerous cells and vascular vessels, each cell type that contributes to the formation of a tumor mass containing cancerous cells and endothelial cells is included within the scope of the present invention. In the present application, tumor mass refers to foci of tumor tissue. The term tumor is generally used to mean benign neoplasm or malignant neoplasm.

[0495] In the present application, examples of inflammatory tissue include the following:

[0496] A joint tissue in rheumatoid arthritis or osteoarthritis,

[0497] Lung tissue (alveolus) in bronchial asthma or COPD,

[0498] A digestive organ tissue in inflammatory bowel disease, Crohn's disease, or ulcerative colitis,

[0499] fibrotic tissue in fibrosis in the liver, kidney or lung, Petition 870260070097, dated 07 / 15 / 2026, page 102 / 455 96 / 202

[0500] a tissue undergoing organ transplant rejection,

[0501] a vascular vessel or heart (cardiac muscle) tissue in arteriosclerosis or heart failure,

[0502] visceral adipose tissue in metabolic syndrome,

[0503] a cutaneous tissue in atopic dermatitis and other dermatitides, and

[0504] spinal nerve tissue in a herniated disc or chronic low back pain.

[0505] Protease specifically expressed or specifically activated, or protease considered to be related to the disease condition of a target tissue (target tissue-specific protease) is known for some target tissues. For example, International Publications Nos. WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846 describes a protease specifically expressed in a cancerous tissue. Additionally, J Inflamm (Lond). 2010; 7: 45, Nat Rev Immunol. July 2006; 6(7): 541-50, Nat Rev Drug Discov. December 2014; 13(12): 904-27, Respir Res. March 4, 2016; 17: 23, Dis Model Mech. February 2014; 7(2): 193-203, and Biochim Biophys Acta. January 2012; 1824(1): 133-45 describe the protease considered to be related to inflammation.

[0506] In addition to protease specifically expressed in a target tissue, there is also protease specifically activated in a target tissue. For example, protease can be expressed in an inactive form and then converted into an active form. Many tissues contain a substance that inhibits the active protease and control activity through the activation process and the presence of the inhibitor (Nat Rev Cancer. July 2003; 3(7):489-501). In a target tissue, the active protease can be specifically activated by escaping inhibition.

[0507] Active protease can be measured using a method employing an antibody that recognizes active protease (PNAS, 2 of Petition 870260070097, dated 07 / 15 / 2026, page 103 / 455 97 / 202 January 2013; 110 (1): 93-98) or a fluorescent labeling method of a protease-recognizable peptide such that fluorescence is extinguished before cleavage but emitted after cleavage (Nat Rev Drug Discov. September 2010; 9 (9): 690-701. doi: 10.1038 / nrd3053).

[0508] From the point of view presented, the term target tissue-specific protease can refer to any of

[0509] (i) protease that is expressed at a higher level in the target tissue than in normal tissues,

[0510] (ii) protease that has greater activity in the target tissue than in normal tissues,

[0511] (iii) protease that is expressed at a higher level in target cells than in normal cells, and

[0512] (iv) protease that has greater activity in target cells than in normal cells.

[0513] Specific examples of a protease include, but are not limited to, cysteine ​​protease (including cathepsin B, L, S, etc. families), aspartyl protease (cathepsins D, E, K, O, etc.), serine protease (including matriptase (including MT-SP1), cathepsins A and G, thrombin, plasmin, urokinase (uPA), tissue plasminogen activator (tPA), elastase, proteinase 3, thrombin, kallikrein, tryptase, and chymase), metalloproteinase (metalloproteinase (MMP1-28) including both membrane-bound forms (MMP14-17 and MMP24-25) and secreted forms (MMP1-13, MMP18-23 and MMP26-28), A-dysintegrin and metalloproteinase (ADAM), A-dysintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), meprine (meprine alpha and meprine beta), CD10 (CALLA), prostate-specific antigen (PSA), legumain, TMPRSS3, TMPRSS4, human neutrophil elastase (HNE), beta secretase (BACE), fibroblast-activating protein alpha (FAP), granzyme B, guanidinobenzoate (GB), hepsin, neprilysin, NS3 / 4A, Petition 870260070097, dated 07 / 15 / 2026, page 104 / 455 98 / 202 HCV-NS3 / 4, calpain, ADAMDEC1, renin, cathepsin C, cathepsin V / L2, cathepsin X / Z / P, cruzipain, otubain 2, kallikrein-related peptidases (KLKs (KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14)), bone morphogenetic protein 1 (BMP-1), activated protein C, blood coagulation-related proteases (Factor Vila, Factor IXa, Factor Xa, Factor XIa, and Factor XIa), HtrA1, lactoferrin, marapsin, PACE4, DESC1, dipeptidyl peptidase 4 (DPP-4), TMPRSS2, cathepsin F, cathepsin H, cathepsin L2, cathepsin O, cathepsin S, granzyme A, gepsin calpain 2, glutamate carboxypeptidase 2, AMSH-like proteases, AMSH, gamma secretase, antiplasmin cleavage enzyme (APCE), decisin 1, alpha N-acetylated acid-like dipeptidase 1 (NAALADL1), and furin.

[0514] From another point of view, target tissue-specific protease may refer to a cancerous tissue-specific protease or an inflammatory tissue-specific protease.

[0515] Examples of cancer tissue-specific proteases include proteases specifically expressed in a cancerous tissue described in International Publications Nos. WO2013 / 128194, WO2010 / 081173 and WO2009 / 025846.

[0516] As for the type of cancer tissue-specific protease, a protease having greater expression specificity in the cancer tissue to be treated is more effective in reducing adverse reactions. Preferred cancer tissue-specific protease has a concentration in cancer tissue at least 5 times, more preferably at least 10 times, most preferably at least 100 times, particularly preferably at least 500 times, most preferably at least 1000 times higher than its concentration in normal tissues. In addition, preferred cancer tissue-specific protease has activity in cancer tissue of at least Petition 870260070097, dated 07 / 15 / 2026, page 105 / 455 99 / 202 less 2 times, more preferably at least 3 times, at least 4 times, at least 5 times, or at least 10 times, more preferably at least 100 times, particularly preferably at least 500 times, more preferably at least 1000 times greater than its activity in normal tissues.

[0517] Cancer tissue-specific protease may be in a form bound to a cancer cell membrane or may be in an extracellularly secreted form not bound to a cell membrane. When cancer tissue-specific protease is not bound to a cancer cell membrane, it is preferable for cancer cell-specific immunocytotoxicity that the cancer tissue-specific protease should exist within or near the cancerous tissue. In the present application, proximity to cancer tissue means encompassing the location where a cancer tissue-specific protease cleavage sequence is cleaved, such that the antigen-binding domain exerts antigen-binding activity. However, it is preferable that damage to normal cells be minimized within the scope of its location.

[0518] From an alternative point of view, a cancerous tissue-specific protease is any of

[0519] (i) protease that is expressed at a higher level in cancerous tissue than in normal tissues,

[0520] (ii) protease that has greater activity in cancerous tissue than in normal tissues,

[0521] (iii) protease that is expressed at a higher level in cancer cells than in normal cells, and

[0522] (iv) protease that has greater activity in cancer cells than in normal cells.

[0523] A type of protease specific to cancerous tissue may Petition 870260070097, dated 07 / 15 / 2026, page 106 / 455 100 / 202 may be used alone, or two or more types of cancer-specific tissue protease may be combined. The number of cancer-specific tissue protease types may be appropriately determined by those skilled in the art in consideration of the type of cancer to be treated.

[0524] From these viewpoints, the specific protease for cancerous tissue is preferably serine protease or metalloproteinase, more preferably matriptase (including MT-SP1), urokinase (uPA), or metalloproteinase, also preferably MTSP1, uPA, MMP2, or MMP9, among the proteases listed above.

[0525] As for the type of protease specific to inflammatory tissue, a protease having greater expression specificity in the inflammatory tissue to be treated is more effective in reducing adverse reactions. Preferred inflammatory tissue-specific protease has a concentration in inflammatory tissue at least 5 times, more preferably at least 10 times, more preferably at least 100 times, particularly preferably at least 500 times, most preferably at least 1000 times greater than its concentration in normal tissues. Furthermore, preferred inflammatory tissue-specific protease has activity in inflammatory tissues at least 2 times, more preferably at least 3 times, at least 4 times, at least 5 times, or at least 10 times, still preferably at least 100 times, particularly preferably at least 500 times, most preferably at least 1000 times greater than its activity in normal tissues.

[0526] The specific inflammatory tissue protease may be in a form bound to an inflammatory cell membrane or it may be in an extracellularly secreted form not bound to a cell membrane. When the specific inflammatory tissue protease is not bound to an inflammatory cell membrane, it is preferred Petition 870260070097, dated 07 / 15 / 2026, page 107 / 455 101 / 202 for specific immunocyte-mediated cytotoxicity to inflammatory cells, requiring that the inflammatory tissue-specific protease exist within or near the inflammatory tissue. In the present application, proximity to inflammatory tissue means encompassing the location where a cleavage sequence of inflammatory tissue-specific protease is cleaved such that the antigen-binding domain exerts antigen-binding activity. However, it is preferable that damage to normal cells be minimized within this location scope.

[0527] From an alternative point of view, inflammatory tissue-specific protease is any of

[0528] (i) protease that is expressed at a higher level in inflammatory tissue than in normal tissues,

[0529] (ii) protease that has greater activity in inflammatory tissue than in normal tissues,

[0530] (iii) protease that is expressed at a higher level in inflammatory cells than in normal cells, and

[0531] (iv) protease that has greater activity in inflammatory cells than in normal cells.

[0532] One type of inflammatory tissue-specific protease may be used alone, or two or more types of inflammatory tissue-specific protease may be combined. The number of inflammatory tissue-specific protease types may be appropriately determined by those skilled in the art in consideration of the pathological condition to be treated.

[0533] From these viewpoints, the specific protease for inflammatory tissue is preferably a metalloproteinase among the proteases listed above. The metalloproteinase is most preferably ADAMTS5, MMP2, MMP7, MMP9, or MMP13.

[0534] A protease cleavage sequence is a sequence Petition 870260070097, dated 07 / 15 / 2026, page 108 / 455 102 / 202 of a particular amino acid that is specifically recognized by target tissue-specific protease when the polypeptide is hydrolyzed by the target tissue-specific protease in an aqueous solution.

[0535] A protease cleavage sequence is preferably an amino acid sequence that is hydrolyzed with high specificity by target tissue-specific protease that is most specifically expressed in the target tissue or cells to be treated or most specifically activated in the target tissue / cells to be treated, from the point of view of reducing adverse reactions.

[0536] Specific examples of a protease cleavage sequence include target sequences that are specifically hydrolyzed by the protease listed above specifically expressed in a cancerous tissue described in International Publications Nos. WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846, a protease specific to an inflammatory tissue, and similar ones. An artificially altered sequence, for example, by introducing an appropriate amino acid mutation to a target sequence that is specifically hydrolyzed by a protease known to also be used. Alternatively, a protease cleavage sequence identified by a method known to those skilled in the art as described in Nature Biotechnology 19, 661-667 (2001) may be used.

[0537] In addition, a naturally occurring protease cleavage sequence may be used. For example, TGFp is converted into a latent protease cleavage form. Likewise, a protease cleavage sequence in a protein that changes its molecular shape by protease cleavage may also be used.

[0538] Examples of a protease cleavage sequence that can be used include, but are not limited to, the sequences described in International Publication No. WO2015 / 116933, International Publication No. WO2015 / 048329, International Publication No. Petition 870260070097, dated 07 / 15 / 2026, page 109 / 455 103 / 202 WO2016 / 118629, International Publication No. WO2016 / 179257, International Publication No. WO2016 / 179285, International Publication No. WO2016 / 179335, International Publication No. WO2016 / 179003, International Publication No. WO2016 / 046778, International Publication No. WO2016 / 014974, United States Patent Publication No. US2016 / 0289324, United States Patent Publication No. US2016 / 0311903, PNAS (2000) 97: 7754-7759, Biochemical Journal (2010) 426: 219-228, and Beilstein J Nanotechnol. (2016) 7: 364-373.

[0539] A protease cleavage sequence is most preferably an amino acid sequence that is specifically hydrolyzed by suitable target tissue-specific protease as mentioned above. The amino acid sequence that is specifically hydrolyzed by target tissue-specific protease is preferably a sequence comprising any of the following amino acid sequences:

[0540] LSGRSDNH (SEQ ID NO: 12, cleavable by MT-SP1 or uPA),

[0541] PLALAG (SEQ ID NO: 25, cleavable by MMP2 or MMP9), and

[0542] VPLSLTMG (SEQ ID NO: 26, cleavable by MMP7).

[0543] Any of the following sequences can also be used as a protease cleavage sequence:

[0544] TSTSGRSANPRG (SEQ ID NO: 74, cleavable by MT-SP1 or uPA),

[0545] ISSGLLSGRSDNH (SEQ ID NO: 75, cleavable by MT-SP1 or uPA),

[0546] AVGLLAPPGGLSGRSDNH (SEQ ID NO: 76, cleavable by MT-SP1 or uPA),

[0547] GAGVPMSMRGGAG (SEQ ID NO: 77, cleavable by MMP1),

[0548] GAGIPVSLRSGAG ​​(SEQ ID NO: 78, cleavable by MMP2),

[0549] GPLGIAGQ (SEQ ID NO: 79, cleavable by MMP2), Petition 870260070097, dated 07 / 15 / 2026, page 110 / 455 104 / 202

[0550] GGPLGMLSQS (SEQ ID NO: 80, cleavable by MMP2),

[0551] PLGLWA (SEQ ID NO: 81, cleavable by MMP2),

[0552] GAGRPFSMIMGAG (SEQ ID NO: 82, cleavable by MMP3),

[0553] GAGVPLSLTMGAG (SEQ ID NO: 83, cleavable by MMP7),

[0554] GAGVPLSLYSGAG (SEQ ID NO: 84, cleavable by MMP9),

[0555] AANLRN (SEQ ID NO: 85, cleavable by MMP11),

[0556] AQAYVK (SEQ ID NO: 86, cleavable by MMP11),

[0557] AANYMR (SEQ ID NO: 87, cleavable by MMP11),

[0558] AAALTR (SEQ ID NO: 88, cleavable by MMP11),

[0559] AQNLMR (SEQ ID NO: 89, cleavable by MMP11),

[0560] AANYTK (SEQ ID NO: 90, cleavable by MMP11),

[0561] GAGPQGLAGQRGIVAG (SEQ ID NO: 91, cleavable by MMP13),

[0562] PRFKIIGG (SEQ ID NO: 92, cleavable by pro-urokinase),

[0563] PRFRIIGG (SEQ ID NO: 93, cleavable by pro-urokinase),

[0564] GAGSGRSAG (SEQ ID NO: 94, cleavable by uPA),

[0565] SGRSA (SEQ ID NO: 95, cleavable by uPA),

[0566] GSGRSA (SEQ ID NO: 96, cleavable by uPA),

[0567] SGKSA (SEQ ID NO: 97, cleavable by uPA),

[0568] SGRSS (SEQ ID NO: 98, cleavable by uPA),

[0569] SGRRA (SEQ ID NO: 99, cleavable by uPA),

[0570] SGRNA (SEQ ID NO: 100, cleavable by uPA),

[0571] SGRKA (SEQ ID NO: 101, cleavable by uPA),

[0572] QRGRSA (SEQ ID NO: 102, cleavable by tPA),

[0573] GAGSLLKSRMVPNFNAG (SEQ ID NO: 103, cleavable by cathepsin B)

[0574]

[0575]

[0576]

[0577] TQGAAA (SEQ ID NO: 104, cleavageable by cathepsin B), GAAAAA (SEQ ID NO: 105, cleavageable by cathepsin B), GAGAAG (SEQ ID NO: 106, cleavageable by cathepsin B), AAAAAG (SEQ ID NO: 107, cleavageable by cathepsin B), Petition 870260070097, dated 07 / 15 / 2026, page 111 / 455 105 / 202

[0578] LCGAAI (SEQ ID NO: 108, cleavable by cathepsin B),

[0579] FAQALG (SEQ ID NO: 109, cleavable with cathepsin B),

[0580] LLQANP (SEQ ID NO: 110, cleavable with cathepsin B),

[0581] LAAANP (SEQ ID NO: 111, cleavable by cathepsin B),

[0582] LYGAQF (SEQ ID NO: 112, cleavable with cathepsin B),

[0583] LSQAQG (SEQ ID NO: 113, cleavable with cathepsin B),

[0584] ASAASG (SEQ ID NO: 114, cleavable with cathepsin B),

[0585] FLGASL (SEQ ID NO: 115, cleavable by cathepsin B),

[0586] AYGATG (SEQ ID NO: 116, cleavable by cathepsin B),

[0587] LAQATG (SEQ ID NO: 117, cleavable by cathepsin B),

[0588] GAGSGVVIATVIVITAG (SEQ ID NO: 118, cleavable by cathepsin L),

[0589] APMAEGGG (SEQ ID NO: 119, cleavable by meprine alfa or meprine beta),

[0590] EAQGDKII (SEQ ID NO: 120, cleavable by meprine alfa or meprine beta),

[0591] LAFSDAGP (SEQ ID NO: 121, cleavable by meprine alfa or meprine beta),

[0592] YVADAPK (SEQ ID NO: 122, cleavable by meprine alfa or meprine beta),

[0593] RRRRR (SEQ ID NO: 123, furin cleavable),

[0594] RRRRRR (SEQ ID NO: 124, furin cleavable),

[0595] GQSSRHRRAL (SEQ ID NO: 125, furin cleavable),

[0596] SSRHRRALD (SEQ ID NO: 126),

[0597] RKSSIIIRMRDVVL (SEQ ID NO: 127, cleavable by plasminogen),

[0598] SSSFDKGKYKKGDDA (SEQ ID NO: 128, cleavable by staphylokinase),

[0599] SSSFDKGKYKRGDDA (SEQ ID NO: 129, cleavable by staphylokinase), Petition 870260070097, dated 07 / 15 / 2026, page 112 / 455 106 / 202

[0600] IEGR (SEQ ID NO: 130, cleavable by Factor Xa),

[0601] IDGR (SEQ ID NO: 131, cleavable by Factor Xa),

[0602] GGSIDGR (SEQ ID NO: 132, cleavable by Factor Xa),

[0603] GPQGIAGQ (SEQ ID NO: 133, cleavable by collagenase),

[0604] GPQGLLGA (SEQ ID NO: 134, cleavable by collagenase),

[0605] GIAGQ (SEQ ID NO: 135, cleavable by collagenase),

[0606] GPLGIAG (SEQ ID NO: 136, cleavable by collagenase),

[0607] GPEGLRVG (SEQ ID NO: 137, cleavable by collagenase),

[0608] YGAGLGVV (SEQ ID NO: 138, cleavable by collagenase),

[0609] AGLGVVER (SEQ ID NO: 139, cleavable by collagenase),

[0610] AGLGISST (SEQ ID NO: 140, cleavable by collagenase),

[0611] EPQALAMS (SEQ ID NO: 141, cleavable by collagenase),

[0612] QALAMSAI (SEQ ID NO: 142, cleavable by collagenase),

[0613] AAYHLVSQ (SEQ ID NO: 143, cleavable by collagenase),

[0614] MDAFLESS (SEQ ID NO: 144, cleavable by collagenase),

[0615] ESLPVVAV (SEQ ID NO: 145, cleavable by collagenase),

[0616] SAPAVESE (SEQ ID NO: 146, cleavable by collagenase),

[0617] DVAQFVLT (SEQ ID NO: 147, cleavable by collagenase),

[0618] VAQFVLTE (SEQ ID NO: 148, cleavable by collagenase),

[0619] AQFVLTEG (SEQ ID NO: 149, cleavable by collagenase),

[0620] PVQPIGPQ (SEQ ID NO: 150, cleavable by collagenase),

[0621] LVPRGS (SEQ ID NO: 151, cleavable by thrombin), and

[0622] TSTSGRSANPRG (SEQ ID NO: 178, cleavable by uPA or MTSP1).

[0623] In one embodiment of the present invention, a flexible linker is also attached to one or both ends of a protease cleavage sequence. The flexible linker at one end of a protease cleavage sequence may be referred to as a first flexible linker, and the flexible linker at the other end may be referred to as a second flexible linker. In Petition 870260070097, dated 07 / 15 / 2026, page 113 / 455 107 / 202 a particular embodiment, a protease cleavage sequence and the flexible ligand have any of the following formulas:

[0624] (protease cleavage sequence),

[0625] (first flexible ligand)-(protease cleavage sequence),

[0626] (protease cleavage sequence)-(second flexible ligand), and

[0627] (first flexible ligand)-(protease cleavage sequence)-(second flexible ligand).

[0628] The flexible linker according to the present embodiment is preferably a peptide linker. The first flexible linker and the second flexible linker each exist independently and arbitrarily and are identical or different flexible linkers each containing at least one flexible amino acid (Gly, etc.). The flexible linker contains, for example, a sufficient number of residues (arbitrarily selected amino acids from Arg, Ile, Gln, Glu, Cys, Tyr, Trp, Thr, Val, His, Phe, Pro, Met, Lys, Gly, Ser, Asp, Asn, Ala, etc., particularly Gly, Ser, Asp, Asn, and Ala, in particular Gly and Ser, especially Gly, etc.) for a protease cleavage sequence to obtain the desired protease accessibility.

[0629] The suitable flexible linker for use at both ends of a protease cleavage sequence is generally a flexible linker that improves protease access to a protease cleavage sequence and increases protease cleavage efficiency. A suitable flexible linker can be easily selected and may preferably be selected within different lengths such as 1 amino acid (Gly, etc.) to 20 amino acids, 2 amino acids to 15 amino acids, or 3 amino acids to 12 amino acids including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids. In Petition 870260070097, dated 07 / 15 / 2026, page 114 / 455 108 / 202 In some embodiments of the present invention, the flexible linker is a peptide linker of 1 to 7 amino acids.

[0630] Examples of flexible linkers include, but are not limited to, glycerin (G)n polymers, glycine-serine polymers (including for example, (GS)n, (GSGGS: SEQ ID NO: 27)ne (GGGS: SEQ ID NO: 28)n, wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers well known in conventional techniques.

[0631] Among them, glycine and glycine-serine polymers are receiving attention because these amino acids are relatively unstructured and readily function as neutral bonds between components.

[0632] Examples of the flexible linker consisting of the glycine-serine polymer include, but are not limited to,

[0633] Ser

[0634] Gly-Ser (GS)

[0635] Ser-Gly (SG)

[0636] Gly-Gly-Ser (GGS)

[0637] Gly-Ser-Gly (GSG)

[0638] Ser-Gly-Gly (SGG)

[0639] Gly-Ser-Ser(GSS)

[0640] Ser-Ser-Gly(SSG)

[0641] Ser-Gly-Ser(SGS)

[0642] Gly-Gly-Gly-Ser (GGGS, SEQ ID NO:28)

[0643] Gly-Gly-Ser-Gly (GGSG, SEQ ID NO:29)

[0644] Gly-Ser-Gly-Gly (GSGG, SEQ ID NO:46)

[0645] Ser-Gly-Gly-Gly (SGGG, SEQ ID NO:47)

[0646] Gly-Ser-Ser-Gly (GSSG, SEQ ID NO:48)

[0647] Gly-Gly-Gly-Gly-Ser (GGGGS, SEQ ID NO: 49) Petição 870260070097, de 15 / 07 / 2026, pág. 115 / 455 109 / 202

[0648] Gly-Gly-Gly-Ser-Gly (GGGSG, SEQ ID NO:33)

[0649] Gly-Gly-Ser-Gly-Gly (GGSGG, SEQ ID NO:30)

[0650] Gly-Ser-Gly-Gly-Gly (GSGGG, SEQ ID NO:32)

[0651] Gly-Ser-Gly-Gly-Ser (GSGGS, SEQ ID NO:27)

[0652] Ser-Gly-Gly-Gly-Gly (SGGGG, SEQ ID NO:51)

[0653] Gly-Ser-Ser-Gly-Gly (GSSGG, SEQ ID NO:52)

[0654] Gly-Ser-Gly-Ser-Gly (GSGSG, SEQ ID NO:31)

[0655] Ser-Gly-Gly-Ser-Gly (SGGSG, SEQ ID NO:53)

[0656] Gly-Ser-Ser-Ser-Gly (GSSSG, SEQ ID NO:34)

[0657] Gly-Gly-Gly-Gly-Gly-Ser (GGGGGS, SEQ ID NO: 50)

[0658] Ser-Gly-Gly-Gly-Gly-Gly (SGGGGG, SEQ ID NO: 54)

[0659] Gly-Gly-Gly-Gly-Gly-Gly-Ser (GGGGGGS, SEQ ID NO: 55)

[0660] Ser-Gly-Gly-Gly-Gly-Gly-Gly (SGGGGGG, SEQ ID NO: 56)

[0661] (Gly-Gly-Gly-Gly-Ser (GGGGS, SEQ ID NO: 49))n

[0662] (Ser-Gly-Gly-Gly-Gly (SGGGG, SEQ ID NO: 51))n

[0663] In the present application, association may refer to, for example, a state where two or more polypeptide regions interact with each other. In general, a hydrophobic bond, a hydrogen bond, an ionic bond, or the like is formed between the polypeptide regions intended to form an associate. As a common example of association, an antibody typified by a natural antibody is known to maintain a paired structure of a variable heavy chain (VH) region and a variable light chain (VL) region through a non-covalent bond or the like between them.

[0664] In some embodiments of the present invention, the inhibitory domain of the carrier portion associates with the antigen-binding domain. The inhibitory domain may constitute a portion of the carrier portion or may constitute the whole of the carrier portion. From another point of view, the inhibitory domain may also be defined Petition 870260070097, dated 07 / 15 / 2026, page 116 / 455 110 / 202 as an association of the portion with the antigen-binding domain, in the carrier portion.

[0665] In a more specific embodiment, the antigen-binding domain that is a single-domain antibody and the binding domain that is VL, VH, or VHH form an association as seen between antibody VH and antibody VL. In another specific embodiment, the antigen-binding domain that is a single-domain antibody and the inhibitory domain that is VL, VH, or VHH form an association as seen between antibody VH and antibody VL, and in a state of the association thus formed, the inhibitory domain conformationally inhibits the binding of the antigen-binding domain to the antigen or conformationally changes the antigen-binding site of the antigen-binding domain so that the antigen-binding activity of the single-domain antibody is inhibited by the VL, VH, or VHH.In one embodiment, using VHH as the single-domain antibody, VHH binding to antigen is considered to be conformationally inhibited by the inhibition domain when CDR3, an important antigen-binding site of VHH, or its neighboring site exists at the binding interface with the inhibition domain.

[0666] The association of the antigen-binding domain with the inhibition domain can be canceled, for example, by cleaving the cleavage site. Cancellation of the association can be used interchangeably with, for example, cancellation of the state where two or more polypeptide regions interact with each other. The interaction between the two or more polypeptide regions can be completely canceled, or the interaction between the two or more polypeptide regions can be partially canceled.

[0667] In the present application, the interface generally refers to the face where two regions are associated or interact with each other. Petition 870260070097, dated 07 / 15 / 2026, page 117 / 455 111 / 202 Amino acid residues forming the interface are generally one or more amino acid residues contained in each polypeptide region undergoing association and more preferably refer to the amino acid residues that approach each other in the association and participate in the interaction. Specifically, the interaction includes a non-covalent bond such as a hydrogen bond, electrostatic interaction, or salt bridge formation between the amino acid residues that approach each other in the association.

[0668] In the present application, the amino acid residues forming the interface specifically refers to the amino acid residues contained in the polypeptide regions constituting the interface. As an example, the polypeptide regions constituting the interface refer to the polypeptide regions responsible for selective intramolecular or intermolecular inhibition in antibodies, ligands, receptors, substrates, etc. Specific examples of such polypeptide regions in antibodies may include a variable heavy chain region and a variable light chain region. In some embodiments of the present invention, examples of such polypeptide regions may include an antigen-binding domain and an inhibition domain.

[0669] Examples of amino acid residues forming the interface include, but are not limited to, amino acid residues that approach each other in the association. Amino acid residues that approach each other in the association can be found, for example, by analyzing the conformations of polypeptides and examining the amino acid sequences of polypeptide regions forming the interface in the association of polypeptides.

[0670] In some embodiments of the present invention, an amino acid residue involved in association in the antigen-binding domain, or an amino acid residue involved in association in the binding domain, can be altered in order to promote association. Petition 870260070097, dated 07 / 15 / 2026, page 118 / 455 112 / 202 of the antigen-binding domain with the inhibitory domain. In another specific embodiment, an amino acid residue forming the interface with the inhibitory domain in the antigen-binding domain, or an amino acid residue forming the interface with the antigen-binding domain in the binding domain, can be altered. In a preferred embodiment, the amino acid residue forming the interface can be altered by a method of introducing a mutation at the amino acid residue interface so that two or more amino acid residues forming the interface have different changes.Changing the amino acid residue to result in different charges includes changing a positively charged amino acid residue to a negatively charged amino acid residue or an uncharged amino acid residue, changing a negatively charged amino acid residue to a positively charged amino acid residue or an uncharged amino acid residue, and changing an uncharged amino acid residue to a positively or negatively charged amino acid residue. Such amino acid alteration is performed for the purpose of promoting association and is not limited by the position of the amino acid alteration or the type of amino acid as long as the purpose of promoting association can be achieved. Examples of alteration include, but are not limited to, substitution.

[0671] In some embodiments of the present invention, VHH functioning as the antigen-binding domain associates with VL functioning as the binding domain. The amino acid residue involved in association with VL in VHH may refer to, for example, an amino acid residue forming the interface between VHH and VL. Examples of the amino acid residue involved in association with VL in VHH include, but are not limited to, amino acid residues at positions 37, 44, 45, and 47 (J. Mol. Biol. (2005) 350, 112 Petition 870260070097, dated 07 / 15 / 2026, p. 119 / 455 113 / 202 125). VHH activity is inhibited by promoting the association between VHH and VL. Similarly, the amino acid residue involved in association with VHH in VL may refer to, for example, an amino acid residue forming the interface between VHH and VL.

[0672] An amino acid residue involved in association with VL, in VHH, can be altered in order to promote the association between VHH and VL. Examples of such amino acid substitution include, but are not limited to, F37V, Y37V, E44G, Q44G, R45L, H45L, G47W, F47W, L47W, T47W, and / or S47W. Instead of altering each residue in VHH, VHH originally having an amino acid residue 37V, 44G, 45L, and / or 47W can be used.

[0673] Instead of the amino acid from VHH, an amino acid residue involved in association with VHH, in VL can be altered, and the amino acid alteration can also be introduced in both VHH and VL, as long as the purpose of promoting the association between VHH and VL can be achieved.

[0674] In some alternative embodiments of the present invention, the antigen-binding domain and the inhibition domain can be associated with each other using VHH as the antigen-binding domain and using VH or VHH as the inhibition domain. An amino acid residue involved in association with VH or VHH functioning as the inhibition domain, in VHH functioning as the antigen-binding domain, can be identified and altered in order to promote the association of the VHH antigen-binding domain with the VH or VHH inhibition domain. Furthermore, an amino acid residue involved in association with VHH functioning as the antigen-binding domain, in VH or VHH functioning as the inhibition domain, can be identified and altered.

[0675] In the case of using a single-domain antibody other than VHH as the antigen-binding domain, a residue Petition 870260070097, dated 07 / 15 / 2026, page 120 / 455 The amino acid 114 / 202 involved in association, in the antigen-binding domain or the inhibition domain, can also be identified and altered similarly as above.

[0676] In some embodiments of the present invention, the carrier portion and the antigen-binding domain are fused by means of a linker. In a more specific embodiment, the carrier portion and the antigen-binding domain are fused by means of a linker containing a cleavage site. In an alternative specific embodiment, the carrier portion and the antigen-binding domain are fused by means of a linker, and the fusion protein thus formed contains a cleavage site.

[0677] In another embodiment of the present invention, the carrier portion and the antigen-binding domain are fused without a linker. In a more specific embodiment, an amino bond is formed between the N-terminal amino acid of the carrier portion and the C-terminal amino acid of the antigen-binding domain to form a fusion protein. The fusion protein formed contains a cleavage site. In a particular embodiment, one to several N-terminal amino acids of the carrier portion and / or one to several C-terminal amino acids of the antigen-binding domain are altered, and the N-terminal of the carrier portion and the C-terminal of the antigen-binding domain are fused to form a cleavage site near the fusion position.More specifically, the cleavage site can be formed, for example, by converting four C-terminal amino acids of the antigen-binding domain into an LSGR sequence and converting four N-terminal amino acids of the carrier portion into an SDNH sequence.

[0678] In some embodiments of the present invention, the polypeptide cleavage site comprising a carrier portion and an antigen-binding domain comprises a sequence of Petition 870260070097, dated 07 / 15 / 2026, page 121 / 455 115 / 202 protease cleavage. A protease cleavage sequence can be placed anywhere in the polypeptide as long as the antigen-binding domain is released by protease cleavage and does not lose its antigen-binding activity after release.

[0679] In some embodiments of the present invention, the carrier portion comprises a constant region of the antibody, and the N-terminal of the constant region of the antibody and the C-terminal of the antigen-binding domain are fused by means of a ligand or without a ligand.

[0680] In a particular embodiment, a protease cleavage sequence is located within the constant region of the antibody contained in the carrier portion. In this case, a protease cleavage sequence may be located within the constant region of the antibody so that the antigen-binding domain is released by protease cleavage. In a specific embodiment, a protease cleavage sequence is located within a constant region of the antibody heavy chain contained in the carrier portion, and more specifically located next to the antigen-binding domain with respect to amino acid position 140 (EU numbering) in the constant region of the antibody heavy chain, preferably next to the antigen-binding domain with respect to amino acid position 122 (EU numbering) in the constant region of the antibody heavy chain.In a specific alternative embodiment, a protease cleavage sequence is located within a constant region of the antibody light chain contained in the carrier portion, and more specifically located next to the antigen-binding domain with respect to amino acid position 130 (EU numbering) (Kabat numbering position 130) in a constant region of the antibody light chain, preferably next to the antigen-binding domain with respect to position 113. Petition 870260070097, dated 07 / 15 / 2026, page 122 / 455 116 / 202 amino acid (EU numbering) (position 113 of Kabat numbering) in the constant region of the antibody light chain.

[0681] In some embodiments of the present invention, the antigen-binding domain is a single-domain antibody, and the C-terminal of the single-domain antibody and the N-terminal of the carrier portion are fused by means of a linker or without a linker.

[0682] In a particular embodiment, a protease cleavage sequence is located within the single-domain antibody. In a more specific embodiment, the single-domain antibody is a single-domain antibody prepared from VH, or VHH, and a protease cleavage sequence is located next to the carrier portion with respect to amino acid position 35b (Kabat numbering) of the single-domain antibody, preferably next to the carrier portion with respect to amino acid position 95 (Kabat numbering) of the single-domain antibody, most preferably next to the carrier portion with respect to amino acid position 109 (Kabat numbering) of the single-domain antibody.In a specific alternative embodiment, the single-domain antibody is a single-domain antibody prepared from VL, and a protease cleavage sequence is located next to the carrier portion with respect to amino acid position 32 (Kabat numbering) of the single-domain antibody, preferably next to the carrier portion with respect to amino acid position 91 (Kabat numbering) of the single-domain antibody, most preferably next to the carrier portion with respect to amino acid position 104 (Kabat numbering) of the single-domain antibody.

[0683] In some embodiments of the present invention, the carrier portion comprises a constant region of the antibody, the antigen-binding domain is a single-domain antibody, and the constant region of the antibody and the single-domain antibody are fused. Petition 870260070097, dated 07 / 15 / 2026, page 123 / 455 117 / 202 via a linker or without a linker. In a more specific embodiment, the N-terminal of the constant region of the antibody and the C-terminal of the single-domain antibody are fused via a linker or without a linker. In an alternative specific embodiment, the C-terminal of the constant region of the antibody and the N-terminal of the single-domain antibody are fused via a linker or without a linker.

[0684] In a particular embodiment, a protease cleavage sequence is located within the constant region of the antibody contained in the carrier portion. In a more specific embodiment, a protease cleavage sequence is located next to the single-domain antibody with respect to amino acid position 140 (EU numbering) in a constant region of the antibody heavy chain, preferably next to the single-domain antibody with respect to amino acid position 122 (EU numbering) in a constant region of the antibody heavy chain.In a specific alternative embodiment, a protease cleavage sequence is located next to the antigen-binding domain with respect to amino acid position 130 (EU numbering) (Kabat numbering position 130) in a constant region of the antibody light chain, preferably next to the antigen-binding domain with respect to amino acid position 113 (EU numbering) (Kabat numbering position 113) in a constant region of the antibody light chain.

[0685] In a particular embodiment, a protease cleavage sequence is located within the single-domain antibody. In a more specific embodiment, the single-domain antibody is located within the single-domain antibody. In a more specific embodiment, the single-domain antibody is a single-domain antibody prepared from VH, or VHH, and a sequence of Petition 870260070097, dated 07 / 15 / 2026, page 124 / 455 118 / 202 protease cleavage is located next to the constant region of the antibody with respect to amino acid position 35b (Kabat numbering) of the single-domain antibody, preferably next to the constant region of the antibody with respect to amino acid position 95 (Kabat numbering) of the single-domain antibody, more preferably next to the constant region of the antibody with respect to amino acid position 109 (Kabat numbering) of the single-domain antibody.In a specific alternative embodiment, the single-domain antibody is a single-domain antibody prepared from VL, and a protease cleavage sequence is located next to the constant region of the antibody with respect to amino acid position 32 (Kabat numbering) of the single-domain antibody, preferably next to the constant region of the antibody with respect to amino acid position 91 (Kabat numbering) of the single-domain antibody, most preferably next to the constant region of the antibody with respect to amino acid position 104 (Kabat numbering) of the single-domain antibody.

[0686] In a particular embodiment, a protease cleavage sequence is located near the boundary between the antigen-binding domain and the carrier portion. The phrase near the boundary between the antigen-binding domain and the carrier portion refers to a portion that resides downstream or upstream of the binding site between the antigen-binding domain and the carrier portion and does not greatly influence the secondary structure of the antigen-binding domain.

[0687] In a more specific embodiment, the antigen-binding domain is linked to the antibody constant region contained in the carrier portion, and a protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody constant region. The phrase near the boundary between the antigen-binding domain and the antibody constant region may refer to Petition 870260070097, dated 07 / 15 / 2026, p. 125 / 455 119 / 202 near the boundary between the antigen-binding domain and an antibody heavy chain constant region, or near the boundary between the antigen-binding domain and an antibody light chain constant region. When the antigen-binding domain is a single-domain antibody prepared from VH, or VHH and is connected to an antibody heavy chain constant region, the phrase near the boundary between the antigen-binding domain and the antibody constant region may refer to between amino acid position 101 (Kabat numbering) of the single-domain antibody and amino acid position 140 (EU numbering) of the antibody heavy chain constant region and may preferably refer to between amino acid position 109 (Kabat numbering) of the single-domain antibody and amino acid position 122 (EU numbering) of the antibody heavy chain constant region.When the antigen-binding domain is a single-domain antibody prepared from VH, or VHH, and is connected to an antibody light chain constant region, the phrase near the boundary between the antigen-binding domain and the antibody light chain constant region may refer to between amino acid position 101 (Kabat numbering) of the single-domain antibody and amino acid position 130 (EU numbering) (Kabat numbering position 130) of the antibody light chain constant region, and may preferably refer to between amino acid position 109 (Kabat numbering) of the single-domain antibody and amino acid position 113 (EU numbering) (Kabat numbering position 113) of the antibody light chain constant region.When the antigen-binding domain is a single-domain antibody prepared from VL, the phrase near the boundary between the antigen-binding domain and the antibody constant region refers to the area between amino acid position 96 (Kabat numbering) of the single-domain antibody and the prescribed position of the antibody constant region, preferably. Petition 870260070097, dated 07 / 15 / 2026, page 126 / 455 120 / 202 between amino acid position 104 (Kabat numbering) of the single-domain antibody and the prescribed position of the antibody constant region.

[0688] In some embodiments of the present invention, the polypeptide is a molecule similar to an IgG antibody. Examples of such embodiments include, but are not limited to: an embodiment in which the carrier portion comprises an IgG antibody constant region, a single-domain antibody functioning as the antigen-binding domain takes the place of VH of an IgG antibody, and the antigen-binding activity is inhibited by VL; an embodiment in which the carrier portion comprises an IgG antibody constant region, a single-domain antibody functioning as the antigen-binding domain takes the place of VL of an IgG antibody, and the antigen-binding activity is inhibited by VH;and an embodiment in which the carrier portion comprises a constant region of IgG antibody, a single-domain antibody functioning as the antigen-binding domain takes the place of one of the VH and VL domains of an IgG antibody, and an additional single-domain antibody inhibits the antigen-binding activity of the antigen-binding domain, which takes the place of the other domain of the IgG antibody.

[0689] The term IgG antibody-like molecule used in the present application is used to define a molecule having portions substantially similar in structure to the constant domains or constant regions as in an IgG antibody, and portions substantially similar in structure to the variable domains or variable regions as in the IgG antibody, and having a conformation substantially similar to that of the IgG antibody. However, in the present application, the IgG antibody-like molecule may or may not exert antigen-binding activity while maintaining structures similar to those of the IgG antibody. Petition 870260070097, dated 07 / 15 / 2026, p. 127 / 455 121 / 202

[0690] The polypeptide may comprise one or more antigen-binding domains. One or more inhibitory domains may inhibit the antigen-binding activity of a plurality of antigen-binding domains. A plurality of antigen-binding domains may each be associated with an inhibitory domain. A plurality of antigen-binding domains may each be fused with the carrier portion. A plurality of antigen-binding domains may each be capable of being released from the polypeptide. The cleavage site(s) for release of a plurality of antigen-binding domains may be a plurality of cleavage sites corresponding to several antigen-binding domains.

[0691] When the polypeptide is a molecule similar to the IgG antibody, antigen-binding domains can be respectively established in portions corresponding to two variable regions of the IgG antibody, as shown in Figure 7. Such embodiment should be understandable to those skilled in the art with reference to the present invention. The incorporated antigen-binding domains and both branches may have the same antigen-binding specificity or may differ in antigen-binding specificity. Such embodiment should be understandable to those skilled in the art with reference to the present invention. It is obvious that these embodiments are included within the scope of the present invention.

[0692] In some embodiments of the present invention, the antigen-binding domain is also linked to a second antigen-binding domain. Examples of the second antigen-binding domain include, but are not limited to, single-domain antibodies, antibody fragments, a module called a domain of approximately 35 amino acids contained in an avimer of cell membrane protein in vivo (International Publications Nos. Petition 870260070097, dated 07 / 15 / 2026, page 128 / 455 122 / 202 WO2004 / 044011 and WO2005 / 040229), adnectin containing a 10Fn3 domain functioning as a protein-binding domain derived from a glycoprotein fibronectin expressed in cell membranes (International Publication No. WO2002 / 032925), Affibody containing an IgG-binding domain scaffold constituting a three-helix bundle composed of 58 amino acids of protein A (International Publication No. WO1995 / 001937), DARPins (ankyrin repeat proteins) which are molecular surface-exposed regions of ankyrin repeats (AR) each having a 33-amino acid residue structure folded into a one-turn subunit, two antiparallel helices, and a loop (International Publication No.WO2002 / 020565), an anticalin having four loop regions connecting eight antiparallel filaments folded towards the central axis at one end of a barrel structure highly conserved in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (International Publication No. WO2003 / 029462), and a depressed region in the inner parallel sheet structure of a horseshoe fold composed of leucine-rich repeat (LRR) modules of a free-flowing immunoglobulin (VLR) structure as observed in the acquired immune systems of jawless vertebrates such as lamprey or myxini (International Publication No. WO2008 / 016854). In a preferred embodiment, the second antigen-binding domain has antigen-binding specificity different from that of the antigen-binding domain.In a preferred embodiment, the molecular weight of the antigen-binding domain and the second bound antigen-binding domain is 60 kDa or less.

[0693] In some more specific embodiments, the antigen-binding domain and the second antigen-binding domain are single-domain antibodies that differ in binding specificity. Petition 870260070097, dated 07 / 15 / 2026, page 129 / 455 123 / 202 to the antigen, the antigen-binding domain and the second bound antigen-binding domain are capable of being released from the polypeptide, and the antigen-binding domain and the second antigen-binding domain form a bispecific antigen-binding molecule after release.Examples of such antigen-binding molecules include, but are not limited to, a bispecific antigen-binding molecule having one antigen-binding domain specifically binding to the target cell surface antigen and a second antigen-binding domain specifically binding to an immunocyte surface antigen; a bispecific antigen-binding molecule having one antigen-binding domain and a second antigen-binding domain binding to different subunits of the same antigen; and a bispecific antigen-binding molecule having one antigen-binding domain and a second antigen-binding domain binding to different epitopes of the same antigen. Such a bispecific antigen-binding molecule can recruit immunocytes to the vicinity of target cells and is thus considered useful in treating a disease caused by target cells.

[0694] The antigen-binding activity of the second antigen-binding domain may or may not be inhibited by the carrier portion. The second antigen-binding domain may or may not be associated with a partial structure of the carrier portion. In particular, when the antigen-binding domain and the second antigen-binding domain differ in antigen-binding specificity, the antigen-binding domain in an unreleased state cannot exert antigen-binding activity, as shown, for example, in Figure 8, even if the antigen-binding activity of the second antigen-binding domain is not inhibited and even if the second antigen-binding domain is not associated with a Petition 870260070097, dated 07 / 15 / 2026, page 130 / 455 124 / 202 Partial structure of the carrier portion. This bispecific antigen-binding molecule comprising the antigen-binding domain linked to the second antigen-binding domain cannot perform a bispecific function if it binds to two types of antigens.

[0695] Figure 8 shows an exemplary form, in which the antigen-binding domain is also linked to the second antigen-binding domain.

[0696] In the present application, the term specificity refers to a property whereby one of the specific binding molecules does not substantially bind to a molecule other than its one or more binding partner molecules. This term is also used when the antigen-binding domain has specificity for an epitope contained in a particular antigen. The term is also used when the antigen-binding domain has specificity for a particular epitope among a plurality of epitopes contained in an antigen. In this context, the term does not substantially bind is determined according to the method described in the section on binding activity and means that the binding activity of a specific binding molecule for a molecule other than its binding partner(s) is 80% or less, generally 50% or less, preferably 30% or less, particularly preferably 15% or less, of its binding activity for binding partner molecule(s).

[0697] The present invention also relates to a pharmaceutical composition (drug) comprising the polypeptide of the present invention and a pharmaceutically acceptable carrier.

[0698] The treatment (and its grammatically derived words, for example, treat and treating) used in this application means clinical intervention intended to alter the natural course of an individual being treated and may be carried out both for prevention and during the course of a clinical pathological condition. The desired effect of Petition 870260070097, dated 07 / 15 / 2026, page 131 / 455 Treatment includes, but is not limited to, preventing the development or recurrence of a disease, relieving symptoms, attenuating any direct or indirect pathological influence of the disease, preventing metastasis, reducing the rate of disease progression, recovering from or alleviating a disease condition, and improving or attenuating prognosis. In some embodiments, the polypeptide of the present invention is used to delay the onset of a disease or slow the progression of the disease.

[0699] In the present invention, the pharmaceutical composition generally refers to a drug for the treatment or prevention of a disease or for examination or diagnosis. In the present invention, the term pharmaceutical composition comprising the polypeptide may be used interchangeably with a method for treating a disease, comprising administering the polypeptide to an individual to be treated, and may be used interchangeably with the use of the polypeptide for the production of a drug for the treatment of a disease. Furthermore, the term pharmaceutical composition comprising the polypeptide may be used interchangeably with the use of the polypeptide for the treatment of a disease.

[0700] The pharmaceutical composition of the present invention can be formulated using a method known to those skilled in the art. For example, the pharmaceutical composition can be parenterally administered in the form of an injection of a sterile solution or suspension with water or any other pharmaceutically acceptable liquid. The pharmaceutical composition can be formulated, for example, by appropriately combining the polypeptide with a pharmacologically acceptable vehicle or medium, specifically, sterile water or physiological saline, a plant oil, an emulsifier, a suspending agent, a surfactant, a stabilizer, a flavoring agent, an excipient, a vehicle, a Petition 870260070097, dated 07 / 15 / 2026, page 132 / 455 126 / 202 antiseptic, a binder, etc., and mixing them into a unit dosage form required for generally accepted pharmaceutical practice. The amount of the active ingredient in these formulations is established so as to provide an appropriate volume within a prescribed range.

[0701] A sterile composition for injection may be formulated according to usual pharmaceutical practice using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose, or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, and sodium chloride). The aqueous solution may be used in combination with an appropriate solubilizer, for example, an alcohol (ethanol, etc.), a polyalcohol (propylene glycol, polyethylene glycol, etc.), or a nonionic surfactant (Polysorbate 80(TM), HCO-50, etc.).

[0702] Examples of oily solutions include sesame oil and soybean oil. The oily solution may also be used in combination with benzyl benzoate and / or benzyl alcohol as a solubilizer. The oily solution may be supplemented with a buffer (e.g., a phosphate buffer solution and a sodium acetate buffer solution), a tranquilizing agent (e.g., procaine hydrochloride), a stabilizer (e.g., benzyl alcohol and phenol), and an antioxidant. The prepared injection solution is usually loaded into an appropriate ampoule.

[0703] The pharmaceutical composition of the present invention is preferably administered via a parenteral route. For example, the composition having a dosage form for injection, transnasal, transpulmonary, or percutaneous administration is administered. The pharmaceutical composition can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. Petition 870260070097, dated 07 / 15 / 2026, page 133 / 455 127 / 202

[0704] The method of administration may be appropriately selected according to the age and symptoms of a patient. The dose of the pharmaceutical composition containing the polypeptide may be set to the range of, for example, 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, the dose of the pharmaceutical composition containing the polypeptide may be set to a dose of, for example, 0.001 to 100000 mg per patient. However, the present invention is not necessarily limited by these numerical values. Although the dose and method of administration vary depending on the body weight, age, symptoms, etc. of a patient, those skilled in the art may establish an appropriate dose and method of administration in consideration of these conditions.

[0705] The present invention also relates to a method for producing a polypeptide comprising a carrier portion having an inhibitory domain, and an antigen-binding domain.

[0706] A method for producing the polypeptide of the present invention is a method comprising: obtaining an antigen-binding domain having antigen-binding activity; linking the antigen-binding domain to a carrier portion such that the antigen-binding activity of the antigen-binding domain is inhibited by an inhibitory domain, to form a polypeptide precursor; and also inserting a cleavage site into the polypeptide precursor or altering a portion of the polypeptide precursor to a cleavage site. The method for introducing the cleavage site can be either the insertion of the cleavage site and the alteration of a portion of the polypeptide precursor, provided that the cleavage site can be introduced into the polypeptide precursor. Alternatively, an alteration site can be introduced into the polypeptide precursor by combining both methods. Such Petition 870260070097, dated 07 / 15 / 2026, page 134 / 455 128 / 202 embodiment, should be obvious to those skilled in the art with reference to the present specification and is included within the scope of the present invention.

[0707] Another method for producing the polypeptide of the present invention is a method comprising: obtaining an antigen-binding domain having antigen-binding activity; and linking the antigen-binding domain to a carrier portion by means of a cleavage site such that the antigen-binding activity of the antigen-binding domain is inhibited by an inhibition domain, to form a polypeptide. When the antigen-binding domain is linked to the carrier portion by means of a cleavage site, the cleavage site may be sandwiched between the antigen-binding domain and the carrier portion, or a portion of the antigen-binding domain and / or a portion of the carrier portion may be altered and used as a portion of the cleavage site.

[0708] In one embodiment, using a single-domain antibody as the antigen-binding domain and using a protease cleavage sequence as the cleavage site, the method for producing the polypeptide will be described below.

[0709] In one embodiment of the present invention, the method for producing a polypeptide comprising a carrier portion having an inhibitory domain and an antigen-binding domain is a production method comprising the following steps:

[0710] (a) obtaining a single-domain antibody that binds to a target antigen;

[0711] (b) linking the single-domain antibody obtained in step (a) to a carrier moiety such that the antigen-binding activity of the single-domain antibody is inhibited by an inhibitory domain of the carrier moiety, to form a polypeptide precursor; and Petition 870260070097, dated 07 / 15 / 2026, page 135 / 455 129 / 202

[0712] (c) introduction of a protease cleavage sequence into the polypeptide precursor.

[0713] In one embodiment of the present invention, the method for producing a polypeptide comprising a carrier portion having an inhibitory domain and an antigen-binding domain is a production method comprising the following steps:

[0714] (a) obtaining a single-domain antibody that binds to a target antigen;

[0715] (b) linking the single-domain antibody obtained in step (a) to a carrier moiety such that the antigen-binding activity of the single-domain antibody is inhibited by an inhibitory domain of the carrier moiety, to form a polypeptide precursor; and

[0716] (c) introduction of a protease cleavage sequence near the boundary between the single-domain antibody and the carrier portion.

[0717] In one embodiment of the present invention, the method for producing a polypeptide comprising a carrier portion having an inhibitory domain and an antigen-binding domain is a production method comprising the following steps:

[0718] (a) obtaining a single-domain antibody that binds to a target antigen; and

[0719] (b) linking the single-domain antibody obtained in step (a) to the carrier portion by means of a protease cleavage sequence such that the antigen-binding activity of the single-domain antibody is inhibited by an inhibitory domain of the carrier portion, to form a polypeptide.

[0720] In a particular embodiment, the method for producing a polypeptide comprising a carrier portion having an inhibitory domain and an antigen-binding domain is the method Petition 870260070097, dated 07 / 15 / 2026, page 136 / 455 130 / 202 of production also comprising the following steps:

[0721] (d) confirmation that the binding activity of the single-domain antibody incorporated into the polypeptide or polypeptide precursor against the target antigen is weakened or lost.

[0722] In the present invention, the phrase "binding activity is weakened" means that the binding activity against the target antigen is diminished as compared to that before binding, and the degree of this decrease is not limited.

[0723] In a particular embodiment, the method for producing a polypeptide comprising a carrier portion having an inhibitory domain and an antigen-binding domain is the production method also comprising the following steps:

[0724] (e) release of single-domain antibody by protease cleavage of a protease cleavage sequence and confirmation that the released single-domain antibody binds to the antigen.

[0725] In one embodiment of the present invention, the method for producing a polypeptide that is a molecule similar to an IgG antibody comprising a carrier portion having an inhibitory domain and an antigen-binding domain is a production method comprising the following steps:

[0726] (a) obtaining a single-domain antibody that binds to a target antigen;

[0727] (b) association of the single-domain antibody obtained in step (a) as a VH substitute for an IgG antibody with VL, or association of the single-domain antibody as a VL substitute for an IgG antibody with VH such that the antigen-binding activity of the single-domain antibody is inhibited, to form a molecule similar to the IgG precursor antibody hosting the single-domain antibody; and Petition 870260070097, dated 07 / 15 / 2026, page 137 / 455 131 / 202

[0728] (c) introduction of a protease cleavage sequence into the molecule similar to the precursor IgG antibody housing the single-domain antibody.

[0729] In one embodiment of the present invention, the method for producing a polypeptide that is a molecule similar to the IgG antibody, a carrier portion having an inhibitory domain, and an antigen-binding domain, is a production method comprising the following steps:

[0730] (a) obtaining a single-domain antibody that binds to a target antigen;

[0731] (b) association of the single-domain antibody obtained in step (a) as a VH substitute for an IgG antibody with VL, or association of the single-domain antibody as a VL substitute for an IgG antibody with VH such that the antigen-binding activity of the single-domain antibody is inhibited, to form a molecule similar to the IgG precursor antibody hosting the single-domain antibody; and

[0732] (c) introduction of a protease cleavage sequence near the boundary between another single-domain antibody and a single-domain antibody and a constant region of the antibody in the IgG antibody-like precursor molecule.

[0733] ​​In one embodiment of the present invention, the method for producing a polypeptide that is a molecule similar to the IgG antibody, a carrier portion having an inhibitory domain, and an antigen-binding domain, is a production method comprising the following steps:

[0734] (a) obtaining a single-domain antibody that binds to a target antigen; and

[0735] (b) binding of the single-domain antibody obtained in step (a) as a surrogate for IgG VH or VL antibody to a region Petition 870260070097, dated 07 / 15 / 2026, page 138 / 455 132 / 202 light chain constant or heavy chain constant region of IgG antibody via a protease cleavage sequence so that the antigen-binding activity of the single-domain antibody is inhibited, to form a molecule similar to the IgG antibody housing the single-domain antibody.

[0736] In a particular embodiment, the method for producing a polypeptide that is a molecule similar to the IgG antibody, a carrier portion having an inhibitory domain, and an antigen-binding domain, is the production method also comprising the following steps:

[0737] (d) confirmation that the binding activity of the single-domain antibody introduced into the IgG antibody-like molecule or into the IgG antibody-like molecule precursor against the target antigen is weakened or lost.

[0738] In the present invention, the phrase "binding activity is weakened" means that the binding activity against the target antigen is diminished as compared to that before association or binding, and the degree of this decrease is not limited.

[0739] In a particular embodiment, the method for producing a polypeptide that is a molecule similar to the IgG antibody, a carrier portion having an inhibitory domain, and an antigen-binding domain, is the production method also comprising the following steps:

[0740] (e) release of single-domain antibody by protease cleavage of a protease cleavage sequence and confirmation that the released single-domain antibody binds to the target antigen.

[0741] In the case of using VH, VL or VHH as the inhibition domain, the method for inhibiting the antigen-binding activity of the single-domain antibody by the inhibition domain portion Petition 870260070097, dated 07 / 15 / 2026, page 139 / 455 133 / 202 carrier includes a method of associating the single-domain antibody within the single-domain antibody with VH, VL, or VHH. The VH, VL, or VHH that inhibits the antigen-binding activity of the supplied single-domain antibody can be analyzed for known VH, VL, or VHH association with the single-domain antibody and by comparing the antigen-binding activity of the single-domain antibody before and after association.

[0742] In another method for inhibiting the antigen-binding activity of the single-domain antibody by particular VH, VL or VHH, an amino acid residue involved in association with VH, VL or VHH in the single-domain antibody can be substituted to promote association, or a pair of binding single-domain antibodies having the desired level of difference in antigen-binding activity between before and after association can also be provided using a single-domain antibody originally having, as such amino acid residue, an amino acid that can promote association.

[0743] In one embodiment of the present invention, the method for producing a polypeptide that is a molecule similar to an IgG antibody, a carrier portion having an inhibitory domain, and an antigen-binding domain, is a production method comprising the following steps:

[0744] (a) replacement of an amino acid residue in a single-domain antibody that engages in association with antibody VH, or replacement of an amino acid residue in a single-domain antibody that engages in association with antibody VL to prepare a single-domain antibody variant while maintaining the binding activity of the single-domain antibody against the target antigen;

[0745] (b) association of the single-domain antibody variant prepared in step (a) with antibody VL, or association of the variant Petition 870260070097, dated 07 / 15 / 2026, page 140 / 455 134 / 202 of single-domain antibody with VH antibody so that the antigen-binding activity of the single-domain antibody variant is inhibited, to form a molecule similar to the IgG precursor antibody harboring the single-domain antibody variant; and

[0746] (c) introduction of a protease cleavage sequence into the molecule similar to the precursor IgG antibody harboring the single-domain antibody variant.

[0747] In one embodiment of the present invention, the method for producing a polypeptide that is a molecule similar to an IgG antibody comprising a carrier portion having an inhibitory domain and an antigen-binding domain is a production method comprising the following steps:

[0748] (a) replacement of an amino acid residue in a single-domain antibody that engages in antibody VH association, or replacement of an amino acid residue in a single-domain antibody that engages in antibody VL association, to prepare a single-domain antibody variant while maintaining the single-domain antibody binding activity against the target antigen;

[0749] (b) association of the single-domain antibody variant prepared in step (a) with antibody VL, or association of the single-domain antibody variant with antibody VH such that the antigen-binding activity of the single-domain antibody variant is inhibited, to form a precursor IgG antibody-like molecule hosting the single-domain antibody variant; and introduction of a protease cleavage sequence near the boundary between the single-domain antibody variant and a constant region in the precursor IgG antibody-like molecule.

[0750] In one embodiment of the present invention, the method for Petition 870260070097, dated 07 / 15 / 2026, page 141 / 455 135 / 202 Production of a polypeptide that is a molecule similar to the IgG antibody comprising a carrier portion having an inhibitory domain and an antigen-binding domain is a production method comprising the following steps:

[0751] (a) substitution of an amino acid residue in a single-domain antibody that engages in association with antibody VH, or substitution of an amino acid residue in a single-domain antibody that engages in association with antibody VL, to prepare a single-domain antibody variant while maintaining the binding activity of the single-domain antibody against the target antigen; and

[0752] (b) linking the single-domain antibody variant prepared in step (a) to a constant region of IgG antibody heavy chain by means of a protease cleavage sequence, or linking the single-domain antibody variant to a constant region of IgG antibody light chain by means of a protease cleavage sequence such that the antigen-binding activity of the single-domain antibody variant is inhibited, to form an IgG antibody-like molecule harboring the single-domain antibody variant.

[0753] In a particular embodiment, the method for producing a polypeptide that is a molecule similar to the IgG antibody comprising a carrier portion having an inhibitory domain, and an antigen-binding domain is the production method also comprising the following steps:

[0754] (d) confirmation that the binding activity of the single-domain antibody variant lodged in the IgG antibody-like molecule or in the IgG antibody-like molecule precursor against the target antigen is weakened or lost.

[0755] In the present invention, the phrase the binding activity is Petition 870260070097, dated 07 / 15 / 2026, page 142 / 455 136 / 202 weakened means that the binding activity against the target antigen is diminished compared to that before association or binding, and the degree of this decrease is not limited.

[0756] In a particular embodiment, the method for producing a polypeptide that is a molecule similar to the IgG antibody comprising a carrier portion having an inhibitory domain, and an antigen-binding domain is the production method also comprising the following steps:

[0757] (e) release of the single-domain antibody variant by protease cleavage of a protease cleavage sequence and confirmation that the released single-domain antibody variant binds to the target antigen.

[0758] The present invention also relates to a polynucleotide encoding the polypeptide comprising a carrier portion having an inhibitory domain, and an antigen-binding domain.

[0759] The polynucleotide according to the present invention is generally transported by (or inserted into) a suitable vector and transfected into host cells. The vector is not particularly limited, provided that the vector can stably retain an inserted nucleic acid. For example, when E. coli is used as the host, a pBluescript vector (manufactured by Stratagene Corp.) or similar is preferred as a vector for cloning. Several commercially available vectors can be used. In the case of using the vector for the purpose of producing the polypeptide of the present invention, an expression vector is particularly useful. The expression vector is not particularly limited, provided that the vector allows the expression of the polypeptide in vitro, in E. coli, in cultured cells, or in individual organisms. The expression vector is preferably, for example, a pBEST vector (manufactured by Promega Corp.) for in vitro expression, a pET vector (manufactured by Invitrogen Corp.)) for. Petition 870260070097, dated 07 / 15 / 2026, pp. 143 / 455 137 / 202 E. coli, a pME18S-FL3 vector (GenBank Accession No. AB009864) for cells, and a pME18S vector (Mol Cell Biol. 8: 466-472 (1988)) for individual organisms. The insertion of the DNA of the present invention into the vector can be performed by a routine method, for example, ligase reaction using restriction sites (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).

[0760] Host cells are not particularly limited, and various host cells are used according to the purpose. Examples of cells for polypeptide expression may include bacterial cells (e.g., Streptococcus, Staphylococcus, E. coli, Streptomyces, and Bacillus subtilis), fungal cells (e.g., yeast and Aspergillus), insect cells (e.g., Drosophila S2 and Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, and Bowes melanoma cells), and plant cells. Vector transfection into host cells can be performed by a method known in the art, for example, a calcium phosphate precipitation method, an electroporation method (Current protocols in Molecular Biology edit. Ausubel et al., (1987) Publish. John Wiley & Sons. Section 9.1-9.9), a Lipofectamine method (manufactured by GIBCO-BRL / Thermo Fisher Scientific Inc.), or a microinjection method.

[0761] An appropriate secretory signal can be incorporated into the polypeptide of interest in order to secrete the polypeptide expressed in host cells into the endoplasmic reticulum lumen, periplasmic space, or an extracellular environment. The signal can be endogenous to the polypeptide of interest or it can be a foreign signal.

[0762] When the polypeptide of the present invention is secreted into a medium, recovery of the polypeptide in the production method is achieved by recovering the medium. When the polypeptide of the present Petition 870260070097, dated 07 / 15 / 2026, page 144 / 455 138 / 202 invention is produced in cells, the cells are first lysed, followed by recovery of the polypeptide.

[0763] A method known in the art including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography can be used to recover and purify the polypeptide of the present invention from recombinant cell cultures.

[0764] Examples of the antigen-binding domain used in some embodiments of the present invention include a single-domain antibody. In these embodiments, the antigen-binding activity of the single-domain antibody can be inhibited by association with a particular VL, association with a particular VH, or association with a particular VHH. The present invention also relates to a method for the analysis of such a single-domain antibody.

[0765] VL, VH, or VHH having a known sequence, for example, VL, VH, or VHH having a sequence registered in the IMGT or Kabat database, can be used as the VL, VH, or VHH that inhibits the antigen-binding activity of the single-domain antibody. In addition, the newly identified VL, VH, or VHH sequence from a human antibody library or similar can be used. The VL, VH, or VHH that inhibits the binding activity of the single-domain antibody can be selected by preparing a protein by combining these sequences and measuring the binding activity using the method described above.

[0766] In some embodiments of the present invention, VL, VH or VHH, having a germline human antibody sequence, can be used as a VL, VH, or VHH that inhibits the antigen-binding activity of the single-domain antibody. Petition 870260070097, dated 07 / 15 / 2026, pp. 145 / 455 139 / 202 In the case of using, for example, VL as the inhibition domain, VL having kappa chain structural sequences or VL having lambda chain structural sequences can be used. Furthermore, VL having modified structural sequences such as combined structural sequences of kappa and lambda chain structural sequences can be used.

[0767] In one embodiment, the present invention provides a method for the analysis of a single-domain antibody whose antigen-binding activity can be inhibited by association with a particular VL, comprising the following steps:

[0768] (a) obtaining a single-domain antibody having target antigen binding activity;

[0769] (b) association of the single-domain antibody obtained in step (a) with a particular VL; and

[0770] (c) confirmation that the single-domain antibody binding activity associated with the particular VL in step (b) against the antigen is weakened or lost.

[0771] In the present invention, the phrase "binding activity is weakened" means that the binding activity against the target antigen is diminished as compared to that before binding, and the degree of this decrease is not limited.

[0772] In one embodiment, the present invention provides a method for the analysis of a single-domain antibody whose antigen-binding activity can be inhibited by association with a particular HV, comprising the following steps:

[0773] (a) obtaining a single-domain antibody having target antigen binding activity;

[0774] (b) association of the single-domain antibody obtained in step (a) with the particular HV; and

[0775] (c) confirmation that the antibody binding activity Petition 870260070097, dated 07 / 15 / 2026, page 146 / 455 The single domain 140 / 202 associated with the particular VH in step (b) against the antigen is weakened or lost.

[0776] In the present invention, the phrase "binding activity is weakened" means that the binding activity against the target antigen is diminished as compared to that before binding, and the degree of this decrease is not limited.

[0777] In one embodiment, the present invention provides a method for the analysis of a single-domain antibody whose antigen-binding activity can be inhibited by association with a particular VHH, comprising the following steps:

[0778] (a) obtaining a single-domain antibody having target antigen binding activity;

[0779] (b) association of the single-domain antibody obtained in step (a) with the particular HHV; and

[0780] (c) confirmation that the binding activity of the single-domain antibody associated with the particular HHV in step (b) against the antigen is weakened or lost.

[0781] In the present invention, the phrase "binding activity is weakened" means that the binding activity against the target antigen is diminished as compared to that before binding, and the degree of this decrease is not limited.

[0782] Examples of the method for associating the single-domain antibody within the single-domain antibody with the particular VL, VH, or VHH include a method of designing a molecule having the single-domain antibody sequence as a surrogate for the sequence of one of VH and VL in an antibody or an antibody fragment comprising both VH and VL, such as a complete antibody, Fab, Fab', or (Fab)2, and expressing a polypeptide having the sequence.

[0783] The present invention also relates to a method for Petition 870260070097, dated 07 / 15 / 2026, page 147 / 455 141 / 202 production of a single-domain antibody whose antigen-binding activity is inhibited by promoting the association of the single-domain antibody without the single-domain antibody with a particular VL, VH or VHH, promoting the association of the single-domain antibody without the single-domain antibody with a particular VL, promoting the association of the single-domain antibody without the single-domain antibody with a particular VH, or promoting the association of the single-domain antibody without the single-domain antibody with a particular VHH, in addition to analysis of a single-domain antibody whose antigen-binding activity is inhibited by association with a particular VL, association with a particular VH, or association with a particular VHH.

[0784] In one embodiment, the present invention provides a method for producing a single-domain antibody whose antigen-binding activity is inhibited by association with a particular VL, comprising the following steps:

[0785] (a) substitution of an amino acid residue in a single-domain antibody that engages in association with antibody VL, to prepare a single-domain antibody variant while maintaining the binding activity of the single-domain antibody against the target antigen.

[0786] In a particular embodiment, the present invention provides a method for producing a single-domain antibody whose antigen-binding activity is inhibited by association with a particular VL, also comprising the following steps:

[0787] (b) association of the variant single-domain antibody prepared in step (a) with the particular VL; and

[0788] (c) confirmation that the antigen-binding activity of the single-domain antibody variant associated with VL is weakened or lost. Petition 870260070097, dated 07 / 15 / 2026, pp. 148 / 455 142 / 202

[0789] In the present invention, the phrase "binding activity is weakened" means that the binding activity against the target antigen is diminished as compared to that before binding, and the degree of this decrease is not limited.

[0790] In one embodiment, the present invention provides a method for producing a single-domain antibody whose antigen-binding activity is inhibited by association with a particular HV, comprising the following steps:

[0791] (a) substitution of an amino acid residue in a single-domain antibody that engages in antibody VH association, to prepare a single-domain antibody variant retaining the single-domain antibody binding activity against the target antigen.

[0792] In a particular embodiment, the present invention provides a method for producing a single-domain antibody whose antigen-binding activity is inhibited by association with a particular HV, also comprising the following steps:

[0793] (b) association of the variant single-domain antibody prepared in step (a) with the particular HV; and

[0794] (c) confirmation that the antigen-binding activity of the HV-associated single-domain antibody variant is weakened or lost.

[0795] In the present invention, the phrase "binding activity is weakened" means that the binding activity against the target antigen is diminished as compared to that before binding, and the degree of this decrease is not limited.

[0796] In one embodiment, the present invention provides a method for producing a single-domain antibody whose antigen-binding activity is inhibited by association with a particular VHH, comprising the following steps: Petition 870260070097, dated 07 / 15 / 2026, page 149 / 455 143 / 202

[0797] (a) substitution of an amino acid residue in a single-domain antibody that is involved in association with VHH, to prepare a single-domain antibody variant maintaining the binding activity of the single-domain antibody against the target antigen.

[0798] In a particular embodiment, the present invention provides a method for producing a single-domain antibody whose antigen-binding activity is inhibited by association with a particular VHH, also comprising the following steps:

[0799] (b) association of the single-domain antibody variant prepared in step (a) with the particular HHV; and

[0800] (c) confirmation that the antigen-binding activity of the HHV-associated single-domain antibody variant is weakened or lost.

[0801] In the present invention, the phrase "binding activity is weakened" means that the binding activity against the target antigen is diminished as compared to that before binding, and the degree of this decrease is not limited.

[0802] The single-domain antibody association step within the single-domain antibody with the particular VL, VH, or VHH is performed by a design method of a molecule having the single-domain antibody sequence as a surrogate for the sequence of one of VH and VL in an antibody or an antibody fragment comprising both VH and VL, such as a complete antibody, Fab, Fab', or (Fab)2, and expressing a polypeptide having the sequence.

[0803] According to a certain embodiment of the present invention, the single-domain antibody of the present invention whose antigen-binding activity is inhibited or lost by association with a particular VL, VH or VHH can be obtained from a library comprising a Petition 870260070097, dated 07 / 15 / 2026, page 150 / 455 144 / 202 plurality of single-domain antibody fusion polypeptides each linked to a first supporting domain by association.

[0804] In the present application, an embodiment of the library can provide a library that allows efficient production of a single-domain antibody whose antigen-binding activity is inhibited or lost by association with a particular VL, VH or VHH.

[0805] In the present application, the library refers to a group of a plurality of fusion polypeptides having different sequences, or nucleic acids or polynucleotides encoding these fusion polypeptides. A plurality of fusion polypeptides contained in the library are fusion polypeptides differing in sequence from one another, not having a single sequence.

[0806] In the present application, the term differing in sequence from one another in a plurality of fusion polypeptides differing in sequence from one another means that the individual fusion polypeptides in the library have distinct sequences. More preferably, the term means that the antibody single-domain portions of the individual fusion polypeptides in the library have distinct sequences. Specifically, the number of distinct sequences in the library reflects the number of independent clones that differ in sequences in the library and is also referred to as a library size. The library size of a typical phage display library is 10⁶ to 10¹² and can be expanded to 10¹⁴ by applying a technique known in the art such as a ribosome display method. However, the number of phage particles for use in panning selection for the phage library is generally 10 to 10,000 times greater than a library size.This excessive multiple, also called the library equivalent number, represents that 10 to 10,000 individual clones may have the same amino acid sequence. Consequently, the term refers to how they differ. Petition 870260070097, dated 07 / 15 / 2026, p. 151 / 455 145 / 202 sequence from each other according to the present invention means that the individual polypeptides in the library, excluding the number of library equivalents, have distinct sequences, and more specifically means that the library has from 10⁶ to 10¹⁴ molecules, preferably 10⁷ to 10¹² molecules, of polypeptides that differ in sequence from each other.

[0807] The term plurality of in the library, which essentially consists of a plurality of fusion polypeptides according to the present invention, generally refers to a group of two or more types of substances, such as, for example, the polypeptide, polynucleotide molecule, vector, or virus of the present invention. Provided that, for example, two or more substances differ in a particular trait from one another, this means that the substances are of two or more types. Examples thereof may include a mutant amino acid observed at a particular amino acid position in an amino acid sequence. For example, two or more polypeptides of the present invention having substantially equal, preferably identical, sequences except for mutant amino acids at particularly diverse amino acid positions exposed to the surface are considered as a plurality of polypeptides of the present invention.In another example, two or more polynucleotide molecules of the present invention having substantially similar, preferably identical, sequences, except for bases encoding mutant amino acids, particularly at highly diverse amino acid positions, exposed on the surface, are considered as a plurality of polynucleotide molecules of the present invention.

[0808] A panning method that uses phage vectors is preferably also used as a method for analyzing fusion polypeptides with binding activity as an index. A Petition 870260070097, dated 07 / 15 / 2026, page 152 / 455 A gene encoding each single-domain antibody and a gene encoding an IgG antibody CH1 domain or a light constant region can be appropriately linked to form a fusion polypeptide. Genes encoding the fusion polypeptides thus formed can be inserted into phage vectors to obtain phages expressing the fusion polypeptides on their surface. After contact of the phages with the desired antigen, the antigen-bound phages can be retrieved to recover the DNAs encoding fusion polypeptides having the binding activity of interest. This operation can be repeated, if necessary, to enrich fusion polypeptides having the desired binding activity.

[0809] In addition to the phage display method, a technique using a cell-free translation system, a technique for presenting fusion polypeptides on the cell or viral surface, a technique using an emulsion, and the like are known as techniques for obtaining fusion polypeptides by panning using a library. For example, a ribosome display method of forming an mRNA-translated protein complex via ribosome by removing a stop codon, etc., a cDNA or mRNA display method of covalently linking a gene sequence to a translated protein using a compound such as puromycin, or a CIS display method of forming a gene-translated protein complex using a nucleic acid-binding protein can be used as the technique using a cell-free translation system. For example, the phage display method, as well as an E. display method.For example, a display method for gram-positive bacteria, a display method for yeast, a display method for mammalian cells, or a display method for viruses can be used as a technique for presenting fused polypeptides on the cell or viral surface. Petition 870260070097, dated 07 / 15 / 2026, page 153 / 455 147 / 202 of virus in vitro using an emulsion containing a gene and a translation-related molecule can be used as the technique using an emulsion. These methods are already known in the art (Nat Biotechnol. December 2000; 18(12): 1287-92, Nucleic Acids Res. 2006; 34(19): e127, Proc Natl Acad Sci US A. March 2, 2004; 101(9): 2806-10, Proc Natl Acad Sci US A. July 22, 2004; 101(25): 9193-8, Protein Eng Des Sel. April 2008; 21(4): 247-55, Proc Natl Acad Sci US A. September 26, 2000; 97(20): 10701-5, MAbs. Sept-Oct 2010; 2(5): 508-18, Methods Mol Biol. 2012; 911: 183-98).

[0810] An association partner of an inhibition domain linked to a second association support domain can be used in a method for obtaining the single-domain antibody of interest from the library comprising a plurality of single-domain antibody fusion polypeptides each linked to a first association support domain.

[0811] In the present application, the first association support domain and the second association support domain refer to domains that interact with each other through a bond such as a hydrophobic bond, a hydrogen bond, or an ionic bond to form an associate. Preferred examples of the first association support domain and the second association support domain include, but are not limited to, an antibody light chain (CL) constant region and a CH1 domain of a heavy chain region.

[0812] The first association support domain and the second association support domain can interact with each other and form the fusion polypeptide association with the association partner, regardless of the degree of associativity between the single-domain antibody and the inhibition domain.

[0813] In an alternative embodiment, the present invention Petition 870260070097, dated 07 / 15 / 2026, page 154 / 455 Patent 148 / 202 provides a library comprising a plurality of single-domain antibody fusion polypeptides linked to an IgG antibody light chain constant region, wherein the single-domain antibodies include a single-domain antibody whose antigen-binding activity is inhibited or lost by association with particular VL, VH, or VHH, and a method for analyzing the library for a single-domain antibody whose antigen-binding activity may be inhibited or could be lost by association with particular VL, VH, or VHH.

[0814] In a specific embodiment, as shown in the Figures 9A(1), 9A(2), 9A(3), 9B, and 9C,

[0815] (1) single domain antibody fusion polypeptides each linked to a first supporting domain by association are displayed on the surface of phages or similar by the display method such as phage display.

[0816] (2) An association partner of an inhibitory domain linked to a second association support domain is provided, and fusion polypeptides are associated with the association partner. A fusion polypeptide that does not bind to the target antigen or has antigen-binding activity of a predetermined value or lower in this state of the fusion polypeptide associated with the association partner is selected.

[0817] (3) The association of the single-domain antibody on the fusion polypeptide selected in (2) with the inhibitory domain on the association partner is cancelled. A fusion polypeptide that binds to the target antigen or has antigen-binding activity of a predetermined value or higher in a state where the single-domain antibody does not associate with the inhibitory domain is selected.

[0818] In this context, for example, a cleavage method of Petition 870260070097, dated 07 / 15 / 2026, page 155 / 455 149 / 202 association partner near the boundary between the inhibition domain and the second association support domain as shown in Figure 9B, or a method of cleaving the fusion polypeptide near the boundary between the single-domain antibody and the first association support domain as shown in Figure 9C can be used as a method for canceling the association of the single-domain antibody with the inhibition domain.

[0819] In another embodiment, the present invention provides a method comprising, as shown in Figure 9D, comparing the difference in single-domain antibody binding activity between when the single-domain antibody and the inhibitory domain are expressed together and when the single-domain antibody is expressed in such a way as not to express the inhibitory domain together with them, instead of comparing the difference in single-domain antibody binding activity between canceled and uncanceled association of the single-domain antibody with the inhibitory domain as shown in Figures 9A to 9C.

[0820] As shown in Figure 9D(1), the single-domain antibody and the inhibitory domain are expressed together to form an association. A fusion polypeptide comprising a single-domain antibody that does not bind to the antigen or has antigen-binding activity of a predetermined value or lower in this state is selected. As shown in Figures 9D(2), 9D(2'), and 9D(2''), the single-domain antibody is expressed so as not to express the inhibitory domain together with it. A fusion polypeptide comprising a single-domain antibody that binds to the antigen or has antigen-binding activity of a predetermined value or higher in this state is selected. As a result, the single-domain antibody whose antigen-binding activity is inhibited or lost by association with a domain of Petition 870260070097, dated 07 / 15 / 2026, page 156 / 455 150 / 202 particular inhibition, for example, VH, VL or VHH, can be analyzed from the library comprising a plurality of single-domain antibody fusion polypeptides, each linked to a primary supporting domain by association. Alternatively, the single-domain antibody is expressed in order not to express the inhibition domain together with it. A polypeptide comprising a single-domain antibody that binds to the antigen or has antigen-binding activity of a predetermined value or higher in this state is selected. Then, the single-domain antibody and the inhibition domain are expressed together to form the association. A polypeptide comprising a single-domain antibody that does not bind to the antigen or has antigen-binding activity of a predetermined value or lower in this state is selected.Also by this method, the single-domain antibody whose antigen-binding activity is inhibited or lost by association with a particular inhibitory domain, for example, VH, VL or VHH, can be analyzed from the library comprising a plurality of single-domain antibody fusion polypeptides each linked to a first holding domain by association.Alternatively, as shown in Figures 9D(2), 9D(2'), and 9D(2''), the single-domain antibody is expressed in such a way as not to express the inhibitory domain together with it (only the single-domain antibody is expressed; only the fusion polypeptide comprising a single-domain antibody and a first supporting domain by association is expressed; or the fusion polypeptide comprising a single-domain antibody and a first supporting domain by association is associated only with the second supporting domain by association), and a fusion polypeptide comprising a single-domain antibody that binds to or has antigen-binding activity of one. Petition 870260070097, dated 07 / 15 / 2026, page 157 / 455 151 / 202 a predetermined value or higher in this state is selected. Then, as shown in Figure 9D(1), the single-domain antibody in the selected fusion polypeptide and the inhibition domain are expressed together to form the association. A fusion polypeptide comprising a single-domain antibody that does not bind to the antigen or has antigen-binding activity of a predetermined value or lower in this state is selected. As a result, the single-domain antibody whose antigen-binding activity is inhibited or lost by association with a particular inhibition domain, for example, VH, VL or VHH, can also be analyzed from the library comprising a plurality of single-domain antibody fusion polypeptides each linked to a first support domain by association.

[0821] Antigen-binding activity of a predetermined value or lower may refer, for example, to antigen-binding activity that falls below a predetermined reference when antigen-binding activity is measured by the method listed in this application. Likewise, antigen-binding activity of a predetermined value or higher may refer, for example, to antigen-binding activity that exceeds a predetermined reference when antigen-binding activity is measured by the method listed in this application. A fusion polypeptide having antigen-binding activity of a predetermined value or higher binds more strongly to the antigen than a fusion polypeptide having antigen-binding activity of a predetermined value or lower.

[0822] The fusion polypeptide selected in (3) described above comprises a single-domain antibody that has no or weak antigen-binding activity in an association state with the inhibitory domain and has (strong) antigen-binding activity in Petition 870260070097, dated 07 / 15 / 2026, page 158 / 455 152 / 202 a state of non-association with the inhibition domain. The fusion polypeptide sequence selected by this method can also be analyzed to elucidate the sequence of the single-domain antibody contained within it. In this way, the single-domain antibody can be produced.

[0823] For the method for analyzing a fusion polypeptide comprising the single-domain antibody of interest using fusion polypeptides and an association partner, it is important to compare the antigen-binding activity of the single-domain antibody between the association and non-association states with the inhibition domain. As shown in Figures 9A(2') and 9A(3'), the antigen-binding activity of the displayed fusion polypeptides is first confirmed, and a fusion polypeptide that binds to the antigen or has antigen-binding activity of a predetermined value or higher is selected. Then, the fusion polypeptides thus selected are associated with the association partner. A fusion polypeptide that does not bind to the antigen or has antigen-binding activity of a predetermined value or lower in this association state is selected.Also by this method, the fusion polypeptide comprising the single-domain antibody of interest can be obtained.

[0824] Here are some options using a domain of The use of IgG antibody CH1 as the first supporting domain by association and using IgG antibody CL as the second supporting domain by association will be described.

[0825] A fusion polypeptide comprising the single-domain antibody of interest can be analyzed from a library comprising a plurality of single-domain antibody fusion polypeptides each linked to an IgG antibody CH1 domain. Petition 870260070097, dated 07 / 15 / 2026, page 159 / 455 153 / 202

[0826] In some embodiments, the present invention provides a library comprising a plurality of single-domain antibody fusion polypeptides each linked to an IgG antibody CH1 domain, wherein the single-domain antibodies include a single-domain antibody whose antigen-binding activity is inhibited or lost by association with particular VL, VH or VHH, and a method for analyzing the library for a fusion polypeptide comprising a single-domain antibody whose antigen-binding activity may be inhibited or could be lost by association with particular VL, VH or VHH.

[0827] In a particular embodiment, the present invention provides a method for the analysis of a fusion polypeptide comprising a single-domain antibody whose antigen-binding activity may be inhibited or could be lost by association with a particular VL, from a library comprising a plurality of single-domain antibody fusion polypeptides each linked to a CH1 domain of IgG antibody. Specifically, the present invention provides a method for the analysis of a single-domain antibody, comprising the following steps:

[0828] (a) In vitro display of library fusion polypeptides according to the present invention;

[0829] (b) providing an association partner of a constant region of IgG antibody light chain fused with the particular VL;

[0830] (c) association of the fusion polypeptides shown in step (a) with the association partner provided in step (b) and selection of a fusion polypeptide that does not bind to the antigen or has antigen-binding activity of a predetermined value or lower in a state where the single-domain antibody associates with the VL; and

[0831] (d) selection of the fusion polypeptides in this way Petition 870260070097, dated 07 / 15 / 2026, page 160 / 455 154 / 202 selected in step (c), a fusion polypeptide that binds to the antigen or has antigen-binding activity of a predetermined value or higher in a state where the single-domain antibody contained therein does not associate with the VL.

[0832] The association partner provided in step (b) also comprises a protease cleavage sequence. In this case, in step (d), the association of the single-domain antibody without the single-domain antibody with the VL is canceled by protease treatment, and the antigen-binding activity of the single-domain antibody can be confirmed in a state where the single-domain antibody does not associate with the VL. A protease cleavage sequence in the association partner is not limited by its position, as long as the association of the single-domain antibody without the single-domain antibody with the VL is canceled by cleavage.As an example of the position, a protease cleavage sequence can be located, for example, near the boundary between the VL and the constant region of the IgG antibody light chain on the associative partner, preferably at any position between amino acid position 96 (Kabat numbering) of the VL and amino acid position 130 (EU numbering) (Kabat numbering position 130) of the antibody light chain constant region, more preferably at any position between amino acid position 104 (Kabat numbering) of the VL and amino acid position 113 (EU numbering) (Kabat numbering position 113) of the antibody light chain constant region.

[0833] Instead of using the association partner comprising a protease cleavage sequence, a protease cleavage sequence can be introduced into the fusion polypeptides in the library, and the fusion polypeptides can be cleaved by protease so that the association of the single-domain antibody without the single-domain antibody with the VL is canceled. A sequence Petition 870260070097, dated 07 / 15 / 2026, page 161 / 455 The 155 / 202 protease cleavage sequence in each fusion polypeptide is not limited by its position, as long as the association of the single-domain antibody without the single-domain antibody with the VL is canceled by cleavage and the single-domain antibody retains its antigen-binding activity even after cleavage. As an example of position, a protease cleavage sequence may be located, for example, near the boundary between the single-domain antibody and the CH1 domain of IgG antibody in the fusion polypeptide.

[0834] In step (d), the complete complements of the fusion polypeptides selected in step (c) or their portions comprising the single-domain antibodies can be displayed again, and the antigen-binding activity of the single-domain antibody can be confirmed in a state where the single-domain antibody does not associate with the VL.

[0835] In a particular embodiment, the present invention provides a method for the analysis of a fusion polypeptide comprising a single-domain antibody whose antigen-binding activity may be inhibited or could be lost by association with a particular HV, from a library comprising a plurality of single-domain antibody fusion polypeptides each linked to a constant region of IgG antibody light chain. Specifically, the present invention provides a method for the analysis of a fusion polypeptide comprising a single-domain antibody, comprising the following steps:

[0836] (a) In vitro display of library fusion polypeptides according to the present invention;

[0837] (b) providing an association partner of a CH1 domain antibody of IgG fused with the particular VH;

[0838] (c) association of the fusion polypeptides shown in step Petition 870260070097, dated 07 / 15 / 2026, page 162 / 455 156 / 202 (a) with the association partner provided in step (b) and selection of a fusion polypeptide that does not bind to the antigen or has antigen-binding activity of a predetermined value or lower in a state where the single-domain antibody associates with HV; and

[0839] (d) selection of the fusion polypeptides thus selected in step (c) a fusion polypeptide that binds to the antigen or has antigen-binding activity of a predetermined value or higher in a state where the single-domain antibody contained therein does not associate with HV.

[0840] The association partner provided in step (b) also comprises a protease cleavage sequence. In this case, in step (d), the association of the single-domain antibody without the single-domain antibody with HV is canceled by protease treatment, and the antigen-binding activity of the single-domain antibody can be confirmed in a state where the single-domain antibody does not associate with HV. A protease cleavage sequence in the association partner is not limited by its position, as long as the association of the single-domain antibody without the single-domain antibody with HV is canceled by cleavage.As an example of location, a protease cleavage sequence can be located, for example, near the boundary between the VH and the CH1 domain of IgG antibody in the association partner, preferably at any position between amino acid position 101 (Kabat numbering) of the VH and amino acid position 140 (EU numbering) of the antibody heavy chain constant region, more preferably at any position between amino acid position 109 (Kabat numbering) of the VH and amino acid position 122 (EU numbering) of the antibody heavy chain constant region.

[0841] Instead of using the association partner comprising a protease cleavage sequence, a cleavage sequence Petition 870260070097, dated 07 / 15 / 2026, page 163 / 455 157 / 202 protease can be introduced into the fusion polypeptides in the library, and the fusion polypeptides can be cleaved by protease so that the association of the single-domain antibody without the single-domain antibody with VH is canceled. A protease cleavage sequence in each fusion polypeptide is not limited by its position, as long as the association of the single-domain antibody without the single-domain antibody with VH is canceled by cleavage and the single-domain antibody retains its antigen-binding activity even after cleavage. As an example of position, a protease cleavage sequence can be located, for example, near the boundary between the single-domain antibody and the constant region of the IgG antibody light chain in the fusion polypeptide.

[0842] In step (d), the complete complements of the fusion polypeptides selected in step (c) or their portions comprising the single-domain antibodies can be displayed again, and the antigen-binding activity of the single-domain antibody can be confirmed in a state where the single-domain antibody does not associate with VH.

[0843] An amino acid contained in each amino acid sequence described in the present invention may be post-translationally modified (for example, the modification of N-terminal glutamine to pyroglutamic acid by pyroglutamylation is a well-known modification to those skilled in the art). Such an amino acid sequence containing the post-translationally modified amino acid is also routinely included in the amino acid sequence described in the present invention.

[0844] It should be understood by those skilled in the art that arbitrary combinations of one or more embodiments described in the present application are also included in the present invention, unless Petition 870260070097, dated 07 / 15 / 2026, page 164 / 455 158 / 202 that there is a technical contradiction based on the common technical understanding of those versed in the art. EXAMPLES

[0845] Hereinafter, examples of the method and composition of the present invention will be described. It should be understood that various other embodiments may be realized in light of the general description mentioned above. Example 1 - Problem of an existing protease-activated antibody

[0846] A method for preparing an antibody that exerts antigen-binding activity only through cleavage by protease expressed at a lesion site such as cancerous tissue or inflammatory tissue has been reported. This antibody, called Probody, is an antibody molecule, as shown in Figure 1, whose antigen-binding activity is inhibited by attaching an antibody to a peptide masking the antibody's antigen-binding site by means of a ligand that is cleaved by protease expressed at a lesion site (Non-Patent Literature 18). The masking peptide is dissociated from Probody by cleavage of the constituent ligand by protease expressed at the target pathological site so that the resulting antibody molecule restores its antigen-binding activity and becomes capable of binding to the antigen in the target pathological tissue.

[0847] It is believed that Probody can selectively bind to antigen at the target pathological site via the mechanism described above, thereby expanding the therapeutic window. However, because antibody cleavage by protease is irreversible in the case of Probody, there may be a possibility that the antibody cleaved at the pathological site could be carried in the blood from the pathological site and bind to antigen expressed in normal tissue as a result of antibody distribution to normal tissues via the bloodstream. Protease-activated Probody retains an Fc region. Petition 870260070097, dated 07 / 15 / 2026, p. 165 / 455 159 / 202 is the same as in Probody before activation and therefore has a long circulation time in the blood. Therefore, protease-activated antibody expressed at a pathological site can circulate in the blood for a long time. The same protease expressed at a high level at a pathological site is also expressed at a low level in normal tissues, and free protease produced at a pathological site can be leaked into the blood (The Chinese-German Journal of Clinical Oncology June 2004, Vol. 3, No. 2 P78-P80). Therefore, Probody can be activated by such free protease. Therefore, there may be a possibility that Probody is activated at a site other than a pathological site. Probody activated in this way also circulates in the blood for a long time. Thus, there is a possibility that Probody is continuously activated at a pathological site, in normal tissues, and in the blood, and the activated Probody, having a long circulation time in the blood, accumulates in the blood.Activated Probody that accumulates in the blood can exhibit adverse reactions by binding to antigen expressed in normal tissues (Figure 2).

[0848] The antigen-binding activity of Probody is inhibited by a masking peptide linked to an antibody via a ligand, however, the antigen-binding activity is not completely inhibited. Probody is in equilibrium between a state where the masking peptide linked via the ligand is bound to the antigen-binding site and a state where the masking peptide is dissociated. A molecule in the dissociated state can bind to the antigen (Figure 3). In fact, anti-EGFR Probody described in the Non-Patent Literature 17 has binding activity against EGFR even before protease cleavage of the ligand. Although antigen-binding activity increases 30 to 100 times by protease cleavage of the ligand, Probody present at a high concentration before activation can exhibit adverse reactions by binding to antigen expressed in normal tissues. Petition 870260070097, dated 07 / 15 / 2026, page 166 / 455 160 / 202 because the Probody before activation has 1 / 30 to 1 / 100 of the binding activity of the activated Probody.

[0849] Probody employs an artificial peptide to mask the antibody's antigen-binding site. The artificial peptide has a sequence absent in natural human proteins and may therefore have immunogenicity in humans. Such immunogenicity is known to diminish the effects of antibody drugs by inducing antidrug antibodies (Blood. March 31, 2016; 127(13):1633-41).

[0850] Possible anti-drug antibodies against Probody are an anti-drug antibody against a complex of the antibody and the masking peptide (Probody before activation), an anti-drug antibody against the antibody dissociated from the masking peptide (activated Probody), an anti-drug antibody against the masking peptide (masking peptide dissociated from activated Probody), and the like. Among these, the anti-drug antibody against the masking peptide (anti-masking peptide antibody) can bind to the masking peptide of Probody before activation and thus activate Probody without protease cleavage (Figure 4). Probody activated by the anti-masking peptide antibody can exhibit adverse reactions by binding to antigen expressed in normal tissues. Example 2 - Concept of protease-activated polypeptide comprising single-domain antibody

[0851] As shown in Example 1, Probody technology has the following problems:

[0852] Probody activated by protease cleavage has a long circulation time in the blood.

[0853] Even Probody before protease cleavage has antigen-binding activity.

[0854] The masking peptide is an artificial non-human sequence and can induce an anti-masking peptide antibody. Petition 870260070097, dated 07 / 15 / 2026, page 167 / 455 161 / 202

[0855] The present inventors believe that a useful way to solve these problems and provide an antibody drug that exerts activity at a pathological site is to satisfy the following conditions:

[0856] 1. An antigen-binding domain activated by protease cleavage has a short half-life in blood.

[0857] 2. The antigen-binding activity of a molecule before protease cleavage is minimized.

[0858] 3. The masking peptide having an artificial non-human sequence should not be used.

[0859] The present inventors designed a molecule shown in Figure 5 as an example of a polypeptide that satisfied the conditions described above. The polypeptide with an antigen-binding domain attached to a carrier moiety has a long half-life and does not bind to the antigen because the antigen-binding activity of the antigen-binding domain is inhibited (A). The antigen-binding domain is released, and the antigen-binding domain thus released restores its antigen-binding activity and also has a short half-life (B).

[0860] The polypeptide shown in Figure 5 has several variations. In the case of using a molecule similar to the IgG antibody, the polypeptide can be produced by a production method as illustrated in Figure 6. First, a single-domain antibody (e.g., VH or VHH) that binds to the target antigen is obtained (A). The obtained single-domain antibody is associated, as a surrogate for one VH and VL of an IgG antibody having a germline sequence, with the other (VL or VH) to form a molecule similar to the IgG antibody (B). A protease cleavage sequence is introduced into the IgG antibody-like molecule (C). Examples of the insertion position include the position near the boundary. Petition 870260070097, dated 07 / 15 / 2026, p. 168 / 455 162 / 202 between the lodged single-domain antibody (VH or VHH) and the constant region (CH1 or CL).

[0861] Single-domain antibodies have antigen-binding activity when existing alone, but lose their antigen-binding activity upon the formation of a variable region with VL, VH, VHH, or similar. VL or VH is a natural human antibody sequence having a germline sequence and therefore has a low risk of immunogenicity and is unlikely to induce an anti-drug antibody recognizing its VL or VH. In the case of the formation of a variable region of the single-domain antibody without the single-domain antibody with VHH, humanization of the VHH reduces the risk of immunogenicity and reduces the likelihood of inducing an anti-drug antibody recognizing this humanized VHH. A protease cleavage sequence inserted into the IgG antibody-like molecule is cleaved by protease so that the single-domain antibody is released. The released single-domain antibody has antigen-binding activity.The IgG antibody-like molecule before protease cleavage is structurally similar to general IgG molecules and therefore has a long circulation time in the blood, whereas the single-domain antibody released by protease cleavage has a molecular weight of approximately 13 kDa without retaining an Fc region and therefore disappears rapidly by renal excretion. In fact, the half-life of full-size IgG is on the order of 2 to 3 weeks (Blood. March 31, 2016; 127(13): 1633-41), while the half-life of the single-domain antibody is approximately 2 hours (Antibodies 2015, 4(3), 141-156). Therefore, the protease-activated antigen-binding molecule has a short half-life in the blood and becomes unlikely to bind to antigen in normal tissues.

[0862] When the single-domain antibody is VL, the same Petition 870260070097, dated 07 / 15 / 2026, page 169 / 455 163 / 202 The concept as described above can be achieved, for example, by introducing a protease cleavage sequence close to the boundary between VL and CL. Example 3 - Preparation of protease-activated polypeptide using VHH binding to IL6R 3-1 Preparation of a polypeptide with VHH binding incorporated into IL6R

[0863] An expression vector encoding IL6R90-G1m (SEQ ID NO: 2) containing IL6R90 (SEQ ID NO: 1), VHH having binding and neutralizing activities against human IL6R as described in International Publication No. WO2010 / 115998, fused with a constant region of human IgG1 (CH1-hinge-CH2-CH3) was prepared by a method known to those skilled in the art.

[0864] Expression vectors encoding VK1-39-k0MT (SEQ ID NO: 3), VK2-28-k0MT (SEQ ID NO: 4), VK3-20-k0MT (SEQ ID NO: 5), VL1-40-lamL (SEQ ID NO: 6), VL1-44-lamL (SEQ ID NO: 7), VL2-14lamL (SEQ ID NO: 8), VL3-21-lamL (SEQ ID NO: 9), k0 (SEQ ID NO: 10), and lamL (SEQ ID NO: 11) as light chains (constant region of variable region) of various subclasses having a human germline sequence were prepared by a method known to those skilled in the art.

[0865] IgG molecules similar to the antibody IL6R90-G1m / VK139-k0MT (heavy chain: SEQ ID NO: 2, light chain: SEQ ID NO: 3), IL6R90-G1m / VK2-28-k0MT (heavy chain: SEQ ID NO: 2, light chain: SEQ ID NO: 4), IL6R90-G1m / VK3-20-k0MT (heavy chain: SEQ ID NO: 2, light chain: SEQ ID NO: 5), IL6R90-G1m / VL1-40-lamL (heavy chain: SEQ ID NO: 2, light chain: SEQ ID NO: 6), IL6R90-G1m / VL144-lamL (heavy chain: SEQ ID NO: 2, light chain: SEQ ID NO: 7), IL6R90-G1m / VL2-14-lamL (heavy chain: SEQ ID NO: 2, light chain: SEQ ID NO: 8), IL6R90-G1m / VL3-21-lamL (heavy chain: SEQ ID NO: Petition 870260070097, dated 07 / 15 / 2026, page 170 / 455 164 / 202 2, light chain: SEQ ID NO: 9), IL6R90-G1m / k0 (heavy chain: SEQ ID NO: 2, light chain: SEQ ID NO: 10), and IL6R90-G1m / lamL (heavy chain: SEQ ID NO: 2, light chain: SEQ ID NO: 11) were expressed by transient expression using FreeStyle 293 cells (Invitrogen Corp.) by a method known to those skilled in the art, and purified by a method known to those skilled in the art using protein A. 3-2 Evaluation of IL6R binding of a polypeptide with VHH binding incorporated into human IL6R

[0866] IL6R90-G1m / VK1-39-k0MT, IL6R90-G1m / VK2-28-k0MT, IL6R90-G1m / VK3-20-k0MT, IL6R90-G1m / VL1-40-lamL, IL6R90G1m / VL1-44-lamL, IL6R90-G1m / VL2-14-lamL, IL6R90-G1m / VL3-21lamL, IL6R90-G1m / k0, and IL6R90-G1m / lamL were evaluated for their binding activity against human IL6R using the following method.

[0867] Recombinant human IL6R used as an antigen was prepared as follows: a stably expressing soluble human IL-6R CHO strain (hereinafter also referred to as hsIL-6R, IL6R or IL-6R) consisting of an amino acid sequence from positions 1 to 357 counted from the N-terminus as reported in J. Immunol. 152, 4958-4968 (1994) was constructed by a method known to those skilled in the art, cultured, and made to express hsIL-6R. From the culture supernatant obtained, hsIL-6R was purified by 2 steps of Blue Sepharose 6 FF column chromatography and gel filtration column chromatography. A fraction eluted as a main peak in the final step was used as a final purified product.

[0868] The hsIL-6R binding assessment of each molecule was conducted using Octet HTX (Pall ForteBio Corp.). Specifically, each molecule was bound to Biosensor / Protein A (ProA) (Pall ForteBio Corp., 18-5013), and hsIL-6R was allowed to act upon it, followed by Petition 870260070097, dated 07 / 15 / 2026, page 171 / 455 165 / 202 Binding evaluation at 30°C. Sensorgrams showing real-time binding responses measured using Octet HTX are shown in Figure 10. IL6R90-G1m / k0 and IL6R90-G1m / lamL without VL bound to hsIL6R, while IL6R90-G1m / VK1-39-k0MT, IL6R90-G1m / VK2-28-k0MT, IL6R90-G1m / VK3-20-k0MT, IL6R90-G1m / VL1-40-lamL, IL6R90G1m / VL1-44-lamL, and IL6R90-G1m / VL2-14-lamL containing a variable region formed with VL were shown to be unable to bind to hsIL-6R. From this, it was discovered that VHH, having binding activity against human IL6R, can lose its IL6R binding activity by forming a variable region through association with VL. 3-3 Introduction of protease cleavage sequence into polypeptide with VHH binding incorporated into IL6R

[0869] The study was conducted to insert a protease cleavage sequence near the boundary between the VHH of antihuman IL6R90 and CH1. Six types of heavy chains shown in Figure 11 were designed so that peptide sequence A (SEQ ID NO: 12), a sequence reported to be cleavable by specifically expressed cancer urokinase (uPA) and MT-SP1, was inserted into 3 sites near the boundary between IL6R90 and CH1 with or without a glycine-serine ligand. Expression vectors encoding IL6R90H1001 (SEQ ID NO: 13), IL6R90H1002 (SEQ ID NO: 14), IL6R90H1003 (SEQ ID NO: 15), IL6R90H1004 (SEQ ID NO: 16), IL6R90H1005 (SEQ ID NO: 17), and IL6R90H1006 (SEQ ID NO: 18) were prepared by a method known to those skilled in the art.

[0870] IgG molecules similar to the antibody IL6R90H1001 / VK139-k0MT (heavy chain: SEQ ID NO: 13, light chain: SEQ ID NO: 3), IL6R90H1002 / VK1-39-k0MT (heavy chain: SEQ ID NO: 14, light chain: SEQ ID NO: 3), IL6R90H1003 / VK1-39-k0MT (heavy chain: SEQ ID NO: 15, light chain: SEQ ID NO: 3), IL6R90H1004 / VK1-39-k0MT (heavy chain: SEQ ID NO: 16, light chain: SEQ ID NO: 3), Petition 870260070097, dated 07 / 15 / 2026, page 172 / 455 166 / 202 IL6R90H1005 / VK1-39-k0MT (heavy chain: SEQ ID NO: 17, light chain: SEQ ID NO: 3), and IL6R90H1006 / VK1-39-k0MT (heavy chain: SEQ ID NO: 18, light chain: SEQ ID NO: 3) were expressed by transient expression using these heavy chains and VK1-39-k0MT (SEQ ID NO: 3) as the light chain and using FreeStyle 293 cells (Invitrogen Corp.) by a method known to those skilled in the art, and purified by a method known to those skilled in the art using protein A. 3-4 Activation of polypeptide hosting protease cleavage sequence by protease cleavage

[0871] If IL6R90H1001 / VK1-39-k0MT, IL6R90H1002 / VK1-39k0MT, IL6R90H1003 / VK1-39-k0MT, IL6R90H1004 / VK1-39-k0MT, IL6R90H1005 / VK1-39-k0MT and IL6R90H1006 / VK1-39-k0MT can release VHH, having binding activity against IL6R by protease cleavage, as has been verified.

[0872] Soluble human IL6R was prepared by a method known to those skilled in the art. The prepared soluble human IL6R was biotinylated by a method known to those skilled in the art.

[0873] For the purpose of biotin ligation to the C-terminal of soluble human IL6R (also referred to as hsIL-6R or soluble human IL6R; SEQ ID NO: 35), a gene fragment encoding a specific sequence (AviTag sequence; SEQ ID NO: 36) to be biotinylated by biotin ligase was ligated via a gene fragment encoding a ligand downstream of a gene fragment encoding hsIL-6R. A gene fragment encoding an hsIL-6R-containing protein linked to the AviTag sequence (hsIL-6R-Avitag; SEQ ID NO: 37) was integrated into a vector for expression in animal cells. The constructed plasmid vector was transfected into FreeStyle 293 cells (Invitrogen Corp.) using 293Fectin (Invitrogen Corp.). Petition 870260070097, dated 07 / 15 / 2026, page 173 / 455 167 / 202 In this operation, cells were co-transfected with a gene for EBNA1 expression (SEQ ID NO: 57) and a gene for biotin ligase expression (BirA; SEQ ID NO: 58), and biotin was also added to the latter for the purpose of biotin labeling with hsIL-6R-Avitag. Cells transfected according to the procedures mentioned above were cultured at 37°C under 8% CO2, and the protein of interest (hsIL-6R-BAP1) was secreted into the culture supernatant. This cell culture solution was filtered through a 0.22 µm bottle top filter to obtain a culture supernatant.

[0874] An anti-human IL-6R antibody was immobilized in HiTrap NHS-activated HP (GE Healthcare Japan Corp.) was prepared according to the manufacturer's protocol to prepare a column (anti-human IL-6R antibody column). The culture supernatant was applied to the TBS-equilibrated anti-human IL-6R antibody column, followed by elution of arginine-bound hsIL-6R at 2 M (pH 4.0). The eluate from the anti-human IL-6R antibody column was then diluted with TBS and applied to the TBS-equilibrated SoftLink Avidin (Promega Corp.) column, followed by elution of hsIL-6R-BAP1 with 5 mM biotin, 50 mM Tris-HCl (pH 8.0), and 2 M arginine (pH 4.0). From this eluate, hsIL-6R-BAP1 aggregates were removed by gel filtration chromatography using Superdex 200 (GE Healthcare Japan Corp.) to obtain purified hsIL-6R-BAP1 with the buffer replaced with DPBS and CHAPS at 0.05%.

[0875] Catalytic Domain of Human Matriptase / ST14 Recombinant (R&D Systems, Inc., 3946-SE-010) was used as the protease. Protease at 12.5 nM and 100 µg / mL of each IgG antibody-like molecule were incubated in PBS at 37°C for 20 hours. Subsequently, protease cleavage was evaluated by SDS-PAGE reduction. The results are shown in Figure 12. As a result, protease cleavage of a sequence of Petition 870260070097, dated 07 / 15 / 2026, page 174 / 455 168 / 202 protease cleavage near the boundary between the VHH and the constant region of the heavy chain was confirmed in IL6R90H1002 / VK1-39k0MT, IL6R90H1004 / VK1-39k0MT, IL6R90H1005 / VK1-39k0MT, and IL6R90H1006 / VK1-39k0MT.

[0876] Next, the IL6R binding assessment of VHH-released VHH was conducted using Octet HTX (Pall ForteBio Corp.). Specifically, hsIL-6R-BAP1 was ligated to a streptavidin sensor (Pall ForteBio Corp., 18-5021), and each cleaved IgG antibody-like molecule was allowed to act on it, followed by binding assessment at 30°C. Sensorgrams showing real-time binding responses measured using Octet HTX are shown in Figure 13. As a result, binding was confirmed at IL6R90H1002 / VK1-39-k0MT, IL6R90H1004 / VK1-39-k0MT, IL6R90H1005 / VK1-39-k0MT, and IL6R90H1006 / VK1-39-k0MT. IL6R90G1m / k0 and IL6R90-G1m / lamL are divalently bound with avidity, while the released VHH is bound with affinity. Therefore, IL6R90H1002 / VK1-39-k0MT, IL6R90H1004 / VK1-39-k0MT, IL6R90H1005 / VK1-39-k0MT and IL6R90H1006 / VK1-39-k0MT treated with protease exhibited a faster IL6R dissociation rate than IL6R90-G1m / k0 and IL6R90-G1m / lamL. Furthermore, VHH had a lower molecular weight than IL6R90-G1m / k0 and IL6R90-G1m / lamL. Therefore, its response, the amount of binding, was lower.

[0877] These results demonstrated that IL6R90H1002 / VK1-39k0MT, IL6R90H1004 / VK1-39-k0MT, IL6R90H1005 / VK1-39-k0MT, or IL6R90H1006 / VK1-39-k0MT do not exhibit binding activity against IL6R as is, while the peptide A sequence inserted near the boundary between the VHH and the constant heavy chain region is cleaved by protease treatment so that the VHH domain is released, and the released VHH can bind to IL6R. From this, it is concluded that the Petition 870260070097, dated 07 / 15 / 2026, p. 175 / 455 The 169 / 202 molecule conforming to the concept described in Example 2 was indeed able to be prepared. Example 4 - Preparation of protease-activated polypeptide by alteration using VHH binding to IL6R 4-1 Evaluation of IL6R binding of polypeptide with VHH-linked incorporated IL6R

[0878] An expression vector encoding 20A11-G1m (SEQ ID NO: 38) containing 20A11 (SEQ ID NO: 19), VHH having binding and neutralizing activities against IL6R as described in International Publication No. WO2010 / 115998, fused with a constant region of human IgG1 (CH1-hinge-CH2-CH3) in the same manner as in Example 3 was prepared by a method known to those skilled in the art.

[0879] The polypeptides 20A11-G1m / VK1-39-k0MT, 20A11G1m / VK2-28-k0MT, 20A11-G1m / VK3-20-k0MT, 20A11-G1m / VL1-40lamL, 20A11-G1m / VL1-44-lamL, 20A11-G1m / VL2-14-lamL, and 20A11G1m / VL3-21-lamL were expressed and purified in the same manner as in Example 3 using their heavy chain and VK1-39-k0MT (SEQ ID NO: 3), VK2-28-k0MT (SEQ ID NO: 4), VK3-20-k0MT (SEQ ID NO: 5), VL1-40-lamL (SEQ ID NO: 6), VL1-44-lamL (SEQ ID NO: 7), VL2-14lamL (SEQ ID NO: 8), and VL3-21-lamL (SEQ ID NO: 9) as light chains.

[0880] O 20A11-G1m / VK1-39-k0MT (heavy chain: SEQ ID NO: 38, light chain: SEQ ID NO: 3), 20A11-G1m / VK2-28-k0MT (heavy chain: SEQ ID NO: 38, light chain: SEQ ID NO: 4), 20A11-G1m / VK320-k0MT (heavy chain: SEQ ID NO: 38, light chain: SEQ ID NO: 5), 20A11-G1m / VL1-40-lamL (heavy chain: SEQ ID NO: 38, light chain: SEQ ID NO: 6), 20A11-G1m / VL1-44-lamL (heavy chain: SEQ ID NO: 38, light chain: SEQ ID NO: 7), 20A11-G1m / VL2-14-lamL (heavy chain: SEQ ID NO: 38, light chain: SEQ ID NO: 8), and 20A11 Petition 870260070097, dated 07 / 15 / 2026, page 176 / 455 170 / 202 The G1m / VL3-21-lamL (heavy chain: SEQ ID NO: 38, light chain: SEQ ID NO: 9) samples obtained were evaluated for their IL6R binding in the same manner as in Example 3. The results are shown in Figure 14. As a result, none of the light chains used in this Example inhibited the IL6R binding activity of 20A11 by association with the light chain containing 20A11 fused with the human germline IgG1 constant region (CH1-hingeCH2-CH3).

[0881] This is probably because 20A11 did not form a stable variable region with VL used in this Example. 4-2 Introduction of an amino acid change at the interface site between VHH and VL in the polypeptide with incorporated VHH without losing antigen binding.

[0882] In order to form a stable variable region between 20A11 and In VL, mutations were introduced into the amino acids present at the interface between 20A11 and VL. An expression vector encoding 20A11hu-G1 m (SEQ ID NO: 39) containing 20A11hu (derived from 20A11 by introducing mutations to replace F at position 37 with V (F37V), R at position 45 with L, and G at position 47 with W (all according to Kabat numbering)) (SEQ ID NO: 20) fused with a constant region of human IgG1 (CH1-hinge-CH2-CH3) in the same manner as in Example 3 was prepared by a method known to those skilled in the art.

[0883] The polypeptides 20A11hu-G1m / VK1-39-k0MT (heavy chain: SEQ ID NO: 39, light chain: SEQ ID NO: 3), 20A11huG1m / VK2-28-k0MT (heavy chain: SEQ ID NO: 39, light chain: SEQ ID NO: 4), 20A11hu-G1m / VK3-20-k0MT (heavy chain: SEQ ID NO: 39, light chain: SEQ ID NO: 5), 20A11hu-G1m / VL1-40-lamL (heavy chain: SEQ ID NO: 39, light chain: SEQ ID NO: 6), 20A11huG1m / VL1-44-lamL (heavy chain: SEQ ID NO: 39, light chain: SEQ Petition 870260070097, dated 07 / 15 / 2026, page 177 / 455 171 / 202 ID NO: 7), 20A11hu-G1m / VL2-14-lamL (heavy chain: SEQ ID NO: 39, light chain: SEQ ID NO: 8), and 20A11hu-G1m / VL3-21-lamL (heavy chain: SEQ ID NO: 39, light chain: SEQ ID NO: 9) were expressed and purified in the same way as in Example 3 using this heavy chain and VK1-39-k0MT (SEQ ID NO: 3), VK2-28-k0MT (SEQ ID NO: 4), VK3-20-k0MT (SEQ ID NO: 5), VL1-40-lamL (SEQ ID NO: 6), VL1-44-lamL (SEQ ID NO: 7), VL2-14-lamL (SEQ ID NO: 8), and VL3-21-lamL (SEQ ID NO: 9) such as light chains. 4-3 Evaluation of IL6R binding of polypeptide with incorporated VHH containing amino acid modification at the interface site between VHH and VL

[0884] The 20A11hu-G1m / VK1-39-k0MT, 20A11hu-G1m / VK2-28k0MT, 20A11hu-G1m / VK3-20-k0MT, 20A11hu-G1m / VL1-40-lamL, The 20A11hu-G1m / VL1-44-lamL, 20A11hu-G1m / VL2-14-lamL, and 20A11huG1m / VL3-21-lamL compounds obtained were evaluated for their bi...

Claims

1. A polypeptide, characterized in that it comprises an antigen-binding domain, a cleavage site, and a carrier portion, the carrier portion having an inhibitory domain that inhibits the antigen-binding activity of the antigen-binding domain, and the antigen-binding domain having a shorter half-life in blood than that of the carrier portion, wherein the cleavage site comprises a protease cleavage sequence, wherein the cleavage site is cleaved so that the association of the inhibitory domain of the carrier portion with the antigen-binding domain is canceled, wherein the antigen-binding domain comprises a single-domain antibody or is a single-domain antibody, wherein the inhibitory domain of the carrier portion associates with the antigen-binding domain and thereby inhibits the antigen-binding activity of the single-domain antibody, wherein the inhibitory domain of the carrier portion is VHH, VH of antibody,or antibody VL, in which the antigen-binding activity of the single-domain antibody is inhibited by VHH, or antibody VH, or antibody VL, in which the carrier portion comprises a constant region of antibody, and in which the protease cleavage sequence is located near the boundary between the antigen-binding domain and the constant region of the antibody.

2. Polypeptide according to claim 1, characterized in that the antigen-binding domain is capable of being released from the polypeptide, and the antigen-binding domain released from the polypeptide has greater antigen-binding activity than that before release.

3. Polypeptide according to claim 2, characterized in that the cleavage site is cleaved in such a way that the antigen-binding domain becomes capable of being released from the polypeptide.

4. Polypeptide according to any one of claims 1 to 3, characterized in that the molecular weight of the antigen-binding domain is less than that of the carrier portion.

5. Polypeptide according to any one of claims 1 to 4, characterized in that the molecular weight of the antigen-binding domain is 60 kDa or less.

6. Polypeptide according to any one of claims 1 to 5, characterized in that the single-domain antibody is VHH, VH with antigen-binding activity on its own, or VL with antigen-binding activity on its own.

7. Polypeptide according to any one of claims 1 to 6, characterized in that the single-domain antibody is VHH or VH with antigen-binding activity on its own, and the inhibitory domain of the carrier portion is the VL antibody, wherein the antigen-binding activity of VHH or VH with antigen-binding activity on its own is inhibited by association with the VL antibody.

8. Polypeptide according to any one of claims 1 to 6, characterized in that the single-domain antibody is VL, which has antigen-binding activity on its own, and the inhibitory domain of the carrier portion is the antibody VH, wherein the antigen-binding activity of VL, which has antigen-binding activity on its own, is inhibited by association with the antibody VH.

9. Polypeptide according to any one of claims 1 to 8, characterized in that the carrier portion has FcRn binding activity, and the antigen-binding domain has no FcRn binding activity or has weaker FcRn binding activity than the carrier portion. Petition 870260070097, dated 07 / 15 / 2026, p. 211 / 455 3 / 4 10. Polypeptide according to any one of claims 1 to 9, characterized in that the protease is a target tissue-specific protease.

11. Polypeptide according to any one of claims 1 to 10, characterized in that the carrier portion comprises an Fc region of an IgG antibody.

12. Polypeptide according to any one of claims 1 to 11, characterized in that the polypeptide is a molecule similar to an IgG antibody.

13. Polypeptide according to claim 12, characterized in that: (i) the single-domain antibody serving as the antigen-binding domain replaces the VH in the IgG antibody-like molecule, and the antigen-binding activity is inhibited by the VL; (ii) the single-domain antibody serving as the antigen-binding domain replaces the VL in the IgG antibody-like molecule, and the antigen-binding activity is inhibited by the VH; or (iii) the single-domain antibody serving as the antigen-binding domain replaces one of the VH or VL domains of the IgG antibody-like molecule, and an additional single-domain antibody inhibits the antigen-binding activity of the antigen-binding domain and replaces the other domain of the IgG antibody.

14. Pharmaceutical composition, characterized by comprising the polypeptide as defined in any one of claims 1 to 13.

15. Method, characterized by being for the production of the polypeptide as defined in any one of claims 1 to 13.

16. Use of the polypeptide as defined in any of claims 1 to 13, or of the composition as defined in claim 14, characterized by being for the production of a composition or medicine or drug for the treatment or prevention of a disease or for examination or diagnosis.

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