Protease-Mediated Target-Specific Cytokine Delivery Using Fusion Polypeptides - Patent application

Abstract

The present invention relates to a fusion protein comprising a ligand binding domain, a protease cleavage site, and a ligand portion. The fusion protein of the present invention comprises a ligand binding domain, a ligand portion, and a protease cleavage site, and restores the biological activity of the ligand when activated by cleavage with a protease. The present invention also relates to a method for producing the fusion protein, their use, and a pharmaceutical composition comprising the fusion protein. The present invention also relates to a method for reducing the association between the heavy chain variable domain (VH) and the light chain variable domain (VL) in the ligand binding domain, which promotes their dissociation from each other. The present disclosure provides a fusion protein in which the ligand is fused to the C-terminus of the constant region or the N-terminus of the ligand binding domain. TIFF2024527582000048.tif43128

Description

[Technical Field]

[0001] The present invention relates to fusion proteins comprising a ligand-binding domain and a ligand-binding moiety having a protease cleavage site, which, when activated by cleavage with a protease, restores the biological activity of the ligand. The present invention also relates to methods for producing the fusion proteins, their uses, and pharmaceutical compositions comprising the fusion proteins. The present invention also relates to methods for reducing the association between the heavy chain variable domain (VH) and the light chain variable domain (VL) within the ligand-binding domain, which promotes their dissociation from each other. [Background technology]

[0002] The innate capabilities of our immune system boast its potency, specificity, and memory. Motivated by these characteristics, immunotherapies have been developed in diverse areas, including infectious diseases, autoimmunity, allergy, transplant rejection, graft-versus-host disease, and cancer. Cytokines and chemokines, small proteins well known for their role in inflammation and the body's immune response to immune attack, are at the center of immunotherapy development.

[0003] However, to date, common concerns about cytokine-mediated immunotherapy remain regarding high systemic toxicity and low to negligible efficacy. Because cytokines are exposed to the entire body upon administration and therefore cause toxicity through systemic effects, cytokines can often only be administered at very low doses to avoid such toxicity. An attractive strategy to overcome this involves linking cytokines to antibodies to locally increase cytokine concentrations at the tumor site. Cytokines delivered to solid tumors by immunocytokines activate the immune system, thereby exerting antitumor effects. Because cytokines, including IL-2, IL-12, and TNF, have strong toxicity, it is expected that the localized effects of these cytokines on cancer can be enhanced while reducing adverse reactions when delivered by antibodies in a localized manner (Non-Patent Documents 1 to 3). However, it has been reported that such immunocytokines diffuse throughout the body and can therefore bind to any cell in the blood or tissues as long as a specific high-affinity cytokine receptor is present, resulting in unwarranted side effects. In certain instances, it has been reported that IL-2 fused to an antibody that binds to a cancer antigen exhibits the same antitumor effect as IL-2 fused to an antibody that does not bind to a cancer antigen, suggesting that the IL-2 moiety, rather than the antibody component, directed its biodistribution (Non-Patent Document 4).

[0004] Another alternative approach involves fusing a cytokine to its receptor via a protease-cleavable linker. In environments where protease expression is high, such as cancer environments, the linker is cleaved, liberating the cytokine from its receptor. Immunocytokines constructed in this manner include TNFα and TNF receptors connected via a linker cleavable by urokinase-type plasminogen activator (uPA) (Non-Patent Document 5), and IL-2 and IL-2 receptors cleavable by matrix metalloproteinase-2 (MMP-2) (Non-Patent Document 6). However, the cytokines in these molecules retain activity while fused to their receptors, and when activated upon protease cleavage, the improvement in activity is limited, i.e., approximately 10-fold.

[0005] More recently, various fusion polypeptides containing cytokines that are released upon protease cleavage have been reported, including, for example, single-chain fragment variable (scFv) fused to IL-2 and IL-12 that are cleavable by matrix metalloproteinases (MMPs) (Non-Patent Document 6, Non-Patent Document 7, Patent Document 2, Patent Document 5) and other fusion polypeptides containing protease-cleavable regions as reported in Patent Documents 1, 3, 4, 6, 7, and 8. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO 2009 / 025846 [Patent Document 2] WO 2011 / 123683 [Patent Document 3] WO 2018 / 097307 [Patent Document 4] WO 2019 / 107380 [Patent Document 5] WO 2019 / 010219 [Patent Document 6] WO 2019 / 010224 [Patent Document 7] WO 2020 / 061526 [Patent Document 8] WO 2021 / 016640 [Non-Patent Document]

[0007] [Non-Patent Document 1] Cyclophosphamide and tucotuzumab (huKS-IL2) following first-line chemotherapy in responding patients with extensive-disease small-cell lung cancer. Gladkov O, Ramlau R, Serwatowski P, Milanowski J, Tomeczko J, Komarnitsky PB, Kramer D, Krzakowski MJ. Anticancer Drugs. 2015 Nov; 26 (10): 1061-8. [Non-Patent Document 2] Defining the Pharmacodynamic Profile and Therapeutic Index of NHS-IL12 Immunocytokine in Dogs with Malignant Melanoma. Paoloni M, Mazcko C, Selting K, Lana S, Barber L, Phillips J, Skorupski K, Vail D, Wilson H, Biller B, Avery A, Kiupel M, LeBlanc A, Bernhardt A, Brunkhorst B, Tighe R, Khanna C. PLoS One.2015 Jun 19; 10 (6): e0129954. [Non-Patent Document 3] Isolated limb perfusion with tumor-targeting human monoclonal antibodycytokine fusion protein L19-TNF plus melphalan and mild hyperthermia in patients with locally advanced extremity melanoma. Papadia F, Basso V, Patuzzo R, MaurichiA, Di Florio A, Zardi L, Ventura E, Gonzalez-Iglesias R, Lovato V, Giovannoni L, Tasciotti A, Neri D, Santinami M, Menssen HD, De Cian F. J Surg Oncol. 2013 Feb; 107(2): 173–9

Outdoor Tools 4

Direct Environment 5

Outdoor Configuration6

[0008] It is well known that cytokines are important immune mediators present in many lesion sites, and when utilized, their effects can significantly improve immune responses. Although many cytokine-mediated immunotherapies have been developed, the problems of high toxicity and low efficacy remain concerns. [Means for solving the problem]

[0009] The inventors reasoned that the ability to deliver high doses of site-specifically activated cytokines or chemokines would overcome the problems of systemic toxicity and low efficacy. To this end, the present inventors have developed a fusion protein comprising a ligand-binding portion comprising a ligand-binding domain and a protease-cleavable site, wherein in a first state, the ligand binds to the ligand-binding domain and its ability to bind to a binding partner is attenuated, and in a second state, the ligand does not bind to the ligand-binding domain and its ability to bind to a binding partner is restored, and it is capable of exerting its biological activity upon binding. In one non-exclusive aspect, the ligand is linked to the C-terminal region of the constant region of the ligand-binding portion of the fusion protein by a non-cleavable peptide linker, and remains bound despite protease cleavage, is able to interact with its binding partner, and exerts its biological activity.

[0010] In one non-exclusive aspect, the fusion protein is a bivalent homodimeric, ligand-binding fusion protein comprising two ligand-binding moieties, each comprising a ligand-binding domain with a protease cleavage site and one ligand bound to the ligand-binding domain, including an IgG antibody-like molecule. Such fusion proteins and pharmaceutical compositions comprising the fusion proteins are useful in treating ligand-mediated diseases. Also included in one non-exclusive aspect are methods for administering the fusion proteins and pharmaceutical compositions comprising the fusion proteins for treating ligand-mediated diseases, or methods for producing the fusion proteins. The inventors have found that the activated form of the fusion protein can accumulate at high concentrations at disease sites and exhibits rapid clearance from the site compared to the native ligand. This provides the advantage of administering the fusion protein at higher doses with fewer side effects compared to the native ligand and other molecular formats described in the prior art that deliver the native ligand in its activated form.

[0011] Exemplary Embodiments Based on such findings, the present invention specifically includes the exemplary embodiments described below. [A-1] Each of them has the general formula (I) from the N-terminus to the C-terminus: [Ligand-binding domain]-[Lx]-[Cx]-[Ly]-[ligand moiety] (I) A bivalent homodimeric fusion protein comprising two polypeptides represented by During the ceremony, Lx represents a peptide linker containing a protease cleavage site; Cx represents a constant region comprising a second peptide linker and, optionally, one or more amino acid residues modified to or from cysteine; Ly represents a third peptide linker; and (a) in a first state, the ligand moiety binds to the ligand-binding domain and the biological activity of the ligand moiety is attenuated, and in a second state, the biological activity of the ligand moiety is restored; and (b) the fusion protein in the first state has a longer serum half-life than in the second state; and (c) the switch from the first state to the second state is mediated by the presence of a protease that catalyzes the protease cleavage site. The bivalent homodimeric fusion protein. [A-2] The fusion protein of [A-1], wherein the ligand-binding domain comprises an antibody variable region. [A-3] The fusion protein of [A-2], wherein the antibody variable region comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). [A-4] A fusion protein according to [A-3], in which the heavy chain variable domain (VH) and light chain variable domain (VL) of the ligand-binding domain associate with each other. [A-5] The fusion protein of [A-4], wherein Cx comprises the CH1 region of the heavy chain and the CL region of the light chain. [A-6] A fusion protein of any of [A-1] to [A-5], wherein the second peptide linker is positioned in the hinge region so as to promote disulfide bond formation between Cys at position 220 (C220) of the heavy chain and Cys at position 214 (C214) (according to EU numbering) of the light chain. [A-7] A fusion protein of any of [A-1] to [A-5], wherein Cx contains at least one amino acid modification, in which amino acid residues in the heavy chain and light chain are modified so that no disulfide bond is formed between position 220 of the heavy chain and position 214 of the light chain (according to EU numbering). [A-8] The fusion protein of [A-7], wherein the light chain contains a C214S mutation and the heavy chain contains a C220S mutation (according to EU numbering). [A-9] A fusion protein of any of [A-1] to [A-5], wherein the heavy chain is modified to enable disulfide bond formation between position 131 of the heavy chain and position 214 of the light chain (according to EU numbering). [A-10] The fusion protein of [A-9], wherein the heavy chain contains S131C and C220S modifications (according to EU numbering). [A-11] A fusion protein of any of [A-1] to [A-10], wherein Cx comprises a sequence selected from the group consisting of SEQ ID NO: 901 (C1), SEQ ID NO: 905 (C2), SEQ ID NO: 908 (C3), SEQ ID NO: 910 (C4), and SEQ ID NO: 932 (C5). [A-12] A fusion protein according to [A-11], wherein Cx comprises the sequence of SEQ ID NO: 910 (C4). [A-13] A fusion protein according to any one of [A-1] to [A-12], wherein Ly comprises a glycine-serine polymer. [A-14] The fusion protein of [A-13], wherein the glycine-serine polymer is selected from the group consisting of (a) to (ee): (a) Ser; (b) Gly Ser (GS); (c) Ser Gly(SG); (d) Gly Gly Ser (GGS); (e) Gly Ser Gly (GSG); (f) Ser Gly Gly (SGG); (g) Gly Ser Ser (GSS); (h) Ser Ser Gly (SSG); (i) Ser Gly Ser (SGS); (j) Gly Gly Gly Ser (GGGS, SEQ ID NO: 136); (k) Gly Gly Ser Gly (GGSG, SEQ ID NO: 137); (l) Gly Ser Gly Gly (GSGG, SEQ ID NO: 138); (m) Ser Gly Gly Gly (SGGG, SEQ ID NO: 139); (n) Gly Ser Ser Gly (GSSG, SEQ ID NO: 140); (o) Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 141); (p) Gly Gly Gly Ser Gly (GGGSG, SEQ ID NO: 142); (q) Gly Gly Ser Gly Gly (GGSGG, SEQ ID NO: 143); (r) Gly Ser Gly Gly Gly (GSGGG, SEQ ID NO: 144); (s) Gly Ser Gly Gly Ser (GSGGS, SEQ ID NO: 145); (t) Ser Gly Gly Gly (SGGGG, SEQ ID NO: 146); (u) Gly Ser Ser Gly Gly (GSSGG, SEQ ID NO: 147); (v) Gly Ser Gly Ser Gly (GSGSG, SEQ ID NO: 148); (w) Ser Gly Gly Ser Gly (SGGSG, SEQ ID NO: 149); (x) Gly Ser Ser Ser Gly (GSSSG, SEQ ID NO: 150); (y) Gly Gly Gly Gly Gly Ser (GGGGGS, SEQ ID NO: 151); (z) Ser Gly Gly Gly Gly (SGGGGG, SEQ ID NO: 152); (aa) Gly Gly Gly Gly Gly Ser (GGGGGGS, SEQ ID NO: 153); (bb) Ser Gly Gly Gly Gly Gly (SGGGGGG, SEQ ID NO: 154); (cc)(Gly Gly Gly Gly Ser(GGGGS, SEQ ID NO: 141))n; (dd) (Ser Gly Gly Gly Gly (SGGGG, SEQ ID NO: 146))n; and (ee)(Gly Gly Ser Gly Gly (GGSGG, SEQ ID NO: 143))n; Here, n is an integer of 1 or more. [A-15] A fusion protein of [A-14], wherein Ly comprises the sequence GGSGGSGGSGGSGGSGGS (sequence number: 903). [A-16] The fusion protein according to any one of [A-1] to [A-15], wherein the ligand portion comprises a cytokine or a chemokine. [A-17] The fusion protein of [A-16], wherein the ligand portion is selected from the group consisting of CXCL9, CXCL10, CXCL11, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-22, IFN-α, IFN-β, IFN-γ, MIG, I-TAC, RANTES, MIP-1a, MIP-1b, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra. [A-18] The fusion protein of [A-17], wherein the ligand moiety is IL-12 or IL-22. [A-19] The fusion protein of [A-18], wherein the IL-12 contains at least one amino acid modification that prevents proteolysis when exposed to a protease. [A-20] A fusion protein of [A-19], wherein the IL-12 does not contain the amino acid sequence of KSKREK (sequence number: 1102). [A-21] The fusion protein of [A-19] or [A-20], wherein the at least one amino acid modification occurs at the interface between the IL-12 and the ligand-binding domain. [A-22] The fusion protein of [A-21], wherein after the at least one amino acid modification is performed, IL-12 comprises a modified sequence selected from the group consisting of (a) to (p): (a) KSHRE (SEQ ID NO: 1052); (b) KSHHE (SEQ ID NO: 1053); (c) KSHKE (SEQ ID NO: 1054); (d) KSHSE (SEQ ID NO: 1055); (e) KSKHRE (SEQ ID NO: 1056); (f) KSKQRE (SEQ ID NO: 1057); (g) KSKERE (SEQ ID NO: 1058); (h) KSKPRE (SEQ ID NO: 1059); (i) KHKE (SEQ ID NO: 1060); (j) KHHE (SEQ ID NO: 1061); (k) KHRE (SEQ ID NO: 1062); (l) KKHE (SEQ ID NO: 1063); (m) KRHE (SEQ ID NO: 1064); (n) KRE (SEQ ID NO: 1065); (o) KHE (SEQ ID NO: 1066); and (p)KKE (SEQ ID NO: 1067). [A-23] The fusion protein of any one of [A-19] to [A-22], wherein the IL-12 comprises a sequence selected from the group consisting of (i) to (xvi): (i) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1068; (ii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1069; (iii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1070; (iv) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1071; (v) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1072; (vi) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1073; (vii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1074; (viii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1075; (ix) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1076; (x) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1077; (xi) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1078; (xii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1079; (xiii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1080; (xiv) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1081; (xv) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1082; and (xvi) An amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1083. [A-24] The fusion protein of [A-23], wherein the IL-12 comprises a sequence selected from the group consisting of (i) to (xvi): (i) an amino acid sequence identical to SEQ ID NO: 1068; (ii) an amino acid sequence identical to SEQ ID NO: 1069; (iii) an amino acid sequence identical to SEQ ID NO: 1070; (iv) an amino acid sequence identical to SEQ ID NO: 1071; (v) an amino acid sequence identical to SEQ ID NO: 1072; (vi) an amino acid sequence identical to SEQ ID NO: 1073; (vii) an amino acid sequence identical to SEQ ID NO: 1074; (viii) an amino acid sequence identical to SEQ ID NO: 1075; (ix) an amino acid sequence identical to SEQ ID NO: 1076; (x) an amino acid sequence identical to SEQ ID NO: 1077; (xi) an amino acid sequence identical to SEQ ID NO: 1078; (xii) an amino acid sequence identical to SEQ ID NO: 1079; (xiii) an amino acid sequence identical to SEQ ID NO: 1080; (xiv) an amino acid sequence identical to SEQ ID NO: 1081; (xv) an amino acid sequence identical to SEQ ID NO: 1082; and (xvi) An amino acid sequence identical to SEQ ID NO: 1083. [A-25] A fusion protein of [A-24], wherein the IL-12 comprises a sequence selected from SEQ ID NO: 1068, or SEQ ID NO: 1069, or SEQ ID NO: 1076, or SEQ ID NO: 1077, or SEQ ID NO: 1078, or SEQ ID NO: 1079, or SEQ ID NO: 1080. [A-26] A fusion protein of any of [A-1] to [A-25], wherein the fusion protein contains two protease cleavage sites, and each protease cleavage site is independently cleavable by a protease specific to a target tissue. [A-27] The fusion protein of [A-26], wherein the target tissue is cancer tissue or inflammatory tissue. [A-28] A fusion protein according to any one of [A-1] to [A-27], wherein each protease cleavage site is cleavable by the same protease. [A-29] The fusion protein of [A-28], wherein each protease cleavage site contains the same protease cleavage sequence. [A-30] A fusion protein of any of [A-1] to [A-29], wherein each protease cleavage site is independently cleavable by a protease selected from the group consisting of matriptase, urokinase-type plasminogen activator (uPA), and matrix metalloproteinase (MMP). [A-31] A fusion protein of any of [A-1] to [A-30], wherein Lx contains a protease cleavage site located near the boundary between the VH region and the CH1 region or near the boundary between the VL region and the CL region. [A-32] A fusion protein of any of [A-1] to [A-31], wherein the ligand-binding domain contains at least one amino acid modification that reduces the association between VH and VL in the second state compared to the first state. [A-33] A fusion protein according to [A-32], wherein the modification is a substitution of an amino acid present at the interface between VH and VL, and the amino acid residue for the modification is in the framework region (FR). [A-34] The fusion protein of [A-33], wherein the substitution is selected from positions 37, 45, 91, or 103 on VH and / or positions 43, 46, 49, or 87 on VL (according to Kabat numbering). [A-34a] The fusion protein of [A-33], wherein the substitution is selected from positions V37, L45, H91, or Y91, or W103 on VH, and / or positions A43, L46, Y49, or Y87 on VL (according to Kabat numbering). [A-35] A fusion protein of [A-34] or [A-34a] in which each of the positions is substituted with either A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y. [A-36] The fusion protein of [A-35], wherein the substitution is selected from positions (according to Kabat numbering) including any one or more of the following: V37S on VH, L45Q, Y91M or H91A, W103I, W103L, or W103M, and / or A43Q on VL, L46Q, Y49A, or Y87L. [A-37] A fusion protein according to any one of [A-34] to [A-36], wherein the substitution further includes at least one modification in an amino acid present at the interface between the ligand-binding domain and the ligand portion, and the amino acid residue for the modification is in a complementarity-determining region (CDR). [A-38] The fusion protein of [A-37], wherein the ligand moiety is IL-12 and the substitution further includes at least one modification selected from position 30 on VL and / or position 100a on VH (according to Kabat numbering). [A-39] The fusion protein of [A-38], wherein the modification is a substitution selected from S30V and / or F100aI (according to Kabat numbering). [A-40] The fusion protein of any one of [A-34] to [A-39], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (z) according to the Kabat numbering system: (a) L46Q and Y49A on VL; (b) H91A on VH and L46Q and Y49A on VL; (c) Y91M on VH and A43Q and Y49A on VL; (d) Y91M on VH and A43Q, L46Q, and Y49A on VL; (e) W103M on VH and A43Q and Y49A on VL; (f) W103M on VH and L46Q and Y49A on VL; (g) V37S on VH and A43Q and Y49A on VL; (h) V37S on VH and L46Q and Y49A on VL; (i) L45Q on VH and A43Q and Y49A on VL; (j) L45Q on VH and L46Q and Y49A on VL; (k) F100aI on VH and A43Q and Y49A on VL; (l) F100aI on VH and A43Q, L46Q, and Y49A on VL; (m) W103L on VH and S30V, L46Q, and Y49A on VL; (n) W103M on VH and S30V, L46Q, and Y49A on VL; (o) V37S and F100aI on VH and S30V, A43Q, and Y49A on VL; (p) V37S and F100aI on VH and S30V, L46Q, and Y49A on VL; (q) W103L on VH and L46Q and Y49A on VL; (r) W103I on VH and L46Q and Y49A on VL; (s) W103M on VH and Y49A and Y87L on VL; (t) W103L on VH and Y49A and Y87L on VL; (u) W103L on VH and S30V, Y49A, and Y87L on VL; (v) V37S and F100aI on VH and L46Q and Y49A on VL; (w) V37S and F100aI on VH and Y49A and Y87L on VL; (x) V37S and F100aI on VH and S30V, Y49A, and Y87L on VL; (y) V37S, F100aI, and W103M on VH and L46Q and Y49A on VL; and (z) V37S, F100aI, and W103L on VH, and L46Q and Y49A on VL. [A-41] The fusion protein of [A-40], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (g) according to the Kabat numbering system: (a) W103M on VH and L46Q and Y49A on VL; (b) W103L on VH and S30V, L46Q, and Y49A on VL; (c) V37S and F100aI on VH and S30V, L46Q, and Y49A on VL; (d) W103L on VH and L46Q and Y49A on VL; (e) V37S and F100aI on VH and L46Q and Y49A on VL; (f) V37S, F100aI, and W103M on VH and L46Q and Y49A on VL; and (g) V37S, F100aI, and W103L on VH, and L46Q and Y49A on VL. [A-42] A fusion protein according to any one of [A-1] to [A-41], wherein the molecular weight of the fusion protein in the second state is smaller than the molecular weight of the fusion protein in the first state. [A-43] A fusion protein according to any one of [A-1] to [A-42], wherein the cleavage site is cleaved so that a portion of the ligand-binding domain is released from the fusion protein. [A-44] The fusion protein of [A-43], wherein the molecular weight of the portion of the ligand-binding domain released from the fusion protein is 26 kDa, 13 kDa, or smaller. [A-45] A fusion protein according to any one of [A-42] to [A-44], wherein the ratio of the molecular weight of the fusion protein in the first state to the molecular weight of the fusion protein in the second state is 10:9. [A-46] A fusion protein of [A-42] to [A-44], wherein the molecular weight of the fusion protein in the second state is 9 / 10 of the molecular weight of the fusion protein in the first state. [A-47] A fusion protein of any of [A-42] to [A-44], wherein the percentage reduction in molecular weight of the fusion protein in the second state compared to the fusion protein in the first state is 10%. [A-48] A fusion protein according to any one of [A-43] to [A-47], wherein the portion of the ligand-binding domain released from the fusion protein comprises VL or VH, or is preferably VL or VH. [A-49] A fusion protein of any of [A-32] to [A-48], wherein the reduction in association between VH and VL in the second state compared to the first state can be expressed by a percentage reduction in maximum RU, when measured under surface plasma resonance (SPR) comparing the response units (RU) of the fusion protein before and after protease cleavage, of 1% or less, or 2% or less, or 3% or less, or 4% or less, or 5% or less, or 6% or less, or 7% or less, or 8% or less, or 9% or less, or 10% or less, or 11% or less, or 12% or less, or 13% or less, or 14% or less, or 15% or less, or 16% or less, or 17% or less, or 18% or less, or 19% or less, or 20% or less. [A-50] A fusion protein of any of [A-32] to [A-49], wherein the reduction in association between VH and VL in the second state compared to the first state can be expressed by a percentage reduction in maximum response units (RU) of 1% or less, or 2% or less, or 3% or less, or 4% or less, or 5% or less, or 6% or less, or 7% or less, when measured under surface plasma resonance (SPR) comparing the RU of the fusion protein before and after protease cleavage. [A-51] The reduction in association between VH and VL in the second state compared to the first state, as measured under surface plasma resonance (SPR) comparing the response units (RU) of the fusion protein before and after protease cleavage, is 15% or less, or 16% or less, or 17% or less, or 18% or less, or 19% or less, or 20% or less, or 21% or less, or 22% or less, or 23% or less, or 24% or less, or 25% or less, or 26% or less. The fusion protein of any of [A-32] to [A-48], which can be expressed by a percentage reduction in maximum RU, which is 27% or less, 28% or less, 29% or less, 30% or less, 31% or less, 32% or less, 33% or less, 34% or less, 35% or less, 36% or less, 37% or less, 38% or less, 39% or less, or 40% or less. [A-52] A fusion protein according to any one of [A-49] to [A-51], wherein the SPR conditions include a contact duration of the fusion protein in the first state with 400 nM uPA for a duration of 30 minutes. [A-53] The percentage of released VH or VL is determined by the following formula (II): % Release of VH or VL = % Reduction in RU × 100 / D (II) is directly proportional to the percentage change in response units (RU) of the fusion protein in the second state compared to the first state, as measured under SPR, according to the formula: where D corresponds to 0.01 × the percentage of the molecular weight of VH or VL, respectively, compared to the molecular weight of the fusion protein in the first state. [A-54] The percentage of released VH or VL is determined by the following formula (II-1): % VH or VL release = % RU reduction × 100 / 10 (II-1) According to [A-53], the fusion protein of [A-53] is directly proportional to the percentage change in response units (RU) of the fusion protein in the second state compared to the first state, as measured under SPR. [A-55] The percentage of released VH or VL is determined by the formula (II-2): % VH or VL release = % RU reduction × 100 / 15.8 (II-2) According to [A-53], the fusion protein of [A-53] is directly proportional to the percentage change in response units (RU) of the fusion protein in the second state compared to the first state, as measured under SPR. [A-56] A fusion protein of any of [A-53] to [A-55], wherein the percentage of released VH or VL is 10% or more, or 20% or more, or 30% or more, or 40% or more, or 50% or more, or 60% or more, or 70% or more, or 80% or more, or 90% or more, or 100% or more. [A-57] A fusion protein of any of [A-1] to [A-56], wherein the ligand portion in the first state and the second state remains bound to the constant region via a third peptide linker. [B-1] Each of them, (i) a ligand-binding portion comprising a ligand-binding domain and a constant region; (ii) a first peptide linker that contains a protease cleavage site and connects the ligand-binding domain to the constant region; (iii) the constant region, including a second peptide linker and, optionally, one or more amino acid residues modified from or to cysteine; and (iv) a ligand portion connected to the C-terminal region of the constant region by a third peptide linker; a bivalent homodimeric fusion protein comprising two polypeptides, comprising: (a) in a first state, the ligand moiety binds to the ligand-binding domain and the biological activity of the ligand moiety is attenuated, and in a second state, the biological activity of the ligand moiety is restored; (b) the fusion protein in the first state has a longer serum half-life than in the second state; and (c) the switch from the first state to the second state is mediated by the presence of a protease that catalyzes the protease cleavage site. The bivalent homodimeric fusion protein. [B-2] The fusion protein of [B-1], wherein the ligand-binding domain comprises an antibody variable region. [B-3] The fusion protein of [B-2], wherein the antibody variable region comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). [B-4] A fusion protein according to [B-3], in which the heavy chain variable domain (VH) and light chain variable domain (VL) of the ligand-binding domain associate with each other. [B-5] The fusion protein of [B-4], wherein the constant region of the ligand-binding portion comprises a heavy chain and a light chain, the heavy chain comprising a CH1 region, and the light chain comprising a CL region. [B-6] A fusion protein according to any one of [B-1] to [B-5], wherein the second peptide linker is positioned in the hinge region so as to promote disulfide bond formation between Cys at position 220 (C220) of the heavy chain and Cys at position 214 (C214) (according to EU numbering) of the light chain. [B-7] A fusion protein according to any one of [B-1] to [B-5], wherein the constant region contains at least one amino acid modification, in which an amino acid residue in the heavy chain and the light chain has been modified so that no disulfide bond is formed between position 220 of the heavy chain and position 214 of the light chain (according to EU numbering). [B-8] The fusion protein of [B-7], wherein the light chain contains a C214S modification and the heavy chain contains a C220S modification (according to EU numbering). [B-9] A fusion protein of any of [B-1] to [B-5], wherein the heavy chain is modified to enable disulfide bond formation between position 131 of the heavy chain and position 214 of the light chain (according to EU numbering). [B-10] The fusion protein of [B-10], wherein the heavy chain contains S131C and C220S modifications (according to EU numbering). [B-11] A fusion protein of any of [B-1] to [B-10], wherein the constant region comprises a sequence selected from the group consisting of SEQ ID NO: 901 (C1), SEQ ID NO: 905 (C2), SEQ ID NO: 908 (C3), SEQ ID NO: 910 (C4), and SEQ ID NO: 932 (C5). [B-12] The fusion protein of [B-11], wherein the constant region comprises the sequence of SEQ ID NO: 910 (C4). [B-13] The fusion protein of any one of [B-1] to [B-12], wherein the third peptide linker contains a glycine-serine polymer. [B-14] The fusion protein of [B-13], wherein the glycine-serine polymer is selected from the group consisting of (a) to (ee): (a) Ser; (b) Gly Ser (GS); (c) Ser Gly(SG); (d) Gly Gly Ser (GGS); (e) Gly Ser Gly (GSG); (f) Ser Gly Gly (SGG); (g) Gly Ser Ser (GSS); (h) Ser Ser Gly (SSG); (i) Ser Gly Ser (SGS); (j) Gly Gly Gly Ser (GGGS, SEQ ID NO: 136); (k) Gly Gly Ser Gly (GGSG, SEQ ID NO: 137); (l) Gly Ser Gly Gly (GSGG, SEQ ID NO: 138); (m) Ser Gly Gly Gly (SGGG, SEQ ID NO: 139); (n) Gly Ser Ser Gly (GSSG, SEQ ID NO: 140); (o) Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 141); (p) Gly Gly Gly Ser Gly (GGGSG, SEQ ID NO: 142); (q) Gly Gly Ser Gly Gly (GGSGG, SEQ ID NO: 143); (r) Gly Ser Gly Gly Gly (GSGGG, SEQ ID NO: 144); (s) Gly Ser Gly Gly Ser (GSGGS, SEQ ID NO: 145); (t) Ser Gly Gly Gly (SGGGG, SEQ ID NO: 146); (u) Gly Ser Ser Gly Gly (GSSGG, SEQ ID NO: 147); (v) Gly Ser Gly Ser Gly (GSGSG, SEQ ID NO: 148); (w) Ser Gly Gly Ser Gly (SGGSG, SEQ ID NO: 149); (x) Gly Ser Ser Ser Gly (GSSSG, SEQ ID NO: 150); (y) Gly Gly Gly Gly Gly Ser (GGGGGS, SEQ ID NO: 151); (z) Ser Gly Gly Gly Gly (SGGGGG, SEQ ID NO: 152); (aa) Gly Gly Gly Gly Gly Ser (GGGGGGS, SEQ ID NO: 153); (bb) Ser Gly Gly Gly Gly Gly (SGGGGGG, SEQ ID NO: 154); (cc)(Gly Gly Gly Gly Ser(GGGGS, SEQ ID NO: 141))n; (dd) (Ser Gly Gly Gly Gly (SGGGG, SEQ ID NO: 146))n; and (ee)(Gly Gly Ser Gly Gly (GGSGG, SEQ ID NO: 143))n; Here, n is an integer of 1 or more. [B-15] The fusion protein of [B-14], wherein the third peptide linker comprises the sequence GGSGGSGGSGGSGGSGGS (sequence number: 903). [B-16] The fusion protein according to any one of [B-1] to [B-15], wherein the ligand portion comprises a cytokine or a chemokine. [B-17] The fusion protein of [B-16], wherein the ligand moiety is selected from the group consisting of CXCL9, CXCL10, CXCL11, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-22, IFN-α, IFN-β, IFN-γ, MIG, I-TAC, RANTES, MIP-1a, MIP-1b, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra. [B-18] The fusion protein of [B-17], wherein the ligand moiety is IL-12 or IL-22. [B-19] The fusion protein of [B-18], wherein the IL-12 contains at least one amino acid modification that prevents proteolysis when exposed to a protease. [B-20] The fusion protein of [B-19], wherein the IL-12 does not contain the amino acid sequence of KSKREK (sequence number: 1102). [B-21] The fusion protein of [B-19] or [B-20], wherein the at least one amino acid modification occurs at the interface between the IL-12 and the ligand-binding domain. [B-22] The fusion protein of [B-21], wherein after the at least one amino acid modification is performed, IL-12 comprises a modified sequence selected from the group consisting of (a) to (p): (a) KSHRE (SEQ ID NO: 1052); (b) KSHHE (SEQ ID NO: 1053); (c) KSHKE (SEQ ID NO: 1054); (d) KSHSE (SEQ ID NO: 1055); (e) KSKHRE (SEQ ID NO: 1056); (f) KSKQRE (SEQ ID NO: 1057); (g) KSKERE (SEQ ID NO: 1058); (h) KSKPRE (SEQ ID NO: 1059); (i) KHKE (SEQ ID NO: 1060); (j) KHHE (SEQ ID NO: 1061); (k) KHRE (SEQ ID NO: 1062); (l) KKHE (SEQ ID NO: 1063); (m) KRHE (SEQ ID NO: 1064); (n) KRE (SEQ ID NO: 1065); (o) KHE (SEQ ID NO: 1066); and (p)KKE (SEQ ID NO: 1067). [B-23] The fusion protein of any one of [B-19] to [B-22], wherein the IL-12 comprises a sequence selected from the group consisting of (i) to (xvi): (i) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1068; (ii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1069; (iii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1070; (iv) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1071; (v) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1072; (vi) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1073; (vii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1074; (viii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1075; (ix) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1076; (x) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1077; (xi) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1078; (xii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1079; (xiii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1080; (xiv) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1081; (xv) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1082; and (xvi) An amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1083. [B-24] The fusion protein of [B-23], wherein the IL-12 comprises a sequence selected from the group consisting of (i) to (xvi): (i) an amino acid sequence identical to SEQ ID NO: 1068; (ii) an amino acid sequence identical to SEQ ID NO: 1069; (iii) an amino acid sequence identical to SEQ ID NO: 1070; (iv) an amino acid sequence identical to SEQ ID NO: 1071; (v) an amino acid sequence identical to SEQ ID NO: 1072; (vi) an amino acid sequence identical to SEQ ID NO: 1073; (vii) an amino acid sequence identical to SEQ ID NO: 1074; (viii) an amino acid sequence identical to SEQ ID NO: 1075; (ix) an amino acid sequence identical to SEQ ID NO: 1076; (x) an amino acid sequence identical to SEQ ID NO: 1077; (xi) an amino acid sequence identical to SEQ ID NO: 1078; (xii) an amino acid sequence identical to SEQ ID NO: 1079; (xiii) an amino acid sequence identical to SEQ ID NO: 1080; (xiv) an amino acid sequence identical to SEQ ID NO: 1081; (xv) an amino acid sequence identical to SEQ ID NO: 1082; and (xvi) An amino acid sequence identical to SEQ ID NO: 1083. [B-25] The fusion protein of [B-24], wherein the IL-12 comprises a sequence selected from SEQ ID NO: 1068, or SEQ ID NO: 1069, or SEQ ID NO: 1076, or SEQ ID NO: 1077, or SEQ ID NO: 1078, or SEQ ID NO: 1079, or SEQ ID NO: 1080. [B-26] A fusion protein according to any one of [B-1] to [B-25], wherein the fusion protein contains two protease cleavage sites, each of which is independently cleavable by a protease specific to a target tissue. [B-27] The fusion protein of [B-26], wherein the target tissue is cancer tissue or inflammatory tissue. [B-28] A fusion protein according to any one of [B-1] to [B-27], wherein each protease cleavage site is cleavable by the same protease. [B-29] The fusion protein of [B-28], wherein each protease cleavage site contains the same protease cleavage sequence. [B-30] A fusion protein of any of [B-1] to [B-29], wherein each protease cleavage site is independently cleavable by a protease selected from the group consisting of matriptase, urokinase-type plasminogen activator (uPA), and matrix metalloproteinase (MMP). [B-31] A fusion protein according to any one of [B-1] to [B-30], wherein the first peptide linker contains a protease cleavage site located near the boundary between the VH region and the CH1 region or near the boundary between the VL region and the CL region. [B-32] A fusion protein according to any one of [B-1] to [B-31], wherein the ligand-binding domain contains at least one amino acid modification that reduces the association between VH and VL in the second state compared to the first state. [B-33] A fusion protein according to [B-32], wherein the modification is a substitution of an amino acid present at the interface between VH and VL, and the amino acid residue for the modification is in the framework region (FR). [B-34] The fusion protein of [B-33], wherein the substitution is selected from positions 37, 45, 91, or 103 on VH and / or positions 43, 46, 49, or 87 on VL (according to Kabat numbering). [B-34a] The fusion protein of [B-33], wherein the substitution is selected from positions V37, L45, H91, or Y91, or W103 on VH, and / or positions A43, L46, Y49, or Y87 on VL (according to Kabat numbering). [B-35] A fusion protein of [B-34] or [B-34a] in which each of the positions is substituted with either A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y. [B-36] The fusion protein of [B-35], wherein the substitution is selected from positions (according to Kabat numbering) including any one or more of the following: V37S on VH, L45Q, Y91M or H91A, W103I, W103L, or W103M, and / or A43Q on VL, L46Q, Y49A, or Y87L. [B-37] A fusion protein according to any one of [B-34] to [B-36], wherein the substitution further includes at least one modification in an amino acid present at the interface between the ligand-binding domain and the ligand portion, and the amino acid residue for the modification is in a complementarity-determining region (CDR). [B-38] The fusion protein of [B-37], wherein the ligand moiety is IL-12 and the substitution further comprises at least one modification selected from position 30 on VL and / or position 100a on VH (according to Kabat numbering). [B-39] The fusion protein of [B-38], wherein the modification is a substitution selected from S30V and / or F100aI (according to Kabat numbering). [B-40] The fusion proteins of [B-34] to [B-39], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (z) according to the Kabat numbering system: (a) L46Q and Y49A on VL; (b) H91A on VH and L46Q and Y49A on VL; (c) Y91M on VH and A43Q and Y49A on VL; (d) Y91M on VH and A43Q, L46Q, and Y49A on VL; (e) W103M on VH and A43Q and Y49A on VL; (f) W103M on VH and L46Q and Y49A on VL; (g) V37S on VH and A43Q and Y49A on VL; (h) V37S on VH and L46Q and Y49A on VL; (i) L45Q on VH and A43Q and Y49A on VL; (j) L45Q on VH and L46Q and Y49A on VL; (k) F100aI on VH and A43Q and Y49A on VL; (l) F100aI on VH and A43Q, L46Q, and Y49A on VL; (m) W103L on VH and S30V, L46Q, and Y49A on VL; (n) W103M on VH and S30V, L46Q, and Y49A on VL; (o) V37S and F100aI on VH and S30V, A43Q, and Y49A on VL; (p) V37S and F100aI on VH and S30V, L46Q, and Y49A on VL; (q) W103L on VH and L46Q and Y49A on VL; (r) W103I on VH and L46Q and Y49A on VL; (s) W103M on VH and Y49A and Y87L on VL; (t) W103L on VH and Y49A and Y87L on VL; (u) W103L on VH and S30V, Y49A, and Y87L on VL; (v) V37S and F100aI on VH and L46Q and Y49A on VL; (w) V37S and F100aI on VH and Y49A and Y87L on VL; (x) V37S and F100aI on VH and S30V, Y49A, and Y87L on VL; (y) V37S, F100aI, and W103M on VH and L46Q and Y49A on VL; and (z) V37S, F100aI, and W103L on VH, and L46Q and Y49A on VL. [B-41] The fusion protein of [B-40], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (g) according to the Kabat numbering system: (a) W103M on VH and L46Q and Y49A on VL; (b) W103L on VH and S30V, L46Q, and Y49A on VL; (c) V37S and F100aI on VH and S30V, L46Q, and Y49A on VL; (d) W103L on VH and L46Q and Y49A on VL; (e) V37S and F100aI on VH and L46Q and Y49A on VL; (f) V37S, F100aI, and W103M on VH and L46Q and Y49A on VL; and (g) V37S, F100aI, and W103L on VH, and L46Q and Y49A on VL. [B-42] A fusion protein according to any one of [B-1] to [B-41], wherein the molecular weight of the fusion protein in the second state is smaller than the molecular weight of the fusion protein in the first state. [B-43] A fusion protein according to any one of [B-1] to [B-42], wherein the cleavage site is cleaved so that a portion of the ligand-binding domain is released from the fusion protein. [B-44] The fusion protein of [B-43], wherein the molecular weight of the portion of the ligand-binding domain released from the fusion protein is 26 kDa, 13 kDa, or smaller. [B-45] A fusion protein according to any one of [B-42] to [B-44], wherein the ratio of the molecular weight of the fusion protein in the first state to the molecular weight of the fusion protein in the second state is 10:9. [B-46] A fusion protein according to any one of [B-42] to [B-44], wherein the molecular weight of the fusion protein in the second state is 9 / 10 of the molecular weight of the fusion protein in the first state. [B-47] A fusion protein according to any one of [B-42] to [B-44], wherein the percentage reduction in molecular weight of the fusion protein in the second state compared to the fusion protein in the first state is 10%. [B-48] The fusion protein of any one of [B-43] to [B-47], wherein the portion of the ligand-binding domain released from the fusion protein comprises VL or VH, or is preferably VL or VH. [B-49] A fusion protein of any of [B-32] to [B-48], wherein the reduction in association between VH and VL in the second state compared to the first state can be expressed by a percentage reduction in maximum RU, when measured under surface plasma resonance (SPR) comparing the reaction units (RU) of the fusion protein before and after protease cleavage, of 1% or less, or 2% or less, or 3% or less, or 4% or less, or 5% or less, or 6% or less, or 7% or less, or 8% or less, or 9% or less, or 10% or less, or 11% or less, or 12% or less, or 13% or less, or 14% or less, or 15% or less, or 16% or less, or 17% or less, or 18% or less, or 19% or less, or 20% or less. [B-50] A fusion protein of any of [B-32] to [B-49], wherein the reduction in association between VH and VL in the second state compared to the first state can be expressed as a percentage reduction in response units of 1% or less, or 2% or less, or 3% or less, or 4% or less, or 5% or less, or 6% or less, or 7% or less, when measured under surface plasma resonance (SPR) comparing the RU of the fusion protein before and after protease cleavage. [B-51] The reduction in association between VH and VL in the second state compared to the first state, as measured under surface plasma resonance (SPR) comparing response units (RU) of the fusion protein before and after protease cleavage, is 15% or less, or 16% or less, or 17% or less, or 18% or less, or 19% or less, or 20% or less, or 21% or less, or 22% or less, or 23% or less, or 24% or less, or 25% or less, or 26% or less. Any of the fusion proteins [B-32] to [B-48], which can be expressed by a percentage reduction in maximum RU, which is 27% or less, 28% or less, 29% or less, 30% or less, 31% or less, 32% or less, 33% or less, 34% or less, 35% or less, 36% or less, 37% or less, 38% or less, 39% or less, or 40% or less. [B-52] A fusion protein according to any one of [B-49] to [B-51], wherein the SPR conditions include a contact duration of the fusion protein in the first state with 400 nM uPA for a duration of 30 minutes. [B-53] The percentage of released VH or VL is determined by measuring the percentage of VH or VL expressed by the formula (II): % Release of VH or VL = % Reduction in RU × 100 / D (II) is directly proportional to the percentage change in response units (RU) of the fusion protein measured under SPR in the second state compared to the first state, according to the formula: where D corresponds to 0.01 × the percentage of the molecular weight of VH or VL, respectively, compared to the molecular weight of the fusion protein in the first state, for any of the fusion proteins [B-49] to [B-51]. [B-54] The percentage of released VH or VL is determined by the following formula (II-1): % VH or VL release = % RU reduction × 100 / 10 (II-1) According to [B-53], the fusion protein of [B-53] is directly proportional to the percentage change in response units (RU) of the fusion protein, measured under SPR, in the second state compared to the first state. [B-55] The percentage of released VH or VL is determined by the formula (II-2): % VH or VL release = % RU reduction × 100 / 15.8 (II-2) According to [B-53], the fusion protein of [B-53] is directly proportional to the percentage change in response units (RU) of the fusion protein, measured under SPR, in the second state compared to the first state. [B-56] A fusion protein of any of [B-53] to [B-55], wherein the percentage of released VH or VL is 20% or more, or 30% or more, or 40% or more, or 50% or more, or 60% or more, or 70% or more, or 80% or more, or 90% or more, or 100% or more. [B-57] A fusion protein of any of [B-1] to [B-56], wherein the ligand portion in the first state and the second state remains bound to the constant region via a third peptide linker. [C-1] A bivalent homodimeric fusion protein comprising an IgG antibody-like polypeptide fused to a ligand portion, (i) a first peptide linker containing a protease cleavage site between the boundary of (ia) the VH region and the CH1 region, or (ib) the VL region and the CL region; (ii) a second peptide linker introduced into the hinge region that connects the CH1 region of the antibody to the Fc region and optionally contains one or more amino acid residues that are modified from or to cysteine; and (iii) a third peptide linker connecting the ligand moiety to the C-terminus of the Fc region of the antibody. Including, (a) in a first state, the ligand moiety binds to the antibody variable region and the biological activity of the ligand moiety is attenuated, and in a second state, the biological activity of the ligand moiety is restored; (b) the fusion protein in the first state has a longer serum half-life than in the second state; and (c) the switch from the first state to the second state is mediated by the presence of a protease that catalyzes the protease cleavage site. The bivalent homodimeric fusion protein. [C-2] A fusion protein according to [C-1], wherein the second peptide linker is positioned in the hinge region so as to promote disulfide bond formation between Cys at position 220 (C220) of the heavy chain and Cys at position 214 (C214) of the light chain (according to EU numbering). [C-3] The fusion protein of [C-1], wherein the constant region contains at least one amino acid modification, wherein an amino acid residue in the heavy chain and the light chain is modified so that no disulfide bond is formed between position 220 of the heavy chain and position 214 of the light chain (according to EU numbering). [C-4] The fusion protein of [C-3], wherein the light chain contains a C214S modification and the heavy chain contains a C220S modification (according to EU numbering). [C-5] A fusion protein of [C-1], in which the heavy chain is modified to enable disulfide bond formation between position 131 of the heavy chain and position 214 of the light chain (according to EU numbering). [C-6] The fusion protein of [C-5], wherein the heavy chain comprises S131C and C220S modifications (according to EU numbering). [C-7] A fusion protein of any of [C-1] to [C-6], wherein the constant region comprises a sequence selected from the group consisting of SEQ ID NO: 901 (C1), SEQ ID NO: 905 (C2), SEQ ID NO: 908 (C3), SEQ ID NO: 910 (C4), and SEQ ID NO: 932 (C5). [C-8] The fusion protein of [C-7], wherein the constant region comprises the sequence of SEQ ID NO: 910 (C4). [C-9] The fusion protein of any one of [C-1] to [C-8], wherein the third peptide linker contains a glycine-serine polymer. [C-10] The fusion protein of [C-9], wherein the glycine-serine polymer is selected from the group consisting of (a) to (ee): (a) Ser; (b) Gly Ser (GS); (c) Ser Gly(SG); (d) Gly Gly Ser (GGS); (e) Gly Ser Gly (GSG); (f) Ser Gly Gly (SGG); (g) Gly Ser Ser (GSS); (h) Ser Ser Gly (SSG); (i) Ser Gly Ser (SGS); (j) Gly Gly Gly Ser (GGGS, SEQ ID NO: 136); (k) Gly Gly Ser Gly (GGSG, SEQ ID NO: 137); (l) Gly Ser Gly Gly (GSGG, SEQ ID NO: 138); (m) Ser Gly Gly Gly (SGGG, SEQ ID NO: 139); (n) Gly Ser Ser Gly (GSSG, SEQ ID NO: 140); (o) Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 141); (p) Gly Gly Gly Ser Gly (GGGSG, SEQ ID NO: 142); (q) Gly Gly Ser Gly Gly (GGSGG, SEQ ID NO: 143); (r) Gly Ser Gly Gly Gly (GSGGG, SEQ ID NO: 144); (s) Gly Ser Gly Gly Ser (GSGGS, SEQ ID NO: 145); (t) Ser Gly Gly Gly (SGGGG, SEQ ID NO: 146); (u) Gly Ser Ser Gly Gly (GSSGG, SEQ ID NO: 147); (v) Gly Ser Gly Ser Gly (GSGSG, SEQ ID NO: 148); (w) Ser Gly Gly Ser Gly (SGGSG, SEQ ID NO: 149); (x) Gly Ser Ser Ser Gly (GSSSG, SEQ ID NO: 150); (y) Gly Gly Gly Gly Gly Ser (GGGGGS, SEQ ID NO: 151); (z) Ser Gly Gly Gly Gly (SGGGGG, SEQ ID NO: 152); (aa) Gly Gly Gly Gly Gly Ser (GGGGGGS, SEQ ID NO: 153); (bb) Ser Gly Gly Gly Gly Gly (SGGGGGG, SEQ ID NO: 154); (cc)(Gly Gly Gly Gly Ser(GGGGS, SEQ ID NO: 141))n; (dd) (Ser Gly Gly Gly Gly (SGGGG, SEQ ID NO: 146))n; and (ee)(Gly Gly Ser Gly Gly (GGSGG, SEQ ID NO: 143))n; Here, n is an integer of 1 or more. [C-11] The fusion protein of [C-10], wherein the third peptide linker comprises the sequence GGSGGSGGSGGSGGSGGS (sequence number: 903). [C-12] The fusion protein according to any one of [C-1] to [C-11], wherein the ligand portion comprises a cytokine or a chemokine. [C-13] The fusion protein of [C-12], wherein the ligand moiety is selected from the group consisting of CXCL9, CXCL10, CXCL11, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-22, IFN-α, IFN-β, IFN-γ, MIG, I-TAC, RANTES, MIP-1a, MIP-1b, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra. [C-14] The fusion protein of [C-13], wherein the ligand moiety is IL-12 or IL-22. [C-15] The fusion protein of [C-14], wherein the IL-12 comprises at least one amino acid modification that prevents proteolysis when exposed to a protease. [C-16] The fusion protein of [C-15], wherein the IL-12 does not contain the amino acid sequence of KSKREK (sequence number: 1102). [C-17] The fusion protein of [C-15] or [C-16], wherein the at least one amino acid modification occurs at the interface between IL-12 and the antibody variable region. [C-18] The fusion protein of [C-17], wherein after the at least one amino acid modification is performed, IL-12 comprises a modified sequence selected from the group consisting of (a) to (p): (a) KSHRE (SEQ ID NO: 1052); (b) KSHHE (SEQ ID NO: 1053); (c) KSHKE (SEQ ID NO: 1054); (d) KSHSE (SEQ ID NO: 1055); (e) KSKHRE (SEQ ID NO: 1056); (f) KSKQRE (SEQ ID NO: 1057); (g) KSKERE (SEQ ID NO: 1058); (h) KSKPRE (SEQ ID NO: 1059); (i) KHKE (SEQ ID NO: 1060); (j) KHHE (SEQ ID NO: 1061); (k) KHRE (SEQ ID NO: 1062); (l) KKHE (SEQ ID NO: 1063); (m) KRHE (SEQ ID NO: 1064); (n) KRE (SEQ ID NO: 1065); (o) KHE (SEQ ID NO: 1066); and (p)KKE (SEQ ID NO: 1067). [C-19] The fusion protein of any one of [C-15] to [C-18], wherein the IL-12 comprises any one of the following (i) to (xvi): (i) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1068; (ii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1069; (iii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1070; (iv) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1071; (v) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1072; (vi) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1073; (vii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1074; (viii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1075; (ix) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1076; (x) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1077; (xi) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1078; (xii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1079; (xiii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1080; (xiv) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1081; (xv) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1082; and (xvi) An amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1083. [C-20] The fusion protein of [C-19], wherein the IL-12 comprises a sequence selected from the group consisting of (i) to (xvi): (i) an amino acid sequence identical to SEQ ID NO: 1068; (ii) an amino acid sequence identical to SEQ ID NO: 1069; (iii) an amino acid sequence identical to SEQ ID NO: 1070; (iv) an amino acid sequence identical to SEQ ID NO: 1071; (v) an amino acid sequence identical to SEQ ID NO: 1072; (vi) an amino acid sequence identical to SEQ ID NO: 1073; (vii) an amino acid sequence identical to SEQ ID NO: 1074; (viii) an amino acid sequence identical to SEQ ID NO: 1075; (ix) an amino acid sequence identical to SEQ ID NO: 1076; (x) an amino acid sequence identical to SEQ ID NO: 1077; (xi) an amino acid sequence identical to SEQ ID NO: 1078; (xii) an amino acid sequence identical to SEQ ID NO: 1079; (xiii) an amino acid sequence identical to SEQ ID NO: 1080; (xiv) an amino acid sequence identical to SEQ ID NO: 1081; (xv) an amino acid sequence identical to SEQ ID NO: 1082; and (xvi) An amino acid sequence identical to SEQ ID NO: 1083. [C-21] The fusion protein of [C-20], wherein the IL-12 comprises a sequence selected from SEQ ID NO: 1068, or SEQ ID NO: 1069, or SEQ ID NO: 1076, or SEQ ID NO: 1077, or SEQ ID NO: 1078, or SEQ ID NO: 1079, or SEQ ID NO: 1080. [C-22] A fusion protein of any of [C-1] to [C-21], wherein the fusion protein contains two protease cleavage sites, and each protease cleavage site is independently cleavable by a protease specific to a target tissue. [C-23] The fusion protein of [C-22], wherein the target tissue is cancer tissue or inflammatory tissue. [C-24] A fusion protein of any of [C-1] to [C-23], wherein each protease cleavage site is cleavable by the same protease. [C-25] The fusion protein of [C-24], wherein each protease cleavage site contains the same protease cleavage sequence. [C-26] A fusion protein of any of [C-1] to [C-25], wherein each protease site is independently cleavable by a protease selected from the group consisting of matriptase, urokinase-type plasminogen activator (uPA), and matrix metalloproteinase (MMP). [C-27] A fusion protein according to any one of [C-1] to [C-26], wherein the antibody variable region contains at least one amino acid modification that reduces the association between VH and VL in the second state compared to the first state. [C-28] A fusion protein according to [C-27], wherein the modification is a substitution of an amino acid present at the interface between VH and VL, and the amino acid residue for the modification is in the framework region (FR). [C-29] The fusion protein of [C-28], wherein the substitution is selected from positions 37, 45, 91, or 103 on VH, and / or positions 43, 46, 49, or 87 on VL (according to Kabat numbering). [C-29a] The fusion protein of [C-28], wherein the substitution is selected from positions V37, L45, H91, Y91, or W103 on VH, and / or positions A43, L46, Y49, or Y87 on VL (according to Kabat numbering). A fusion protein of [C-29] or [C-29a] in which each of the [C-30] positions is substituted with either A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y. [C-31] The fusion protein of [C-30], wherein the substitution is selected from positions (according to Kabat numbering) including any one or more of the following: V37S on VH, L45Q, Y91M or H91A, W103I, W103L, or W103M, and / or A43Q on VL, L46Q, Y49A, or Y87L. [C-32] A fusion protein according to any one of [C-29] to [C-31], wherein the substitution further includes at least one modification in an amino acid present at the interface between the ligand-binding domain and the ligand portion, and the amino acid residue for the modification is in a complementarity-determining region (CDR). [C-33] The fusion protein of [C-32], wherein the ligand moiety is IL-12 and the substitution further comprises at least one alteration selected from position 30 on VL and / or position 100a on VH (according to Kabat numbering). [C-34] The fusion protein of [C-33], wherein the modification is a substitution selected from S30V and / or F100aI (according to Kabat numbering). [C-35] The fusion protein of [C-29] to [C-34], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (z) according to the Kabat numbering system: (a) L46Q and Y49A on VL; (b) H91A on VH and L46Q and Y49A on VL; (c) Y91M on VH and A43Q and Y49A on VL; (d) Y91M on VH and A43Q, L46Q, and Y49A on VL; (e) W103M on VH and A43Q and Y49A on VL; (f) W103M on VH and L46Q and Y49A on VL; (g) V37S on VH and A43Q and Y49A on VL; (h) V37S on VH and L46Q and Y49A on VL; (i) L45Q on VH and A43Q and Y49A on VL; (j) L45Q on VH and L46Q and Y49A on VL; (k) F100aI on VH and A43Q and Y49A on VL; (l) F100aI on VH and A43Q, L46Q, and Y49A on VL; (m) W103L on VH and S30V, L46Q, and Y49A on VL; (n) W103M on VH and S30V, L46Q, and Y49A on VL; (o) V37S and F100aI on VH and S30V, A43Q, and Y49A on VL; (p) V37S and F100aI on VH and S30V, L46Q, and Y49A on VL; (q) W103L on VH and L46Q and Y49A on VL; (r) W103I on VH and L46Q and Y49A on VL; (s) W103M on VH and Y49A and Y87L on VL; (t) W103L on VH and Y49A and Y87L on VL; (u) W103L on VH and S30V, Y49A, and Y87L on VL; (v) V37S and F100aI on VH and L46Q and Y49A on VL; (w) V37S and F100aI on VH and Y49A and Y87L on VL; (x) V37S and F100aI on VH and S30V, Y49A, and Y87L on VL; (y) V37S, F100aI, and W103M on VH and L46Q and Y49A on VL; and (z) V37S, F100aI, and W103L on VH, and L46Q and Y49A on VL. [C-36] The fusion protein of [C-35], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (g) according to the Kabat numbering system: (a) W103M on VH and L46Q and Y49A on VL; (b) W103L on VH and S30V, L46Q, and Y49A on VL; (c) V37S and F100aI on VH and S30V, L46Q, and Y49A on VL; (d) W103L on VH and L46Q and Y49A on VL; (e) V37S and F100aI on VH and L46Q and Y49A on VL; (f) V37S, F100aI, and W103M on VH and L46Q and Y49A on VL; and (g) V37S, F100aI, and W103L on VH, and L46Q and Y49A on VL. [C-37] A fusion protein according to any one of [C-1] to [C-36], wherein the molecular weight of the fusion protein in the second state is smaller than the molecular weight of the fusion protein in the first state. [C-38] A fusion protein of any of [C-1] to [C-37], wherein the cleavage site is cleaved so that a portion of the polypeptide is released from the fusion protein. [C-39] The fusion protein of [C-38], wherein the molecular weight of the portion released from the fusion protein is 26 kDa, 13 kDa, or smaller. [C-40] A fusion protein of any one of [C-37] to [C-39], wherein the ratio of the molecular weight of the fusion protein in the first state to the molecular weight of the fusion protein in the second state is 10:9. [C-41] A fusion protein of [C-37] to [C-39], wherein the molecular weight of the fusion protein in the second state is 9 / 10 of the molecular weight of the fusion protein in the first state. [C-42] A fusion protein of any one of [C-37] to [C-39], wherein the percentage reduction in molecular weight of the fusion protein in the second state compared to the fusion protein in the first state is 10%. [C-43] The fusion protein of any one of [C-38] to [C-42], wherein the portion released from the fusion protein comprises VL or VH, or is preferably VL or VH. [C-44] A fusion protein of any of [C-27] to [C-43], wherein the reduction in the association between VH and VL in the second state compared to the first state can be expressed by a percentage reduction in maximum RU, when measured under surface plasma resonance (SPR) comparing the reaction units (RU) of the fusion protein before and after protease cleavage, of 1% or less, or 2% or less, or 3% or less, or 4% or less, or 5% or less, or 6% or less, or 7% or less, or 8% or less, or 9% or less, or 10% or less, or 11% or less, or 12% or less, or 13% or less, or 14% or less, or 15% or less, or 16% or less, or 17% or less, or 18% or less, or 19% or less, or 20% or less. [C-45] A fusion protein of any of [C-27] to [C-44], wherein the reduction in association between VH and VL in the second state compared to the first state can be expressed as a percentage reduction in response units of 1% or less, or 2% or less, or 3% or less, or 4% or less, or 5% or less, or 6% or less, or 7% or less, when measured under surface plasma resonance (SPR) comparing the RU of the fusion protein before and after protease cleavage. [C-46] The reduction in association between VH and VL in the second state compared to the first state, as measured under surface plasma resonance (SPR) comparing response units (RU) of the fusion protein before and after protease cleavage, is 15% or less, or 16% or less, or 17% or less, or 18% or less, or 19% or less, or 20% or less, or 21% or less, or 22% or less, or 23% or less, or 24% or less, or 25% or less, or 26% or less. The fusion protein of any of [C-27] to [C-43], which can be expressed by a percentage reduction in maximum RU, which is 27% or less, 28% or less, 29% or less, 30% or less, 31% or less, 32% or less, 33% or less, 34% or less, 35% or less, 36% or less, 37% or less, 38% or less, 39% or less, or 40% or less. [C-47] A fusion protein of any one of [C-27] to [C-46], wherein the SPR conditions include a contact duration of the fusion protein in the first state with 400 nM uPA for a duration of 30 minutes. [C-48] The percentage of released VH or VL is determined by the formula (II): % Release of VH or VL = % Reduction in RU × 100 / D (II) is directly proportional to the percentage change in response units (RU) of the fusion protein measured under SPR in the second state compared to the first state, according to the formula: where D corresponds to 0.01 × the percentage of the molecular weight of VH or VL, respectively, compared to the molecular weight of the fusion protein in the first state, for any of the fusion proteins [C-27] to [A-47]. [C-49] The percentage of released VH or VL is determined by the formula (II-1): % VH or VL release = % RU reduction × 100 / 10 (II-1) According to [C-48], the fusion protein is directly proportional to the percentage change in response units (RU) of the fusion protein, measured under SPR, in the second state compared to the first state. [C-50] The percentage of released VH or VL is determined by the formula (II-2): % VH or VL release = % RU reduction × 100 / 15.8 (II-2) According to [C-48], the fusion protein is directly proportional to the percentage change in response units (RU) of the fusion protein, measured under SPR, in the second state compared to the first state. [C-51] A fusion protein of any of [C-48] to [C-50], wherein the percentage of released VH or VL is 10% or more, or 20% or more, or 30% or more, or 40% or more, or 50% or more, or 60% or more, or 70% or more, or 80% or more, or 90% or more, or 100% or more. [C-52] A fusion protein of any one of [C-1] to [C-51], in which the ligand portion in the first state and the second state remains bound to the constant region via a third peptide linker. [D-1] A bivalent homodimeric fusion protein comprising IL-12, comprising any one of the following sequences (i) to (v): (i) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1084, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1085; (ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1084, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1086; (iii) a heavy chain variable domain (VH) comprising an amino acid sequence identical to SEQ ID NO: 1084, and a light chain variable domain (VL) comprising an amino acid sequence identical to SEQ ID NO: 1085; (iv) a heavy chain variable domain (VH) comprising an amino acid sequence identical to SEQ ID NO: 1084, and a light chain variable domain (VL) comprising an amino acid sequence identical to SEQ ID NO: 1086; and (v) A heavy chain variable domain and a light chain variable domain that compete with the heavy chain variable domain and the light chain variable domain described in (i) or (iv). [D-2] A bivalent homodimeric fusion protein comprising IL-12, comprising any one of the following sequences (i) to (x): (i) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1009, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1012; (ii) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1016, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1012; (iii) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1017, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1012; (iv) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1009, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1050; (v) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1016, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1050; (vi) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1017, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1050; (vii) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1009, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1088; (viii) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1016, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1088; (ix) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1017, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1088; and (x) A heavy chain variable and a light chain that compete with the heavy chain and the light chain described in any one of (i) to (ix). [D-3] A bivalent homodimeric fusion protein comprising IL-12, comprising any one of the following sequences (i) to (x): (i) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1009, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1012; (ii) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1016, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1012; (iii) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1017, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1012; (iv) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1009, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1050; (v) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1016, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1050; (vi) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1017, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1050; (vii) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1009, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1088; (viii) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1016, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1088; (ix) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1017, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1088; and (x) A heavy chain and a light chain that compete with the heavy chain and the light chain described in any one of (i) to (ix). [D-4] A bivalent homodimeric fusion protein comprising IL-22, comprising any one of the following sequences (i) to (iv): (i) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to, or identical to, SEQ ID NO: 1095, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1096; (ii) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to, or identical to, SEQ ID NO: 1097, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1098; (iii) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to, or identical to, SEQ ID NO: 1099, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1100; and (iv) A heavy chain and a light chain that compete with the heavy chain and the light chain described in any one of (i) to (iii). [D-5] A bivalent homodimeric fusion protein comprising IL-22, comprising any one of the following sequences (i) to (iii): (i) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to, or identical to, SEQ ID NO: 1091, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to, or identical to, SEQ ID NO: 1092; (ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to, or identical to, SEQ ID NO: 1093, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to, or identical to, SEQ ID NO: 1094; and (iii) A heavy chain variable domain and a light chain variable domain that compete with the heavy chain variable domain and the light chain variable domain described in (i) or (ii). [E-1] Any of the fusion proteins [A-1] to [A-57], [B-1] to [B-57], and [C-1] to [C-52], which contain any one of the following sequences (i) to (ix): (i) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1084, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1085; (ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1084, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1086; (iii) a heavy chain variable domain (VH) comprising an amino acid sequence identical to SEQ ID NO: 1084, and a light chain variable domain (VL) comprising an amino acid sequence identical to SEQ ID NO: 1085; (iv) a heavy chain variable domain (VH) comprising an amino acid sequence identical to SEQ ID NO: 1084, and a light chain variable domain (VL) comprising an amino acid sequence identical to SEQ ID NO: 1086; (v) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1091, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1092; (vi) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1093, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1094; (vii) a heavy chain variable domain (VH) comprising an amino acid sequence identical to SEQ ID NO: 1091, and a light chain variable domain (VL) comprising an amino acid sequence identical to SEQ ID NO: 1092; (viii) a heavy chain variable domain (VH) comprising an amino acid sequence identical to SEQ ID NO: 1093, and a light chain variable domain (VL) comprising an amino acid sequence identical to SEQ ID NO: 1094; and (ix) A heavy chain variable domain and a light chain variable domain that compete with the heavy chain variable domain and the light chain variable domain described in any one of (i) to (viii). [E-2] Any one of [A-1] to [A-57], [B-1] to [B-57], and [C-1] to [C-52], which contains any one of the following sequences (i) to (xiii): (i) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1009, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1012; (ii) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1016, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1012; (iii) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1017, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1012; (iv) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1009, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1050; (v) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1016, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1050; (vi) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1017, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1050; (vii) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1009, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1088; (viii) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1016, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1088; (ix) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1017, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1088; (x) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1095, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1096; (xi) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1097, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1098; (xii) a light chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1099, and a heavy chain comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1100; (xiii) A heavy chain and a light chain that compete with the heavy chain and the light chain described in any one of (i) to (xii). [E-3] A fusion protein selected from the group consisting of [A-1] to [A-57], [B-1] to [B-57], and [C-1] to [C-52], which contains any one of the following sequences (i) to (xiii): (i) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1009, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1012; (ii) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1016, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1012; (iii) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1017, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1012; (iv) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1009, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1050; (v) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1016, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1050; (vi) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1017, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1050; (vii) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1009, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1088; (viii) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1016, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1088; (ix) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1017, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1088; (x) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1095, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1096; (xi) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1097, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1098; (xii) a light chain comprising an amino acid sequence identical to SEQ ID NO: 1099, and a heavy chain comprising an amino acid sequence identical to SEQ ID NO: 1100; and (xiii) A heavy chain and a light chain that compete with the heavy chain and the light chain described in any one of (i) to (xii). A pharmaceutical composition comprising any one of the fusion proteins [E-4][A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-1] to [D-5] and [E-1] to [E-3], and [J-1] to [J-55], and a pharmaceutically acceptable carrier. [E-5] A pharmaceutical composition of any of the fusion proteins [A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-1] to [D-5] and [E-1] to [E-3], and [J-1] to [J-55], or [E-4], for use as a pharmaceutical. [E-6] A pharmaceutical composition of any of the fusion proteins [A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-1] to [D-3] and [E-1] to [E-3], and [J-1] to [J-55], or [E-4], for use in an IL-12-mediated disease or disorder. [E-7] A pharmaceutical composition of any of the fusion proteins [A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-4] or [D-5] and [E-1] to [E-3], and [J-1] to [J-47], or [E-4], for use in an IL-22-mediated disease or disorder. [E-8] A pharmaceutical composition of any of the fusion proteins [A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-1] to [D-5] and [E-3] to [E-4], and [J-1] to [J-55], or [E-4], for use in the treatment of cancer. [E-9] A pharmaceutical composition of any of the fusion proteins [A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-1] to [D-5] and [E-1] to [E-3], and [J-1] to [J-55], or [E-4], for use in the treatment of an inflammatory disease or disorder. [E-10] Use of any of the fusion proteins [A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-1] to [D-3] and [E-1] to [E-3], and [J-1] to [J-55], or the pharmaceutical composition [E-4], in the manufacture of a pharmaceutical for the treatment of an IL-12-mediated disease or disorder. [E-11] Use of any of the fusion proteins [A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-4] or [D-5] and [E-1] to [E-3], and [J-1] to [J-47], or a pharmaceutical composition of [E-4] in the manufacture of a pharmaceutical for the treatment of an IL-22-mediated disease or disorder. [E-12] Use of any of the fusion proteins [A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-1] to [D-5] and [E-1] to [E-3], and [J-1] to [J-55], or the pharmaceutical composition [E-4], in the manufacture of a pharmaceutical for the treatment of cancer. [E-13] Use of any of the fusion proteins of [A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-1] to [D-5] and [E-1] to [E-3], and [J-1] to [J-55], or the pharmaceutical composition of [E-4], in the manufacture of a pharmaceutical for the treatment of an inflammatory disease or disorder. [E-14] A method for treating an individual having an IL-12-mediated disease or disorder, comprising the step of administering an effective amount of any of the fusion proteins [A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-1] to [D-3] and [E-1] to [E-3], and [J-1] to [J-55], or the pharmaceutical composition [E-4]. [E-15] A method for treating an individual having an IL-22-mediated disease or disorder, comprising the step of administering an effective amount of any of the fusion proteins [A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-4] or [D-5] and [E-1] to [E-3], and [J-1] to [J-47], or the pharmaceutical composition [E-4]. [E-16] A method for treating an individual having cancer, comprising the step of administering an effective amount of any of the fusion proteins [A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-1] to [D-5] and [E-1] to [E-3], and [J-1] to [J-55], or the pharmaceutical composition [E-4]. [E-17] A method for treating an individual having an inflammatory disease or disorder, comprising the step of administering an effective amount of any of the fusion proteins [A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-1] to [D-5] and [E-1] to [E-3], and [J-1] to [J-55], or the pharmaceutical composition [E-4]. An isolated polynucleotide encoding any of the fusion proteins [E-18][A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-1] to [D-5] and [E-1] to [E-3], and [J-1] to [J-55]. A vector comprising the polynucleotide of [E-19][E-18]. [E-20] [E-18] A host cell containing the polynucleotide or [E-19]. [E-21] A method for producing any one of the fusion proteins [A-1] to [A-57], [B-1] to [B-57] and [C-1] to [C-52], [D-1] to [D-5] and [E-1] to [E-3], and [J-1] to [J-55], the method comprising a step of culturing the host cell of [E-20] so that the fusion protein is produced. [E-22] The method described in [E-21], comprising a step of introducing a substitution into an amino acid present at the interface between VH and VL to reduce the association between VH and VL in a second state compared to a first state, and wherein the amino acid residue for the substitution is in the framework region (FR). [E-23] The method described in [E-22], wherein the amino acid position for the substitution is selected from positions 37, 45, 91, or 103 on VH, and / or positions 43, 46, 49, or 87 on VL (according to Kabat numbering). [E-23a] The method described in [E-22], wherein the amino acid position for the substitution is selected from positions V37, L45, H91, Y91, or W103 on VH, and / or positions A43, L46, Y49, or Y87 on VL (according to Kabat numbering). [E-24] The method of [E-23] or [E-23a], wherein each of the positions is substituted with either A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y. [E-25] The method of [E-24], wherein the substitution is selected from any one or more of the following (according to Kabat numbering): V37S on VH, L45Q, Y91M or H91A, W103I, W103L, or W103M, and / or A43Q on VL, L46Q, Y49A, or Y87L. [E-26] The method described in any of [E-23] to [E-25], wherein the substitution further includes at least one substitution in an amino acid present at the interface between the ligand-binding domain and the ligand portion, and the amino acid residue for the modification is in a complementarity-determining region (CDR). [E-27] The method described in [E-26], wherein the ligand moiety is IL-12 and the substitution further comprises at least one substitution selected from position 30 on VL and / or position 100a on VH (according to Kabat numbering). [E-28] The method according to [E-27], wherein the substitution is selected from S30V and / or F100aI (according to Kabat numbering). [E-29] The method according to any one of [E-23] to [E-28], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (bb) according to the Kabat numbering system: (a) L46Q and Y49A on VL; (b) H91A on VH and L46Q and Y49A on VL; (c) Y91M on VH and A43Q and Y49A on VL; (d) Y91M on VH and A43Q, L46Q, and Y49A on VL; (e) W103M on VH and A43Q and Y49A on VL; (f) W103M on VH and L46Q and Y49A on VL; (g) W103I on VH and L46Q and Y49A on VL; (h) W103L on VH and L46Q and Y49A on VL; (i) V37S on VH and A43Q and Y49A on VL; (j) V37S on VH and L46Q and Y49A on VL; (k) L45Q on VH and A43Q and Y49A on VL; (l) L45Q on VH and L46Q and Y49A on VL; (m) F100aI on VH and A43Q and Y49A on VL; (n) F100aI on VH and A43Q, L46Q, and Y49A on VL; (o) W103L on VH and S30V, L46Q, and Y49A on VL; (p) W103M on VH and S30V, L46Q, and Y49A on VL; (q) V37S and F100aI on VH and S30V, A43Q, and Y49A on VL; (r) V37S and F100aI on VH and S30V, L46Q, and Y49A on VL; (s) W103L on VH and L46Q and Y49A on VL; (t) W103I on VH and L46Q and Y49A on VL; (u) W103M on VH and Y49A and Y87L on VL; (v) W103L on VH and Y49A and Y87L on VL; (w) W103L on VH and S30V, Y49A, and Y87L on VL; (x) V37S and F100aI on VH and L46Q and Y49A on VL; (y) V37S and F100aI on VH and Y49A and Y87L on VL; (z) V37S and F100aI on VH and S30V, Y49A, and Y87L on VL; (aa) V37S, F100aI, and W103M on VH and L46Q and Y49A on VL; and (bb) V37S, F100aI, and W103L on VH, and L46Q and Y49A on VL. [E-30] The method according to [E-29], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (g) according to the Kabat numbering system: (a) W103M on VH and L46Q and Y49A on VL; (b) W103L on VH and S30V, L46Q, and Y49A on VL; (c) V37S and F100aI on VH and S30V, L46Q, and Y49A on VL; (d) W103L on VH and L46Q and Y49A on VL; (e) V37S and F100aI on VH and L46Q and Y49A on VL; (f) V37S, F100aI, and W103M on VH and L46Q and Y49A on VL; and (g) V37S, F100aI, and W103L on VH, and L46Q and Y49A on VL. [E-31] The method described in [E-30], further comprising a step of recovering the fusion protein from the host cell. [E-32] The method according to [E-21], comprising the following steps: (a) introducing at least one amino acid modification or at least one pair of amino acid modifications at the VH / VL interface of the fusion protein, which enhances dissociation of the VH or VL from the fusion protein, and optionally introducing at least one amino acid mutation at the interface between the ligand and the ligand-binding domain; (b) confirming that step (a) does not disrupt binding of the ligand to VH and VL; (c) confirming that step (a) reduces the association of VH and VL upon protease cleavage at the protease cleavage site; and (d) linking the VH or VL of step (a) to an IgG heavy chain constant region via a protease cleavage sequence; (e) obtaining a polynucleotide encoding the fusion protein of step (e); (f) culturing a host cell containing the polynucleotide of step (e); and (g) producing and recovering the fusion protein from the host cells of step (f). [F-1] A polypeptide comprising at least one antigen-binding domain comprising a protease cleavage site, wherein upon cleavage at the protease cleavage site, antibody domains adjacent to the protease cleavage site dissociate, and the dissociation is promoted by at least one amino acid modification made at the interface between the antibody domain and a corresponding interacting domain. [F-2] The polypeptide according to [F-1], which is an antibody or an antibody fragment. [F-3] The polypeptide according to [F-2], wherein the antibody is an IgG antibody selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgG-IgG, IgG-Fab, or CrossMab antibodies. [F-4] The polypeptide described in [F-3], wherein the antibody is monovalent or bivalent. [F-5] The polypeptide according to [F-4], wherein the antibody is monospecific or bispecific. [F-6] The polypeptide described in [F-5], wherein the antibody fragment comprises an antigen-binding domain. [F-7] The polypeptide according to [F-6], wherein the antibody fragment is selected from the group consisting of scFv, scFv-Fc, tandem scFv, Fab, tandem Fab, F(ab')2, Fab2, Fab-scFv-Fc, F(ab')2-scFv2, bispecific Fab2, trispecific Fab2, bispecific diabody, trispecific diabody, tandem diabody, triabody, tetrabody, minibody, bibody, or tribody. [F-8] The polypeptide described in [F-7], wherein the antigen-binding domain comprises an antibody variable region. [F-9] A polypeptide described in [F-8], wherein the antibody variable region comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) that are associated with each other, and optionally, the VH is associated with a CH1 region and / or the VL is associated with a CL region. [F-10] The polypeptide described in [F-9], wherein the protease cleavage site is located at the boundary between the VH region and the CH1 region, or the boundary between the VL region and the CL region, or the boundary between VH and VL. [F-11] A polypeptide described in [F-10], wherein at least one amino acid modification is made at the interface between VH and VL, and the amino acid modification reduces the association between VH and VL in the cleaved state compared to the uncleaved state. [F-12] A polypeptide described in [F-11], in which at least one pair of amino acid modifications is made at the interface between VH and VL, and the amino acid modifications reduce the association between VH and VL in the cleaved state compared to the uncleaved state. [F-13] A polypeptide described in [F-11], wherein the at least one amino acid modification is a substitution of an amino acid present at the interface between VH and VL, and the amino acid residue for the substitution is in the framework region (FR). [F-14] The polypeptide described in [F-12], wherein the at least one pair of amino acid modifications is a substitution of an amino acid pair present at the interface between VH and VL. [F-15] The polypeptide described in [F-13], wherein the at least one amino acid substitution includes an amino acid substitution to achieve the same charge as the corresponding interacting amino acid at the interface between VH and VL or a neutral charge. [F-16] The polypeptide described in [F-14], wherein the pair of amino acid substitutions includes substitution of both amino acids to have the same charge or a neutral charge. [F-17] A polypeptide according to any one of [F-11] to [F-16], wherein the substitutions are selected from positions 37, 39, 44, 45, 47, 91, and 103 on VH and / or positions 38, 43, 44, 46, 49, 87, and 98 on VL (according to Kabat numbering). [F-17a] A polypeptide according to any one of [F-11] to [F-16], wherein the substitutions are selected from positions V37, Q39, G44, L45, W47, H91, Y91, and W103 on VH, and / or positions R38, A43, P44, L46, Y49, Y87, and F98 on VL (according to Kabat numbering). [F-18] The method of [F-17] or [F-17a], wherein each of the positions is substituted with either A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y. [F-19] The polypeptide of [F-18], wherein the substitution is selected from positions (according to Kabat numbering) including any one or more of the following: Q39D on VH, W47A, W47L, or W47M, Y91A, Y91L, Y91M, or H91A, W103A, W103I, W103L, or W103M, V37S or V37Q, G44Q, L45A or L45Q, and / or R38E on VL, Y49A, Y87A, Y87L, or Y87M, F98A, F98L, or F98M, A43Q, P44A, P44S, or P44Q, L46E or L46Q. [F-20] The polypeptide of [F-19], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (pp) according to the Kabat numbering system: (a) L46Q and Y49A on VL; (b) Q39D on VH and R38E on VL; (c) H91A on VH and L46Q and Y49A on VL; (d) Y91A on VH and A43Q and Y49A on VL; (e) Y91A on VH and P44A and Y49A on VL; (f) Y91A on VH and L46Q and Y49A on VL; (g) Y91A on VH and Y49A and Y87L on VL; (h) Y91M on VH and A43Q and Y49A on VL; (i) Y91M on VH and P44A and Y49A on VL; (j) Y91M on VH and L46Q and Y49A on VL; (k) Y91M on VH and Y49A and Y87L on VL; (l) Y91M on VH and Y49A and F98L on VL; (m) W103L on VH and A43Q and Y49A on VL; (n) W103L on VH and P44A and Y49A on VL; (o) W103L on VH and L46Q and Y49A on VL; (p) W103L on VH and Y49A and Y87L on VL; (q) W103I on VH and A43Q and Y49A on VL; (r) W103I on VH and P44A and Y49A on VL; (s) W103I on VH and L46Q and Y49A on VL; (t) W103M on VH and A43Q and Y49A on VL; (u) W103M on VH and P44A and Y49A on VL; (v) W103M on VH and L46Q and Y49A on VL; (w) W103M on VH and Y49A and Y87L on VL; (x) V37S on VH and A43Q and Y49A on VL; (y) V37S on VH and P44A and Y49A on VL; (z) V37S on VH and L46Q and Y49A on VL; (aa) V37S on VH and Y49A and Y87L on VL; (bb) V37S on VH and Y49A and F98L on VL; (cc) L45Q on VH and A43Q and Y49A on VL; (dd) L45Q on VH and P44A and Y49A on VL; (ee) L45Q on VH and L46Q and Y49A on VL; (ff) L45Q on VH and Y49A and Y87L on VL; (gg) L45Q on VH and Y49A and F98M on VL; (hh) Y91M on VH and A43Q, P44A, and Y49A on VL; (ii) Y91M on VH and A43Q, L46Q, and Y49A on VL; (jj) Y91M on VH and L46Q, Y49A, and Y87M on VL; (kk) V37S on VH and L46Q, Y49A, and Y87M on VL; (ll) V37S and L45Q on VH and A43Q and Y49A on VL; (mm) V37S and Y91M on VH and A43Q and Y49A on VL; (nn) V37S and W103M on VH and A43Q and Y49A on VL; (oo) V37S and Y91M on VH and L46Q and Y49A on VL; and (pp) V37S and L45Q on VH, and Y49A and Y87M on VL. [F-21] A pharmaceutical composition comprising any one of the polypeptides [F-1] to [F-20] and a pharmaceutically acceptable carrier. [F-22] A pharmaceutical composition according to [F-21] or a polypeptide according to any one of [F-1] to [F-20] for use as a pharmaceutical. [F-23] A pharmaceutical composition according to [F-21] or a polypeptide according to any one of [F-1] to [F-20] for use in treating a disease or disorder. [F-24] Use of the pharmaceutical composition according to [F-21] or the polypeptide according to any one of [F-1] to [F-20] in the manufacture of a pharmaceutical for the treatment of a disease or disorder. [F-25] A method for treating an individual having a disease or disorder, comprising the step of administering an effective amount of a pharmaceutical composition described in [F-21] or a polypeptide described in any of [F-1] to [F-20]. [F-26] An isolated polynucleotide encoding the polypeptide according to any one of [F-1] to [F-20]. A vector comprising the polynucleotide of [F-27][F-26]. [F-28] A host cell containing the polynucleotide of [F-26] or the vector of [F-27]. [F-29] A method for producing a polypeptide according to any one of [F-1] to [F-20], comprising the step of culturing the host cell of [F-28] so as to produce the polypeptide. [F-30] The method according to [F-29], comprising the following steps: (a) introducing a peptide linker comprising a protease cleavage site, wherein the protease-cleavable peptide linker connects the VH region to the CH1 region, or the VL region to the CL region, or the VH to the VL; (b) introducing at least one substitution mutation into at least one amino acid present at the interface between VH and VL to promote dissociation of VH from VL or dissociation of VL from VH; (c) confirming that step (b) does not disrupt binding of the antigen to the VH and VL; and (d) confirming that step (b) reduces the association of VH and VL upon protease cleavage at the protease cleavage site; (e) obtaining a polynucleotide encoding the polypeptide of step (d); (f) culturing a host cell containing the polynucleotide of step (e); and (g) producing and recovering the fusion protein from the host cells of step (f). [F-31] The method of [F-30], wherein the substitutions are selected from positions 37, 39, 44, 45, 47, 91, and 103 on VH and / or positions 38, 43, 44, 46, 49, 87, and 98 on VL (according to Kabat numbering). [F-31a] The method of [F-30], wherein the substitutions are selected from positions V37, Q39, G44, L45, W47, H91, Y91, and W103 on VH, and / or positions R38, A43, P44, L46, Y49, Y87, and F98 on VL (according to Kabat numbering). [F-32] The method of [F-31] or [F-31a], wherein each of the positions is substituted with either A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y. [F-33] The method of [F-32], wherein the substitution is selected from positions (according to Kabat numbering) including any one or more of the following: Q39D on VH, W47A, W47L, or W47M, Y91A, Y91L, Y91M, or H91A, W103A, W103I, W103L, or W103M, V37S or V37Q, G44Q, L45A or L45Q, and / or R38E on VL, Y49A, Y87A, Y87L, or Y87M, F98A, F98L, or F98M, A43Q, P44A, P44S, or P44Q, L46E or L46Q. [F-34] The method according to [F-33], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (pp) according to the Kabat numbering system: (a) L46Q and Y49A on VL; (b) Q39D on VH and R38E on VL; (c) H91A on VH and L46Q and Y49A on VL; (d) Y91A on VH and A43Q and Y49A on VL; (e) Y91A on VH and P44A and Y49A on VL; (f) Y91A on VH and L46Q and Y49A on VL; (g) Y91A on VH and Y49A and Y87L on VL; (h) Y91M on VH and A43Q and Y49A on VL; (i) Y91M on VH and P44A and Y49A on VL; (j) Y91M on VH and L46Q and Y49A on VL; (k) Y91M on VH and Y49A and Y87L on VL; (l) Y91M on VH and Y49A and F98L on VL; (m) W103L on VH and A43Q and Y49A on VL; (n) W103L on VH and P44A and Y49A on VL; (o) W103L on VH and L46Q and Y49A on VL; (p) W103L on VH and Y49A and Y87L on VL; (q) W103I on VH and A43Q and Y49A on VL; (r) W103I on VH and P44A and Y49A on VL; (s) W103I on VH and L46Q and Y49A on VL; (t) W103M on VH and A43Q and Y49A on VL; (u) W103M on VH and P44A and Y49A on VL; (v) W103M on VH and L46Q and Y49A on VL; (w) W103M on VH and Y49A and Y87L on VL; (x) V37S on VH and A43Q and Y49A on VL; (y) V37S on VH and P44A and Y49A on VL; (z) V37S on VH and L46Q and Y49A on VL; (aa) V37S on VH and Y49A and Y87L on VL; (bb) V37S on VH and Y49A and F98L on VL; (cc) L45Q on VH and A43Q and Y49A on VL; (dd) L45Q on VH and P44A and Y49A on VL; (ee) L45Q on VH and L46Q and Y49A on VL; (ff) L45Q on VH and Y49A and Y87L on VL; (gg) L45Q on VH and Y49A and F98M on VL; (hh) Y91M on VH and A43Q, P44A, and Y49A on VL; (ii) Y91M on VH and A43Q, L46Q, and Y49A on VL; (jj) Y91M on VH and L46Q, Y49A, and Y87M on VL; (kk) V37S on VH and L46Q, Y49A, and Y87M on VL; (ll) V37S and L45Q on VH and A43Q and Y49A on VL; (mm) V37S and Y91M on VH and A43Q and Y49A on VL; (nn) V37S and W103M on VH and A43Q and Y49A on VL; (oo) V37S and Y91M on VH and L46Q and Y49A on VL; and (pp) V37S and L45Q on VH, and Y49A and Y87M on VL. [F-35] The method according to any one of [F-28] to [F-34], further comprising a step of recovering the polypeptide from the host cell. [G-1] A bivalent homodimeric fusion protein comprising a full-length IgG antibody comprising an antigen-binding domain, wherein the antigen-binding domain comprises a variable region, the variable region comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) that associate with each other, and (a) a protease cleavage site at the boundary between the VH and CH1 domains or the boundary between the VL and CL domains of the variable regions, and (b) a ligand that binds to the variable regions, wherein upon protease cleavage, (i) either the VH or VL dissociates from the fusion protein, and (ii) the ligand dissociates from the variable regions, and the dissociation described in (i) is promoted by at least one amino acid modification made at the interface between the VH and VL that reduces the association between the VH and VL in the cleaved state compared to the uncleaved state. [G-2] The fusion protein of [G-1], wherein the full-length IgG antibody is an IgG antibody-like polypeptide. [G-3] The fusion protein of [G-2], wherein the modification is a substitution of an amino acid present at the interface between VH and VL. [G-4] A fusion protein of [G-3], in which at least one pair of amino acid substitutions is made at the interface between VH and VL, and the amino acid residues for the substitution are in the framework region (FR). [G-5] A fusion protein of [G-4], wherein the pair of amino acid substitutions includes substitution of both amino acids to have the same charge or a neutral charge. [G-6] A fusion protein according to [G-3] to [G-5], wherein the substitutions are selected from positions 37, 39, 44, 45, 47, 91, and 103 on VH and / or positions 38, 43, 44, 46, 49, 87, and 98 on VL (according to Kabat numbering). [G-6a] A fusion protein of [G-3] to [G-5], wherein the substitutions are selected from positions V37, Q39, G44, L45, W47, H91, Y91, and W103 on VH, and / or positions R38, A43, P44, L46, Y49, Y87, and F98 on VL (according to Kabat numbering). [G-7] A fusion protein of [G-6] or [G-6a] in which each of the positions is substituted with either A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y. [G-8] The fusion protein of [G-7], wherein the substitution is selected from positions (according to Kabat numbering) including any one or more of the following: Q39D on VH, W47A, W47L, or W47M, Y91A, Y91L, Y91M, or H91A, W103A, W103I, W103L, or W103M, V37S or V37Q, G44Q, L45A or L45Q, and / or R38E on VL, Y49A, Y87A, Y87L, or Y87M, F98A, F98L, or F98M, A43Q, P44A, P44S, or P44Q, L46E or L46Q. [G-9] A fusion protein of any of [G-6] to [G-8], wherein the substitution further includes at least one modification in an amino acid present at the interface between the variable region and the ligand, and the amino acid residue for the modification is in the complementarity-determining region (CDR). [G-10] A fusion protein of [G-9], wherein the ligand is IL-12 and the substitution further includes at least one modification selected from position 30 on VL and / or position 100a on VH (according to Kabat numbering). [G-11] The fusion protein of [G-10], wherein the modification is a substitution selected from S30V and / or F100aI (according to Kabat numbering). [G-12] The fusion protein of [G-11], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (hhh) according to the Kabat numbering system: (a) L46Q and Y49A on VL; (b) Q39D on VH and R38E on VL; (c) H91A on VH and L46Q and Y49A on VL; (d) Y91A on VH and A43Q and Y49A on VL; (e) Y91A on VH and P44A and Y49A on VL; (f) Y91A on VH and L46Q and Y49A on VL; (g) Y91A on VH and Y49A and Y87L on VL; (h) Y91M on VH and A43Q and Y49A on VL; (i) Y91M on VH and P44A and Y49A on VL; (j) Y91M on VH and L46Q and Y49A on VL; (k) Y91M on VH and Y49A and Y87L on VL; (l) Y91M on VH and Y49A and F98L on VL; (m) W103L on VH and A43Q and Y49A on VL; (n) W103L on VH and P44A and Y49A on VL; (o) W103L on VH and L46Q and Y49A on VL; (p) W103L on VH and Y49A and Y87L on VL; (q) W103I on VH and A43Q and Y49A on VL; (r) W103I on VH and P44A and Y49A on VL; (s) W103I on VH and L46Q and Y49A on VL; (t) W103M on VH and A43Q and Y49A on VL; (u) W103M on VH and P44A and Y49A on VL; (v) W103M on VH and L46Q and Y49A on VL; (w) W103M on VH and Y49A and Y87L on VL; (x) V37S on VH and A43Q and Y49A on VL; (y) V37S on VH and P44A and Y49A on VL; (z) V37S on VH and L46Q and Y49A on VL; (aa) V37S on VH and Y49A and Y87L on VL; (bb) V37S on VH and Y49A and F98L on VL; (cc) L45Q on VH and A43Q and Y49A on VL; (dd) L45Q on VH and P44A and Y49A on VL; (ee) L45Q on VH and L46Q and Y49A on VL; (ff) L45Q on VH and Y49A and Y87L on VL; (gg) L45Q on VH and Y49A and F98M on VL; (hh) F100aI on VH and A43Q and Y49A on VL; (ii) F100aI on VH and P44A and Y49A on VL; (jj) F100aI on VH and L46Q and Y49A on VL; (kk) F100aI on VH and Y49A and Y87L on VL; (ll) F100aI on VH and Y49A and F98L on VL; (mm) Y91M on VH and A43Q, P44A, and Y49A on VL; (nn) Y91M on VH and A43Q, L46Q, and Y49A on VL; (oo) Y91M on VH and L46Q, Y49A, and Y87M on VL; (pp) V37S on VH and L46Q, Y49A, and Y87M on VL; (qq) F100aI on VH and A43Q, L46Q, and Y49A on VL; (rr) F100aI on VH and L46Q, Y49A, and Y87M on VL; (ss) V37S and L45Q on VH and A43Q and Y49A on VL; (tt) V37S and Y91M on VH and A43Q and Y49A on VL; (uu) V37S and F100aI on VH and A43Q and Y49A on VL; (vv) V37S and W103M on VH and A43Q and Y49A on VL; (ww) V37S and Y91M on VH and L46Q and Y49A on VL; (xx) V37S and F100aI on VH and L46Q and Y49A on VL; (yy) V37S and L45Q on VH and Y49A and Y87M on VL; (zz) W103L on VH and S30V, L46Q, and Y49A on VL; (aaa) W103M on VH and S30V, L46Q, and Y49A on VL; (bbb) V37S and F100aI on VH and S30V, A43Q, and Y49A on VL; (ccc) V37S and F100aI on VH and S30V, L46Q, and Y49A on VL; (ddd) W103L on VH and S30V, Y49A, and Y87L on VL; (eee) V37S and F100aI on VH and Y49A and Y87L on VL; (fff) V37S and F100aI on VH and S30V, Y49A, and Y87L on VL; (ggg) V37S, F100aI, and W103M on VH and L46Q and Y49A on VL; and (hhh) V37S, F100aI, W103L on VH, and L46Q and Y49A on VL. [G-13] A fusion protein of any one of [G-1] to [G-12], in which the molecular weight of the fusion protein is smaller after protease cleavage at the protease cleavage site than before said cleavage. [G-14] The reduction in association between VH and VL in the cleaved state compared to the uncleaved state, as measured by surface plasma resonance (SPR) comparing the response units (RU) of the fusion protein before and after protease cleavage, is 15% or less, or 16% or less, or 17% or less, or 18% or less, or 19% or less, or 20% or less, or 21% or less, or 22% or less, or 23% or less, or 24% or less, or 25% or less, or 26% or less. The fusion protein of any of [G-1] to [G-13], which can be expressed by a percentage reduction in maximum RU, is 27% or less, 28% or less, 29% or less, 30% or less, 31% or less, 32% or less, 33% or less, 34% or less, 35% or less, 36% or less, 37% or less, 38% or less, 39% or less, or 40% or less. [G-15] A fusion protein of any of [G-1] to [G-13], wherein the reduction in the association between VH and VL in the cleaved state compared to the uncleaved state can be expressed as a percentage reduction in maximum RU, when measured under surface plasma resonance (SPR) comparing the reaction units (RU) of the fusion protein before and after protease cleavage, of 1% or less, or 2% or less, or 3% or less, or 4% or less, or 5% or less, or 6% or less, or 7% or less, or 8% or less, or 9% or less, or 10% or less, or 11% or less, or 12% or less, or 13% or less, or 14% or less, or 15% or less, or 16% or less, or 17% or less, or 18% or less, or 19% or less, or 20% or less. [G-16] The fusion protein of either [G-14] or [G-15], wherein the SPR conditions include a contact duration of the fusion protein in its uncleaved state with 400 nM uPA for a duration of 30 minutes. [G-17] The percentage of released VH-ligands or VL-ligands is determined by the following formula (II): % Release of VH-ligand or VL-ligand = % Reduction in RU × 100 / D (II) is directly proportional to the percentage change in response units (RU) of the fusion protein measured under SPR in the cleaved state compared to the uncleaved state, according to the formula: where D corresponds to 0.01 × the percentage of the molecular weight of the VH-ligand or VL-ligand, respectively, compared to the molecular weight of the fusion protein in the uncleaved state for any of the fusion proteins [G-14] to [G-16]. [G-18] The fusion protein of [G-17], wherein the percentage of released VH or VL is 10% or more, or 20% or more, or 30% or more, or 40% or more, or 50% or more, or 60% or more, or 70% or more, or 80% or more, or 90% or more, or 100% or more. [G-19] A pharmaceutical composition comprising any one of the fusion proteins [G-1] to [G-18] and a pharmaceutically acceptable carrier. [G-20] A pharmaceutical composition according to [G-19] or a fusion protein according to any one of [G-1] to [G-18] for use as a pharmaceutical. [G-21] A pharmaceutical composition according to [G-19] or a fusion protein according to any one of [G-1] to [G-18] for use in treating a disease or disorder. [G-22] Use of the pharmaceutical composition described in [G-19] or the fusion protein described in any of [G-1] to [G-18] in the manufacture of a pharmaceutical for the treatment of a disease or disorder. [G-23] A method for treating an individual having a disease or disorder, comprising the step of administering an effective amount of the pharmaceutical composition described in [G-19] or a fusion protein described in any of [G-1] to [G-18]. An isolated polynucleotide encoding any one of the fusion proteins [G-24] [G-1] to [G-18]. A vector comprising the polynucleotides [G-25] and [G-24]. [G-26] A host cell containing the polynucleotide of [G-24] or the vector of [G-25]. [G-27] A method for producing any one of the fusion proteins of [G-1] to [G-24], the method comprising a step of culturing the host cell of [G-26]. [G-28] The method according to [G-27], comprising the following steps: (a) introducing at least one amino acid modification or at least one pair of amino acid modifications at the VH and VL interface of the fusion protein, which facilitates dissociation of the VH or VL from the fusion protein, and optionally introducing at least one amino acid modification at the ligand-variable region interface; (b) confirming that step (a) does not disrupt binding of the ligand to VH and VL; (c) confirming that step (a) reduces the association of VH and VL upon protease cleavage at the protease cleavage site; and (d) linking the VH or VL of step (a) to an IgG heavy chain constant region via a protease cleavage sequence; (e) obtaining a polynucleotide encoding the fusion protein of step (e); (f) culturing a host cell containing the polynucleotide of step (e); and (g) producing and recovering the fusion protein from the host cells of step (f). [G-29] The method of [G-28], wherein the modification is a substitution and the substitution is selected from positions 37, 39, 44, 45, 47, 91, and 103 on VH and / or positions 38, 43, 44, 46, 49, 87, and 98 on VL (according to Kabat numbering). [G-29a] The fusion protein of [G-28], wherein the substitutions are selected from positions V37, Q39, G44, L45, W47, H91, Y91, and W103 on VH, and / or positions R38, A43, P44, L46, Y49, Y87, and F98 on VL (according to Kabat numbering). A fusion protein of [G-29] or [G-29a] in which each of the [G-30] positions is substituted with either A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y. [G-31] The method of [G-30], wherein the substitution is selected from positions (according to Kabat numbering) including any one or more of the following: Q39D on VH, W47A, W47L, or W47M, Y91A, Y91L, Y91M, or H91A, W103A, W103I, W103L, or W103M, V37S or V37Q, G44Q, L45A or L45Q, and / or R38E on VL, Y49A, Y87A, Y87L, or Y87M, F98A, F98L, or F98M, A43Q, P44A, P44S, or P44Q, L46E or L46Q. [G-32] A method described in [G-28] to [G-31], wherein the substitution further includes at least one modification in an amino acid present at the interface between the variable region and the ligand, and the amino acid residue for the modification is in the complementarity-determining region (CDR). [G-33] The method described in [G-32], wherein the ligand is IL-12 and the substitution further includes at least one modification selected from position 30 on VL and / or position 100a on VH (according to Kabat numbering). [G-34] The method according to [G-33], wherein the modification is a substitution selected from S30V and / or F100aI (according to Kabat numbering). [G-35] The method according to [G-34], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (hhh) according to the Kabat numbering system: (a) L46Q and Y49A on VL; (b) Q39D on VH and R38E on VL; (c) H91A on VH and L46Q and Y49A on VL; (d) Y91A on VH and A43Q and Y49A on VL; (e) Y91A on VH and P44A and Y49A on VL; (f) Y91A on VH and L46Q and Y49A on VL; (g) Y91A on VH and Y49A and Y87L on VL; (h) Y91M on VH and A43Q and Y49A on VL; (i) Y91M on VH and P44A and Y49A on VL; (j) Y91M on VH and L46Q and Y49A on VL; (k) Y91M on VH and Y49A and Y87L on VL; (l) Y91M on VH and Y49A and F98L on VL; (m) W103L on VH and A43Q and Y49A on VL; (n) W103L on VH and P44A and Y49A on VL; (o) W103L on VH and L46Q and Y49A on VL; (p) W103L on VH and Y49A and Y87L on VL; (q) W103I on VH and A43Q and Y49A on VL; (r) W103I on VH and P44A and Y49A on VL; (s) W103I on VH and L46Q and Y49A on VL; (t) W103M on VH and A43Q and Y49A on VL; (u) W103M on VH and P44A and Y49A on VL; (v) W103M on VH and L46Q and Y49A on VL; (w) W103M on VH and Y49A and Y87L on VL; (x) V37S on VH and A43Q and Y49A on VL; (y) V37S on VH and P44A and Y49A on VL; (z) V37S on VH and L46Q and Y49A on VL; (aa) V37S on VH and Y49A and Y87L on VL; (bb) V37S on VH and Y49A and F98L on VL; (cc) L45Q on VH and A43Q and Y49A on VL; (dd) L45Q on VH and P44A and Y49A on VL; (ee) L45Q on VH and L46Q and Y49A on VL; (ff) L45Q on VH and Y49A and Y87L on VL; (gg) L45Q on VH and Y49A and F98M on VL; (hh) F100aI on VH and A43Q and Y49A on VL; (ii) F100aI on VH and P44A and Y49A on VL; (jj) F100aI on VH and L46Q and Y49A on VL; (kk) F100aI on VH and Y49A and Y87L on VL; (ll) F100aI on VH and Y49A and F98L on VL; (mm) Y91M on VH and A43Q, P44A, and Y49A on VL; (nn) Y91M on VH and A43Q, L46Q, and Y49A on VL; (oo) Y91M on VH and L46Q, Y49A, and Y87M on VL; (pp) V37S on VH and L46Q, Y49A, and Y87M on VL; (qq) F100aI on VH and A43Q, L46Q, and Y49A on VL; (rr) F100aI on VH and L46Q, Y49A, and Y87M on VL; (ss) V37S and L45Q on VH and A43Q and Y49A on VL; (tt) V37S and Y91M on VH and A43Q and Y49A on VL; (uu) V37S and F100aI on VH and A43Q and Y49A on VL; (vv) V37S and W103M on VH and A43Q and Y49A on VL; (ww) V37S and Y91M on VH and L46Q and Y49A on VL; (xx) V37S and F100aI on VH and L46Q and Y49A on VL; (yy) V37S and L45Q on VH and Y49A and Y87M on VL; (zz) W103L on VH and S30V, L46Q, and Y49A on VL; (aaa) W103M on VH and S30V, L46Q, and Y49A on VL; (bbb) V37S and F100aI on VH and S30V, A43Q, and Y49A on VL; (ccc) V37S and F100aI on VH and S30V, L46Q, and Y49A on VL; (ddd) W103L on VH and S30V, Y49A, and Y87L on VL; (eee) V37S and F100aI on VH and Y49A and Y87L on VL; (fff) V37S and F100aI on VH and S30V, Y49A, and Y87L on VL; (ggg) V37S, F100aI, and W103M on VH and L46Q and Y49A on VL; and (hhh) V37S, F100aI, and W103L on VH, and L46Q and Y49A on VL. [G-36] The method according to any one of [G-27] to [G-35], further comprising a step of recovering the polypeptide from the host cell. [H-1] Screening for any of the fusion proteins or polypeptides [A-1] to [A-57], [B-1] to [B-57], [C-1] to [C-52], [D-1] to [D-5], [E-1] to [E-3], [F-1] to [F-20], [G-1] to [G-18], and [J-1] to [J-55], which have a mutation that reduces the association between VH and VL in a cleaved state or a second state compared to an uncleaved state or a first state. The method comprises the steps of comparing the maximum response units recorded for any of the fusion proteins or polypeptides [A-1] to [A-57], [B-1] to [B-57], [C-1] to [C-52], [D-1] to [D-5], [E-1] to [E-3], [F-1] to [F-20], and [G-1] to [G-18] before and after protease cleavage under surface plasma resonance (SPR), and and later, 1% or less, or 2% or less, or 3% or less, or 4% or less, or 5% or less, or 6% or less, or 7% or less, or 8% or less, or 9% or less, or 10% or less, or 11% or less, or 12% or less, or 13% or less, or 14% or less, or 15% or less, or 16% or less, or 17% or less, or 18% or less, or 19% or less, or 20% or less, or 21% or less, or 22% or less, and selecting mutations that result in a reduction in response units of 23% or less, or 24% or less, or 25% or less, or 26% or less, or 27% or less, or 28% or less, or 29% or less, or 30% or less, or 31% or less, or 32% or less, or 33% or less, or 34% or less, or 35% or less, or 36% or less, or 37% or less, or 38% or less, or 39% or less, or 40% or less. [H-2] A method for screening for any of the fusion proteins or polypeptides [A-1] to [A-57], [B-1] to [B-57], [C-1] to [C-52], [D-1] to [D-5], [E-1] to [E-3], [F-1] to [F-20], [G-1] to [G-18], and [J-1] to [J-55], which have a mutation that reduces the association between VH and VL in a cleaved state or a second state compared to an uncleaved state or a first state, the method comprising the steps of: (a) introducing at least one amino acid mutation or at least one pair of amino acid mutations at the VH / VL interface of a fusion protein or polypeptide, which mutation promotes dissociation of the VH or VL domain from the fusion protein or polypeptide, and optionally introducing at least one amino acid mutation at the interface between a ligand or antigen and a ligand-binding domain or antigen-binding domain; (b) measuring the first response unit (RU1) of the immobilized fusion protein or polypeptide of step (a) in a BIACORE surface plasma resonance (SPR) assay in the absence of proteases; (c) measuring the second response unit (RU2) of the immobilized fusion protein or polypeptide of step (a) in the same BIACORE surface plasma resonance (SPR) assay in the presence of a protease; (d) The percentage difference between RU1 and RU2 before and after protease cleavage is 1% or less, or 2% or less, or 3% or less, or 4% or less, or 5% or less, or 6% or less, or 7% or less, or 8% or less, or 9% or less, or 10% or less, or 11% or less, or 12% or less, or 13% or less, or 14% or less, or 15% or less, or 16% or less, or 17% or less, or 18% or less, or 19% or less, or 20% or less. or is 21% or less, or is 22% or less, or is 23% or less, or is 24% or less, or is 25% or less, or is 26% or less, or is 27% or less, or is 28% or less, or is 29% or less, or is 30% or less, or is 31% or less, or is 32% or less, or is 33% or less, or is 34% or less, or is 35% or less, or is 36% or less, or is 37% or less, or is 38% or less, or is 39% or less, or is 40% or less. [H-3] The method of [H-1] or [H-2], wherein the percentage reduction in reaction units corresponds to the percentage reduction in molecular weight resulting from the release of VH or VL from the fusion protein or polypeptide. [H-4] A method for screening for any of the fusion proteins or polypeptides [A-1] to [A-57], [B-1] to [B-57], [C-1] to [C-52], [D-1] to [D-5], [E-1] to [E-3], [F-1] to [F-20], [G-1] to [G-18], and [J-1] to [J-55], which have a mutation that reduces the association between VH and VL in a cleaved state or a second state compared to an uncleaved state or a first state, the method comprising the steps of: (a) introducing at least one amino acid mutation or at least one pair of amino acid mutations at the VH / VL interface of a fusion protein or polypeptide, which mutation promotes dissociation of the VH or VL domain from the fusion protein or polypeptide, and optionally introducing at least one amino acid mutation at the interface between a ligand or antigen and a ligand-binding domain or antigen-binding domain; (b) subjecting the first set of fusion proteins or polypeptides before protease cleavage to size exclusion chromatography (SEC) to obtain a first chromatograph containing peak A1 (first peak); (c) subjecting the second set of fusion proteins or polypeptides after protease cleavage to SEC to obtain a second chromatograph containing peak A2 (second peak) and an additional peak A2' (third peak) (A2' is a shoulder peak of A2); (d) determining the percentage resulting from the area under the curve (AUC) of peak A2' (the third peak) divided by the AUC of peak A1 (the first peak); (e) the percentage obtained in step (d) is 1% or less, or 2% or less, or 3% or less, or 4% or less, or 5% or less, or 6% or less, or 7% or less, or 8% or less, or 9% or less, or 10% or less, or 11% or less, or 12% or less, or 13% or less, or 14% or less, or 15% or less, or 16% or less, or 17% or less, or 18% or less, or 19% or less, or 20% or less, or Selecting the mutation in step (a) if the variance in the nucleotide sequence is 1% or less, or 22% or less, or 23% or less, or 24% or less, or 25% or less, or 26% or less, or 27% or less, or 28% or less, or 29% or less, or 30% or less, or 31% or less, or 32% or less, or 33% or less, or 34% or less, or 35% or less, or 36% or less, or 37% or less, or 38% or less, or 39% or less, or 40% or less. [H-5] The method of [H-4], wherein the percentage determined in (d) corresponds to the percentage of VH or VL dissociated from the fusion protein or polypeptide after protease cleavage. [H-6] Any of the methods [H-1] to [H-5], wherein when screening any of the fusion proteins [A-1] to [A-57], [B-1] to [B-57], [C-1] to [C-52], [D-1] to [D-5], [E-1] to [E-3], [F-1] to [F-20], [G-1] to [G-18], or [J-1] to [J-55], the percentage is 10% or less. [H-7] Any of the methods [H-1] to [H-5], wherein when screening any of the fusion proteins or polypeptides of [A-1] to [A-57], [B-1] to [B-57], [C-1] to [C-52], [D-1] to [D-5], [E-1] to [E-3], [F-1] to [F-20], [G-1] to [G-18], or [J-1] to [J-55], the percentage is 10% or less, or 16% or less, or 20% or less, or 30% or less, or 37% or less. [H-8] Any of the methods [H-1] to [H-6], further comprising the following step: i. measuring the biological activity of any of the fusion proteins or polypeptides [A-1] to [A-57], [B-1] to [B-57], [C-1] to [C-52], [D-1] to [D-5], [E-1] to [E-3], [F-1] to [F-20], [G-1] to [G-18], and [J-1] to [J-55] before protease cleavage; ii. measuring the biological activity of the fusion protein or polypeptide of step (i) after protease cleavage; iii. introducing at least one amino acid modification or at least one pair of amino acid modifications at the VH / VL interface in the fusion protein or polypeptide of step (i), and optionally introducing at least one amino acid modification at the interface between the ligand or antigen and the ligand-binding domain or antigen-binding domain, wherein the amino acid modifications promote dissociation of the VH or VL from the fusion protein or polypeptide upon protease cleavage in the presence of a protease; iv. measuring the biological activity of the fusion protein or polypeptide of step (iii) prior to protease cleavage; v. measuring the biological activity of the fusion protein or polypeptide in step (iii) after protease cleavage; and vi. Selecting amino acid modifications that result in a biological activity of the fusion protein or polypeptide in step (v) that is greater than the biological activity of the fusion protein or polypeptide in step (iv). [H-9] The method of [H-8], further comprising the steps of: (a) determining "V1," the difference in biological activity of the fusion protein or polypeptide between (i) and (ii), and "V2," the difference in biological activity of the fusion protein or polypeptide between (iv) and (v); and (b) selecting amino acid modifications for which the value of V2 is greater than V1; A library of amino acid mutations that reduces the association between VH and VL in any of the fusion proteins or polypeptides [A-1] to [A-57], [B-1] to [B-57], [C-1] to [C-52], [D-1] to [D-5], [E-1] to [E-3], [F-1] to [F-20], [G-1] to [G-18], and [J-1] to [J-55], including mutations selected in [H-10][H-1] to [H-9]. [H-11] The library described in [H-10], wherein the mutations are substitutions selected from positions 37, 39, 44, 45, 47, 91, and 103 on VH and / or positions 38, 43, 44, 46, 49, 87, and 98 on VL (according to Kabat numbering). [H-11a] The library described in [H-10], wherein the mutations are substitutions selected from positions V37, Q39, G44, L45, W47, H91, Y91, and W103 on VH, and / or positions R38, A43, P44, L46, Y49, Y87, and F98 on VL (according to Kabat numbering). [H-12] The library of [H-11] or [H-11a], wherein each of the positions is substituted with either A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y. [H-13] The library of [H-12], wherein the mutation is a substitution selected from positions (according to Kabat numbering) including any one or more of the following: Q39D on VH, W47A, W47L, or W47M, Y91A, Y91L, Y91M, or H91A, W103A, W103L, or W103M, V37S or V37Q, G44Q, L45A or L45Q, and / or R38E on VL, Y49A, Y87A, Y87L, or Y87M, F98A, F98L, or F98M, A43Q, P44A, P44S, or P44Q, L46E or L46Q. [H-14] The library according to [H-13], wherein the mutation is additionally selected from position 30 on VL or position 100a on VH (according to Kabat numbering). [H-15] The library according to [H-14], wherein the mutation is a substitution selected from S30V and F100aI (according to Kabat numbering). [H-16] The library according to [H-15], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (hhh) according to the Kabat numbering system: (a) L46Q and Y49A on VL; (b) Q39D on VH and R38E on VL; (c) H91A on VH and L46Q and Y49A on VL; (d) Y91A on VH and A43Q and Y49A on VL; (e) Y91A on VH and P44A and Y49A on VL; (f) Y91A on VH and L46Q and Y49A on VL; (g) Y91A on VH and Y49A and Y87L on VL; (h) Y91M on VH and A43Q and Y49A on VL; (i) Y91M on VH and P44A and Y49A on VL; (j) Y91M on VH and L46Q and Y49A on VL; (k) Y91M on VH and Y49A and Y87L on VL; (l) Y91M on VH and Y49A and F98L on VL; (m) W103L on VH and A43Q and Y49A on VL; (n) W103L on VH and P44A and Y49A on VL; (o) W103L on VH and L46Q and Y49A on VL; (p) W103L on VH and Y49A and Y87L on VL; (q) W103I on VH and A43Q and Y49A on VL; (r) W103I on VH and P44A and Y49A on VL; (s) W103I on VH and L46Q and Y49A on VL; (t) W103M on VH and A43Q and Y49A on VL; (u) W103M on VH and P44A and Y49A on VL; (v) W103M on VH and L46Q and Y49A on VL; (w) W103M on VH and Y49A and Y87L on VL; (x) V37S on VH and A43Q and Y49A on VL; (y) V37S on VH and P44A and Y49A on VL; (z) V37S on VH and L46Q and Y49A on VL; (aa) V37S on VH and Y49A and Y87L on VL; (bb) V37S on VH and Y49A and F98L on VL; (cc) L45Q on VH and A43Q and Y49A on VL; (dd) L45Q on VH and P44A and Y49A on VL; (ee) L45Q on VH and L46Q and Y49A on VL; (ff) L45Q on VH and Y49A and Y87L on VL; (gg) L45Q on VH and Y49A and F98M on VL; (hh) F100aI on VH and A43Q and Y49A on VL; (ii) F100aI on VH and P44A and Y49A on VL; (jj) F100aI on VH and L46Q and Y49A on VL; (kk) F100aI on VH and Y49A and Y87L on VL; (ll) F100aI on VH and Y49A and F98L on VL; (mm) Y91M on VH and A43Q, P44A, and Y49A on VL; (nn) Y91M on VH and A43Q, L46Q, and Y49A on VL; (oo) Y91M on VH and L46Q, Y49A, and Y87M on VL; (pp) V37S on VH and L46Q, Y49A, and Y87M on VL; (qq) F100aI on VH and A43Q, L46Q, and Y49A on VL; (rr) F100aI on VH and L46Q, Y49A, and Y87M on VL; (ss) V37S and L45Q on VH and A43Q and Y49A on VL; (tt) V37S and Y91M on VH and A43Q and Y49A on VL; (uu) V37S and F100aI on VH and A43Q and Y49A on VL; (vv) V37S and W103M on VH and A43Q and Y49A on VL; (ww) V37S and Y91M on VH and L46Q and Y49A on VL; (xx) V37S and F100aI on VH and L46Q and Y49A on VL; (yy) V37S and L45Q on VH and Y49A and Y87M on VL; (zz) W103L on VH and S30V, L46Q, and Y49A on VL; (aaa) W103M on VH and S30V, L46Q, and Y49A on VL; (bbb) V37S and F100aI on VH and S30V, A43Q, and Y49A on VL; (ccc) V37S and F100aI on VH and S30V, L46Q, and Y49A on VL; (ddd) W103L on VH and S30V, Y49A, and Y87L on VL; (eee) V37S and F100aI on VH and Y49A and Y87L on VL; (fff) V37S and F100aI on VH and S30V, Y49A, and Y87L on VL; (ggg) V37S, F100aI, and W103M on VH and L46Q and Y49A on VL; and (hhh) V37S, F100aI, and W103L on VH, and L46Q and Y49A on VL. [I-1] Isolated protease-resistant interleukin-12 (IL-12). [I-2] The protease-resistant IL-12 according to [I-1], wherein the protease is selected from the group consisting of matriptase, urokinase-type plasminogen activator (uPA), and matrix metalloproteinase (MMP). [I-3] Protease-resistant IL-12 according to [I-2], wherein the protease is urokinase-type plasminogen activator (uPA). [I-4] A protease-resistant IL-12 of any of [I-1] to [I-3], which contains at least one amino acid modification that prevents proteolysis of IL-12 when exposed to a protease. [I-5] The protease-resistant IL-12 of [I-4], which does not contain the amino acid sequence of KSKREK (sequence number: 1102). [I-6] The protease-resistant IL-12 of [I-5], wherein the at least one amino acid modification occurs at the interface between IL-12 and the heparin-binding site of IL-12. [I-7] The protease-resistant IL-12 of [I-6], wherein after the at least one amino acid modification is performed, the IL-12 comprises a modified sequence selected from the group consisting of (a) to (p): (a) KSHRE (SEQ ID NO: 1052); (b) KSHHE (SEQ ID NO: 1053); (c) KSHKE (SEQ ID NO: 1054); (d) KSHSE (SEQ ID NO: 1055); (e) KSKHRE (SEQ ID NO: 1056); (f) KSKQRE (SEQ ID NO: 1057); (g) KSKERE (SEQ ID NO: 1058); (h) KSKPRE (SEQ ID NO: 1059); (i) KHKE (SEQ ID NO: 1060); (j) KHHE (SEQ ID NO: 1061); (k) KHRE (SEQ ID NO: 1062); (l) KKHE (SEQ ID NO: 1063); (m) KRHE (SEQ ID NO: 1064); (n) KRE (SEQ ID NO: 1065); (o) KHE (SEQ ID NO: 1066); and (p)KKE (SEQ ID NO: 1067). [I-8] The protease-resistant IL-12 of any one of [I-1] to [I-7], wherein the IL-12 comprises any one of the following (i) to (xvi): (i) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1068; (ii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1069; (iii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1070; (iv) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1071; (v) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1072; (vi) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1073; (vii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1074; (viii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1075; (ix) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1076; (x) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1077; (xi) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1078; (xii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1079; (xiii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1080; (xiv) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1081; (xv) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1082; and (xvi) An amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1083. [I-9] The protease-resistant IL-12 of any one of [I-1] to [I-8], wherein the IL-12 comprises any one of the following (i) to (xvi): (i) an amino acid sequence identical to SEQ ID NO: 1068; (ii) an amino acid sequence identical to SEQ ID NO: 1069; (iii) an amino acid sequence identical to SEQ ID NO: 1070; (iv) an amino acid sequence identical to SEQ ID NO: 1071; (v) an amino acid sequence identical to SEQ ID NO: 1072; (vi) an amino acid sequence identical to SEQ ID NO: 1073; (vii) an amino acid sequence identical to SEQ ID NO: 1074; (viii) an amino acid sequence identical to SEQ ID NO: 1075; (ix) an amino acid sequence identical to SEQ ID NO: 1076; (x) an amino acid sequence identical to SEQ ID NO: 1077; (xi) an amino acid sequence identical to SEQ ID NO: 1078; (xii) an amino acid sequence identical to SEQ ID NO: 1079; (xiii) an amino acid sequence identical to SEQ ID NO: 1080; (xiv) an amino acid sequence identical to SEQ ID NO: 1081; (xv) an amino acid sequence identical to SEQ ID NO: 1082; and (xvi) An amino acid sequence identical to SEQ ID NO: 1083. [J-1] Each of them has the general formula (I) from the N-terminus to the C-terminus: [Ligand-binding domain]-[Lx]-[Cx]-[Ly]-[ligand moiety] (I) A bivalent homodimeric fusion protein comprising two polypeptides represented by During the ceremony, Lx represents a peptide linker containing a protease cleavage site; Cx represents a constant region comprising a second peptide linker and, optionally, one or more amino acid residues modified to or from cysteine; Ly represents a third peptide linker; and the ligand-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), and the ligand-binding domain comprises at least one amino acid modification that reduces association between VH and VL in the presence of a protease that catalyzes cleavage of the protease cleavage site (the "cleaved state") compared to the absence of the protease (the "uncleaved state"). The bivalent homodimeric fusion protein. [J-2] A fusion protein according to [J-1], wherein the modification is a substitution of an amino acid present at the interface between VH and VL, and the amino acid residue for the modification is in the framework region (FR). [J-3] The fusion protein of [J-2], wherein the substitution is selected from positions 37, 45, 91, or 103 on VH and / or positions 43, 46, 49, or 87 on VL (according to Kabat numbering). [J-3a] The fusion protein of [J-2], wherein the substitution is selected from positions V37, L45, H91, Y91, or W103 on VH, and / or positions A43, L46, Y49, or Y87 on VL (according to Kabat numbering). [J-4] A fusion protein of [J-3] or [J-3a] in which each of the positions is substituted with either A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y. [J-5] The fusion protein of [J-4], wherein the substitution is selected from positions (according to Kabat numbering) including any one or more of the following: V37S on VH, L45Q, Y91M or H91A, W103I, W103L, or W103M, and / or A43Q on VL, L46Q, Y49A, or Y87L. [J-6] A fusion protein of [J-5], wherein the substitution further includes at least one modification in an amino acid present at the interface between the ligand-binding domain and the ligand, and the amino acid residue for the modification is in the complementarity-determining region (CDR). [J-7] The fusion protein of [J-6], wherein the ligand moiety is IL-12 and the substitution further includes at least one modification selected from position 30 on VL and / or position 100a on VH (according to Kabat numbering). [J-8] The fusion protein of [J-7], wherein the modification is a substitution selected from S30V and / or F100aI (according to Kabat numbering). [J-9] The fusion proteins of [J-2] to [J-8], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (z) according to the Kabat numbering system: (a) L46Q and Y49A on VL; (b) H91A on VH and L46Q and Y49A on VL; (c) Y91M on VH and A43Q and Y49A on VL; (d) Y91M on VH and A43Q, L46Q, and Y49A on VL; (e) W103M on VH and A43Q and Y49A on VL; (f) W103M on VH and L46Q and Y49A on VL; (g) V37S on VH and A43Q and Y49A on VL; (h) V37S on VH and L46Q and Y49A on VL; (i) L45Q on VH and A43Q and Y49A on VL; (j) L45Q on VH and L46Q and Y49A on VL; (k) F100aI on VH and A43Q and Y49A on VL; (l) F100aI on VH and A43Q, L46Q, and Y49A on VL; (m) W103L on VH and S30V, L46Q, and Y49A on VL; (n) W103M on VH and S30V, L46Q, and Y49A on VL; (o) V37S and F100aI on VH and S30V, A43Q, and Y49A on VL; (p) V37S and F100aI on VH and S30V, L46Q, and Y49A on VL; (q) W103L on VH and L46Q and Y49A on VL; (r) W103I on VH and L46Q and Y49A on VL; (s) W103M on VH and Y49A and Y87L on VL; (t) W103L on VH and Y49A and Y87L on VL; (u) W103L on VH and S30V, Y49A, and Y87L on VL; (v) V37S and F100aI on VH and L46Q and Y49A on VL; (w) V37S and F100aI on VH and Y49A and Y87L on VL; and (x) V37S and F100aI on VH and S30V, Y49A, and Y87L on VL; (y) V37S, F100aI, and W103M on VH and L46Q and Y49A on VL; and (z) V37S, F100aI, and W103L on VH, and L46Q and Y49A on VL. [J-10] The fusion protein of [J-9], wherein the substitution is selected from the group consisting of any one of the following combinations (a) to (g) according to the Kabat numbering system: (a) W103M on VH and L46Q and Y49A on VL; (b) W103L on VH and S30V, L46Q, and Y49A on VL; (c) V37S and F100aI on VH and S30V, L46Q, and Y49A on VL; (d) W103L on VH and L46Q and Y49A on VL; and (e) V37S and F100aI on VH and L46Q and Y49A on VL; (f) V37S, F100aI, and W103M on VH and L46Q and Y49A on VL; and (g) V37S, F100aI, and W103L on VH, and L46Q and Y49A on VL. [J-11] Any of the fusion proteins [J-1] to [J-10], wherein the molecular weight of the fusion protein in the cleaved state is smaller than the molecular weight of the fusion protein in the uncleaved state. [J-12] A fusion protein of any of [J-1] to [J-11], in which the cleavage site is cleaved so that a portion of the ligand-binding domain is released from the fusion protein in the cleaved state. [J-13] The fusion protein of [J-12], wherein the molecular weight of the portion of the ligand-binding domain released from the fusion protein is 26 kDa, 13 kDa, or smaller. [J-14] A fusion protein according to any one of [J-1] to [J-13], wherein the ratio of the molecular weight of the fusion protein in a cleaved state to the molecular weight of the fusion protein in an uncleaved state is 10:9. [J-15] A fusion protein according to any one of [J-1] to [J-14], wherein the molecular weight of the fusion protein in the cleaved state is 9 / 10 of the molecular weight of the fusion protein in the uncleaved state. [J-16] A fusion protein according to any one of [J-1] to [J-15], wherein the percentage reduction in molecular weight of the fusion protein in a cleaved state compared to the fusion protein in an uncleaved state is 10%. [J-17] A fusion protein according to any one of [J-1] to [J-16], wherein the portion of the ligand-binding domain released from the fusion protein upon protease cleavage comprises VL or VH. [J-18] A fusion protein of any of [J-1] to [J-17], wherein the reduction in the association between VH and VL in the cleaved state compared to the uncleaved state can be expressed as a percentage reduction in maximum RU, when measured under surface plasma resonance (SPR) comparing the reaction units (RU) of the fusion protein in the absence and presence of protease, of 1% or less, or 2% or less, or 3% or less, or 4% or less, or 5% or less, or 6% or less, or 7% or less, or 8% or less, or 9% or less, or 10% or less, or 11% or less, or 12% or less, or 13% or less, or 14% or less, or 15% or less, or 16% or less, or 17% or less, or 18% or less, or 19% or less, or 20% or less. [J-19] A fusion protein of any of [J-1] to [J-18], wherein the reduction in the association between VH and VL in the cleaved state compared to the uncleaved state can be expressed as a percentage reduction in maximum RU of 1% or less, or 2% or less, or 3% or less, or 4% or less, or 5% or less, or 6% or less, or 7% or less, when measured under surface plasma resonance (SPR) comparing the RU of the fusion protein in the absence and presence of a protease. [J-20] The reduction in association between VH and VL in the cleaved state compared to the uncleaved state, as measured by surface plasma resonance (SPR) comparing the response units (RU) of the fusion protein in the absence and presence of protease, is 15% or less, or 16% or less, or 17% or less, or 18% or less, or 19% or less, or 20% or less, or 21% or less, or 22% or less, or 23% or less, or 24% or less, or 25% or less; or any of the fusion proteins [J-1] to [J-19], which can be expressed by a percentage reduction in maximum RU of 26% or less, or 27% or less, or 28% or less, or 29% or less, or 30% or less, or 31% or less, or 32% or less, or 33% or less, or 34% or less, or 35% or less, or 36% or less, or 37% or less, or 38% or less, or 39% or less, or 40% or less. [J-21] A fusion protein selected from any of [J-1] to [J-20], wherein the SPR conditions include a duration of contact between the fusion protein in an uncleaved state and 400 nM uPA protease for 30 minutes. [J-22] The percentage of released VH or VL is determined by measuring the percentage of VH or VL expressed by the formula (II): % Release of VH or VL = % Reduction in RU × 100 / D (II) is directly proportional to the percentage change in response units (RU) of the fusion protein measured under SPR in the cleaved state compared to the uncleaved state, according to the formula: where D corresponds to 0.01 × the percentage of the molecular weight of VH or VL, respectively, compared to the molecular weight of the fusion protein in the uncleaved state of any of the fusion proteins [J-19] to [J-20]. [J-23] The percentage of released VH or VL is determined by the following formula (II-1): % VH or VL release = % RU reduction × 100 / 10 (II-1) According to [J-22], the fusion protein is directly proportional to the percentage change in response units (RU) of the fusion protein in the cleaved state compared to the uncleaved state, as measured under SPR. [J-24] The percentage of released VH or VL is determined by the following formula (II-2): % VH or VL release = % RU reduction × 100 / 15.8 (II-2) According to [J-23], the fusion protein is directly proportional to the percentage change in response units (RU) of the fusion protein in the cleaved state compared to the uncleaved state, as measured under SPR. [J-25] Any one of the fusion proteins [J-22] to [J-24], wherein the percentage of released VH or VL is 10% or more, or 20% or more, or 30% or more, or 40% or more, or 50% or more, or 60% or more, or 70% or more, or 80% or more, or 90% or more, or 100% or more. [J-26] A fusion protein of any one of [J-1] to [J-25], in which the ligand portion in the uncleaved state and the cleaved state remains linked to the constant region via a third peptide linker. [J-27] A fusion protein of any of [J-1] to [J-26], in which the binding between the ligand portion and the ligand-binding domain is weakened in the cleaved state compared to the uncleaved state. [J-28] A fusion protein according to any one of [J-1] to [J-27], in which, in an uncleaved state, the ligand portion binds to the ligand-binding domain and the biological activity of the ligand portion is attenuated, and in a cleaved state, the biological activity of the ligand is restored. [J-29] A fusion protein of any one of [J-1] to [J-28], wherein Cx comprises the CH1 region of the heavy chain and the CL region of the light chain. [J-30] A fusion protein of any of [J-1] to [J-29], in which a second peptide linker is positioned in the hinge region so as to promote disulfide bond formation between Cys at position 220 (C220) of the heavy chain and Cys at position 214 (C214) of the light chain (according to EU numbering). [J-31] A fusion protein of any of [J-1] to [J-29], wherein Cx contains at least one amino acid modification, in which amino acid residues in the heavy chain and light chain are modified so that no disulfide bond is formed between position 220 of the heavy chain and position 214 of the light chain (according to EU numbering). [J-32] The fusion protein of [J-31], wherein the light chain comprises a C214S modification and the heavy chain comprises a C220S modification (according to EU numbering). [J-33] A fusion protein of any of [J-1] to [J-29], in which the heavy chain is modified to enable disulfide bond formation between position 131 of the heavy chain and position 214 of the light chain (according to EU numbering). [J-34] The fusion protein of [J-33], wherein the heavy chain comprises S131C and C220S modifications (according to EU numbering). [J-35] A fusion protein of any of [J-1] to [J-34], wherein Cx comprises an array selected from the group consisting of SEQ ID NO: 901 (C1), SEQ ID NO: 905 (C2), SEQ ID NO: 908 (C3), SEQ ID NO: 910 (C4), and SEQ ID NO: 932 (C5). [J-36] A fusion protein of [J-35], wherein Cx comprises the sequence of SEQ ID NO: 910 (C4). [J-37] A fusion protein according to any one of [J-1] to [J-36], wherein Ly comprises a glycine-serine polymer. [J-38] The fusion protein of [J-37], wherein the glycine-serine polymer is selected from the group consisting of (a) to (ee): (a) Ser; (b) Gly Ser (GS); (c) Ser Gly(SG); (d) Gly Gly Ser (GGS); (e) Gly Ser Gly (GSG); (f) Ser Gly Gly (SGG); (g) Gly Ser Ser (GSS); (h) Ser Ser Gly (SSG); (i) Ser Gly Ser (SGS); (j) Gly Gly Gly Ser (GGGS, SEQ ID NO: 136); (k) Gly Gly Ser Gly (GGSG, SEQ ID NO: 137); (l) Gly Ser Gly Gly (GSGG, SEQ ID NO: 138); (m) Ser Gly Gly Gly (SGGG, SEQ ID NO: 139); (n) Gly Ser Ser Gly (GSSG, SEQ ID NO: 140); (o) Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 141); (p) Gly Gly Gly Ser Gly (GGGSG, SEQ ID NO: 142); (q) Gly Gly Ser Gly Gly (GGSGG, SEQ ID NO: 143); (r) Gly Ser Gly Gly Gly (GSGGG, SEQ ID NO: 144); (s) Gly Ser Gly Gly Ser (GSGGS, SEQ ID NO: 145); (t) Ser Gly Gly Gly (SGGGG, SEQ ID NO: 146); (u) Gly Ser Ser Gly Gly (GSSGG, SEQ ID NO: 147); (v) Gly Ser Gly Ser Gly (GSGSG, SEQ ID NO: 148); (w) Ser Gly Gly Ser Gly (SGGSG, SEQ ID NO: 149); (x) Gly Ser Ser Ser Gly (GSSSG, SEQ ID NO: 150); (y) Gly Gly Gly Gly Gly Ser (GGGGGS, SEQ ID NO: 151); (z) Ser Gly Gly Gly Gly (SGGGGG, SEQ ID NO: 152); (aa) Gly Gly Gly Gly Gly Ser (GGGGGGS, SEQ ID NO: 153); (bb) Ser Gly Gly Gly Gly Gly (SGGGGGG, SEQ ID NO: 154); (cc)(Gly Gly Gly Gly Ser(GGGGS, SEQ ID NO: 141))n; (dd) (Ser Gly Gly Gly Gly (SGGGG, SEQ ID NO: 146))n; and (ee)(Gly Gly Ser Gly Gly (GGSGG, SEQ ID NO: 143))n; Here, n is an integer of 1 or more. [J-39] A fusion protein of [J-38], wherein Ly comprises the sequence GGSGGSGGSGGSGGSGGS (sequence number: 903). [J-40] A fusion protein of any of [J-1] to [J-39], wherein the fusion protein contains two protease cleavage sites, and each protease cleavage site is independently cleavable by a protease specific to a target tissue. [J-41] The fusion protein of [J-40], wherein the target tissue is cancer tissue or inflammatory tissue. [J-42] A fusion protein of any of [J-1] to [J-41], in which each protease cleavage site is cleavable by the same protease. [J-43] The fusion protein of [J-42], wherein each protease cleavage site contains the same protease cleavage sequence. [J-44] A fusion protein of any of [J-1] to [J-43], wherein each protease cleavage site is independently cleavable by a protease selected from the group consisting of matriptase, urokinase-type plasminogen activator (uPA), and matrix metalloproteinase (MMP). [J-45] A fusion protein of any of [J-1] to [J-44], wherein Lx contains a protease cleavage site located near the boundary between the VH region and the CH1 region or near the boundary between the VL region and the CL region. [J-46] A fusion protein according to any one of [J-1] to [J-45], wherein the ligand portion comprises a cytokine or a chemokine. [J-47] The fusion protein of [J-46], wherein the ligand portion is selected from the group consisting of CXCL9, CXCL10, CXCL11, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-22, IFN-α, IFN-β, IFN-γ, MIG, I-TAC, RANTES, MIP-1a, MIP-1b, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra. [J-48] The fusion protein of [J-47], wherein the ligand moiety is IL-12. [J-49] The fusion protein of [J-48], wherein the IL-12 comprises at least one amino acid modification that prevents proteolysis when exposed to a protease that catalyzes the cleavage of IL-12. [J-50] A fusion protein of [J-49], wherein the IL-12 does not contain the amino acid sequence of KSKREK (sequence number: 1102). [J-51] The fusion protein of [J-49] or [J-50], wherein the at least one amino acid modification occurs at the interface between the IL-12 and the ligand-binding domain. [J-52] The fusion protein of [J-51], wherein after the at least one amino acid modification is performed, IL-12 comprises a modified sequence selected from the group consisting of (a) to (p): (a) KSHRE (SEQ ID NO: 1052); (b) KSHHE (SEQ ID NO: 1053); (c) KSHKE (SEQ ID NO: 1054); (d) KSHSE (SEQ ID NO: 1055); (e) KSKHRE (SEQ ID NO: 1056); (f) KSKQRE (SEQ ID NO: 1057); (g) KSKERE (SEQ ID NO: 1058); (h) KSKPRE (SEQ ID NO: 1059); (i) KHKE (SEQ ID NO: 1060); (j) KHHE (SEQ ID NO: 1061); (k) KHRE (SEQ ID NO: 1062); (l) KKHE (SEQ ID NO: 1063); (m) KRHE (SEQ ID NO: 1064); (n) KRE (SEQ ID NO: 1065); (o) KHE (SEQ ID NO: 1066); and (p)KKE (SEQ ID NO: 1067). [J-53] The fusion protein of [J-49] to [J-52], wherein the IL-12 comprises a sequence selected from the group consisting of (i) to (xvi): (i) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1068; (ii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1069; (iii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1070; (iv) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1071; (v) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1072; (vi) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1073; (vii) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1074; (viii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1075; (ix) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1076; (x) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1077; (xi) an amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1078; (xii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1079; (xiii) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1080; (xiv) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1081; (xv) an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 1082; and (xvi) An amino acid sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1083. [J-54] The fusion protein of [J-53], wherein the IL-12 comprises a sequence selected from the group consisting of (i) to (xvi): (i) an amino acid sequence identical to SEQ ID NO: 1068; (ii) an amino acid sequence identical to SEQ ID NO: 1069; (iii) an amino acid sequence identical to SEQ ID NO: 1070; (iv) an amino acid sequence identical to SEQ ID NO: 1071; (v) an amino acid sequence identical to SEQ ID NO: 1072; (vi) an amino acid sequence identical to SEQ ID NO: 1073; (vii) an amino acid sequence identical to SEQ ID NO: 1074; (viii) an amino acid sequence identical to SEQ ID NO: 1075; (ix) an amino acid sequence identical to SEQ ID NO: 1076; (x) an amino acid sequence identical to SEQ ID NO: 1077; (xi) an amino acid sequence identical to SEQ ID NO: 1078; (xii) an amino acid sequence identical to SEQ ID NO: 1079; (xiii) an amino acid sequence identical to SEQ ID NO: 1080; (xiv) an amino acid sequence identical to SEQ ID NO: 1081; (xv) an amino acid sequence identical to SEQ ID NO: 1082; and (xvi) An amino acid sequence identical to SEQ ID NO: 1083. [J-55] The fusion protein of [J-54], wherein the IL-12 comprises a sequence selected from SEQ ID NO: 1068, or SEQ ID NO: 1069, or SEQ ID NO: 1076, or SEQ ID NO: 1077, or SEQ ID NO: 1078, or SEQ ID NO: 1079, or SEQ ID NO: 1080. [K-1] A library comprising a fusion protein of any of the preceding embodiments, wherein the library is obtained by a method of screening for fusion proteins comprising one or more amino acid modifications that reduce the association between VH and VL in the presence of a protease compared to in the absence of the protease, and the screening method is as exemplified in any of the preceding embodiments. [K-2] A library comprising a fusion protein of any of the aforementioned embodiments, wherein the library is obtained by a method for producing a fusion protein comprising one or more amino acid modifications that reduce the association between VH and VL in the presence of a protease compared to in the absence of the protease, and the one or more amino acid modifications that reduce the association between VH and VL in the presence of a protease compared to in the absence of the protease are identified by a screening method as exemplified in any of the aforementioned embodiments. [K-3] A method for releasing VH or VL from a fusion protein of any of the aforementioned embodiments or a polypeptide of any of the aforementioned embodiments, the method comprising a step of introducing at least one amino acid modification at the interface between VH and VL that reduces the association between VH and VL, and the at least one amino acid modification is selected from the screening methods exemplified in any of the aforementioned embodiments. [K-4] A library comprising a plurality of bivalent homodimeric fusion proteins, each fusion protein in the library comprising a protease cleavage site and a ligand-binding domain, wherein the ligand-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) that associate with each other, and wherein the ligand-binding domain comprises at least one amino acid modification that reduces the association between the VH and VL before and after protease cleavage at the cleavage site. [K-5] A method for releasing VH or VL from a bivalent homodimeric fusion protein, wherein the fusion protein comprises a protease cleavage site and a ligand-binding domain, wherein the ligand-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) that associate with each other, and wherein the ligand-binding domain comprises at least one amino acid modification that reduces the association between the VH and VL after protease cleavage at the cleavage site compared to before protease cleavage at the cleavage site, and wherein the VH or VL is released from the fusion protein after protease cleavage at the cleavage site, the method comprising the step of introducing at least one amino acid modification at the interface between the VH and VL, and wherein the amino acid is in a framework region (FR). [K-6] A method for screening for a bivalent homodimeric fusion protein, the fusion protein comprising a protease cleavage site and a ligand-binding domain, the ligand-binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) that associate with each other, and the ligand-binding domain comprising at least one amino acid modification that reduces association between the VH and VL after protease cleavage at the cleavage site (the "cleaved state") compared to before protease cleavage at the cleavage site (the "uncleaved state"), and the VH or VL is released from the fusion protein after protease cleavage at the cleavage site, and the method comprises: (a) introducing at least one amino acid modification or at least one pair of amino acid modifications at the VH / VL interface, and optionally introducing at least one amino acid modification at the interface between the ligand and the ligand-binding domain, that promotes dissociation of the VH or VL; (b) measuring the first response unit (RU1) of the immobilized fusion protein of step (a) in an uncleaved BIACORE surface plasma resonance (SPR) assay; (c) measuring the second response unit (RU2) of the immobilized fusion protein of step (a) in the same BIACORE surface plasma resonance (SPR) assay in a cleaved state; and (d) selecting the modification in step (a) if the percentage difference between RU1 and RU2 is 1% or less, or 5% or less, or 10% or less, or 15% or less, or 20% or less, or 30% or less, or 40% or less. Including, and the percentage reduction in reaction units corresponds to the percentage reduction in molecular weight due to the release of VH or VL from the fusion protein. The method. [K-7] A method for screening for a bivalent homodimeric fusion protein, the fusion protein comprising a protease cleavage site and a ligand-binding domain, the ligand-binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) that associate with each other, and the ligand-binding domain comprising at least one amino acid modification that reduces association between the VH and VL after protease cleavage at the cleavage site (the "cleaved state") compared to before protease cleavage at the cleavage site (the "uncleaved state"), and the VH or VL is released from the fusion protein after protease cleavage at the cleavage site, and the method comprises: (a) introducing at least one amino acid modification or at least one pair of amino acid modifications at the VH / VL interface, and optionally introducing at least one amino acid modification at the interface between the ligand and the ligand-binding domain, that promotes dissociation of the VH or VL; (b) subjecting the first set of fusion proteins in an uncleaved state to size exclusion chromatography (SEC) to obtain a first chromatograph containing peak A1 (first peak); (c) subjecting the second set of fusion proteins in the cleaved state to SEC to obtain a second chromatograph containing peak A2 (the second peak) and an additional peak A2' (the third peak) (A2' is a shoulder peak of A2); (d) determining the percentage resulting from the area under the curve (AUC) of peak A2' (the third peak) divided by the AUC of peak A1 (the first peak); and (e) selecting the modifications in step (a) such that the percentage obtained in step (d) is 1% or less, or 5% or less, or 10% or less, or 15% or less, or 20% or less, or 30% or less, or 40% or less; Including, and the percentage reduction determined in step (d) corresponds to the percentage reduction in molecular weight resulting from the release of VH or VL from the fusion protein. The method. [Brief explanation of the drawings]

[0012] [Figure 1] Expected profile of IL-12 fusion proteins: As an inactive molecule, IL-12 biological activity should be inhibited, and the IL-12 fusion protein should have a long systemic half-life. As an activated molecule, IL-12 biological activity is restored upon cleavage by a disease-specific protease. In addition, the fusion protein should be retained at high concentrations in diseased tissues and exhibit a short systemic half-life. [Figure 2] Figure 2A shows the molecular format of the monovalent IL-12 fusion protein used to evaluate the effect of the ligand-binding domain on the pharmacokinetics of the inactive IL-12 fusion protein. Figure 2B shows the pharmacokinetics of the inactive IL-12 fusion protein in tumor-free mice. The top graph shows the plasma concentrations of monovalent IL-12 free FP1 (filled circles), FP2 (filled triangles), and FP3 (crosses) after a single intravenous dose in tumor-free mice (n=3). The bottom table shows the pharmacokinetic parameters of each fusion protein. CO: backward extrapolated concentration immediately after intravenous injection; t1 / 2: elimination half-life; AUCinf: area under the plasma concentration-time curve extrapolated from time zero to infinity; CL: total clearance; Vss: volume of distribution at steady state. [Figure 3]Various formats of IL-12 fusion proteins. Figure 3A shows an IL-12 release fusion protein in which a cleavable linker was introduced into the elbow hinge region between the VH and CH1 regions. Single-chain IL-12 was added to the C-terminus of the Fc domain via the cleavable linker. Digestion of the cleavable linker results in the release of active IL-12. Figure 3B shows an IL-12 fusion protein in which a cleavable linker was introduced into the elbow hinge region between the VH and CH1 regions. A GS linker was inserted into the hinge region, and single-chain IL-12 was added to the C-terminus of the Fc domain via the GS linker. Digestion of the cleavable linker results in the release of active IL-12 fused to the Fc. [Figure 4] Bivalent IL-12-free FP4 and bivalent IL-12-fused FP5 were subjected to IL-12 luciferase assays. Both variants exhibited lower IL-12 bioactivity than hIL-12_His tag in the absence of MT-SP1, and IL-12 bioactivity was restored to the same level as hIL-12_His tag upon MT-SP1 treatment. [Figure 5] Various forms of activated IL-12 fusion proteins upon protease cleavage. (A) In the free form, freely dissociated IL-12 molecules are a representative activated molecule, i.e., recombinant IL-12. (B) In the fusion form, the KLH bivalent fusion FP6 is a representative activated molecule. [Figure 6] IL-12 concentrations in tumor lysates and tumor interstitial fluid of recombinant IL-12 or KLH-bivalent IL12 fusion FP6 in a mouse model bearing LS1034 tumors injected with human T cells after a total of six repeated intratumoral injections. Tumor retention levels comparing the free and fusion forms of activated IL-12 show higher retention concentrations for the fusion form than for the free form in both tumor lysates and interstitial fluid. [Figure 7]Time course of plasma KLH-bivalent IL-12 fusion FP6 concentrations after intravenous administration in cynomolgus monkeys. KLH-bivalent IL-12 fusion FP6 was rapidly eliminated, with a clearance of 1975 mL / day / kg, approximately 13-fold faster than the reported clearance of recombinant IL-12 of 6.23 mL / hour / kg (150 mL / day / kg) (Pharmacology 2010;85:319-327). [Figure 8] (A) Time course analysis of KLH bivalent fusion FP7 showed a clearance of 335 ml / day / kg in SCID mice. (B) Time course analysis of inactive and active forms of the IL-12 fusion protein. Clearance levels were similar between the inactive and active forms of the IL-12 fusion protein. [Figure 9] (A) As the activated and inactivated IL-12 fusion proteins showed similar clearance in Figure 8, this phenomenon could potentially have been observed because the VH domain, VL domain, and IL-12 moiety may exhibit binding affinity and did not completely dissociate after protease cleavage. (B) The activity of the inactivated and activated forms of the IL-12 fusion proteins indicates that the activated form remains capable of binding to the IL-12 receptor after protease digestion and activates IL-12 signaling to the same extent as recombinant IL-12, regardless of the mean clearance observed in (A). [Figure 10A] Schematic representation of a Biacore assay to assess the percentage dissociation of VH from the fusion protein. Representation of an assay performed on an anti-IL-12 antibody that binds to IL-12. [Figure 10B] Schematic representation of a Biacore assay to assess the percentage dissociation of VH from the fusion protein. Representation of an assay performed on a bivalent IL-12 fusion protein. [Figure 11A] Screening of amino acid modifications at the VH / VL interface to promote VH dissociation from anti-IL-12 antibodies. Evaluation of single amino acid modifications and the percentage of VH dissociation. [Figure 11B] Screening of amino acid modifications at the VH / VL interface to promote VH dissociation from anti-IL-12 antibodies. Combinations of amino acid modifications and evaluation of the percentage of VH dissociation. [Figure 12A] Screening and evaluation of amino acid modifications at the VH / VL interface that promote VH dissociation from bivalent IL-12 fusion proteins. [Figure 12B] Screening and evaluation of amino acid modifications at the VH / VL interface that promote VH dissociation from bivalent IL-12 fusion proteins. [Figure 13] Plasma concentration time course of IL-12 fusion proteins with VH-releasing modifications in SCID mice. Inclusion of modifications at the VH / VL interface resulted in greater dissociation of IL-12 from the digestion products of the IL-12 fusion proteins and faster clearance compared to digestion products of IL-12 fusion proteins without any modifications at the VH / VL interface. [Figure 14A] Profile of CXCL10 fusion proteins. As inactive molecules, the biological activity of CXCL10 should be inhibited, and the CXCL10 fusion protein should have a long systemic half-life. As activated molecules, CXCL10 biological activity is restored upon cleavage by disease-specific proteases. In addition, the fusion protein should be retained at high concentrations in diseased tissues and exhibit a short systemic half-life. [Figure 14B] Evaluation of amino acid modifications to promote VH dissociation from the fusion protein. [Figure 15] Screening and evaluation of amino acid modifications at the VH / VL interface that promote VH dissociation from bivalent IL-22 fusion proteins. [Figure 16] Time course analysis of KLH bivalent fusion FP7 with and without MT-SP1 digestion. Digestion with MT-SP1 unexpectedly resulted in slower clearance of KLH bivalent fusion FP7, which could affect the profile of activated IL12 molecules. [Figure 17]SDS-PAGE analysis showing MT-SP1-mediated digestion using a KLH bivalent fusion variant with a protease-resistant modification in the heparin-binding domain of p40. The top panel shows undigested samples and samples digested with MT-SP1 after 1 hour of incubation. The bottom panel shows samples digested with MT-SP1 after 4 and 24 hours of incubation. [Figure 18] SDS-PAGE analysis showing MT-SP1-mediated digestion using a KLH bivalent fusion variant with a protease-resistant modification in the heparin-binding domain of p40. The top panel shows undigested samples and samples digested with MT-SP1 after 1 hour of incubation. The bottom panel shows samples digested with MT-SP1 after 4 and 24 hours of incubation. [Figure 19] The IL-12 activity of the protease-resistant IL-12 variants was evaluated using a luciferase assay. All protease-resistant IL-12 variants showed activity similar to that of hIL12_His tag, regardless of protease treatment. [Figure 20] Time course of plasma concentrations of protease-resistant IL-12 variants as KLH-bivalent fusions in SCID mice. All protease-resistant variants showed slower elimination than the control (KLH-bivalent IL12006v1). [Figure 21] The bivalent IL-12 fusion proteins FP8, FP11, and FP12 were subjected to IL-12 luciferase assays. All three fusion proteins showed lower IL-12 bioactivity than hIL-12_His tag in the absence of MT-SP1, but IL-12 bioactivity was restored to the same level as hIL-12_His tag upon treatment with MT-SP1. [Figure 22A] Schematic diagram of IL-22 release from the fusion protein "FP14" from which VH-IL-22 is released. [Figure 22B]Assessment of the activity of IL-22 fusion proteins with and without protease digestion. To assess whether amino acid modifications at the VH / VL interface promoted IL-22 release, IL-22 activity was assayed using the concentration of IL-10 secreted from cells in response to IL-22. A total of three assay plates were assayed, and Figure 22B corresponds to the evaluation results for plate 1. For each assay plate, an IL-22 fusion protein without any modifications at the VH / VL interface, i.e., a control fusion protein ("Ab4H / Ab4L FP14"), was included as a reference. To compare IL-22 activity among IL-22 fusion proteins, the IL-10 response curve was interpolated at a concentration of 200 pg / mL. The activity window was calculated as the ratio of the concentrations of each IL-22 fusion protein that induced 200 pg / mL IL-10 with and without uPA protease. [Figure 22C] Assessment of the activity of IL-22 fusion proteins with and without protease digestion. To assess whether amino acid modifications at the VH / VL interface promoted IL-22 release, IL-22 activity was assayed using the concentration of IL-10 secreted from cells in response to IL-22. A total of three assay plates were assayed, and Figure 22C corresponds to the evaluation results for plate 1. For each assay plate, an IL-22 fusion protein without any modifications at the VH / VL interface, i.e., a control fusion protein ("Ab4H / Ab4L FP14"), was included as a reference. To compare IL-22 activity among IL-22 fusion proteins, the IL-10 response curve was interpolated at a concentration of 200 pg / mL. The activity window was calculated as the ratio of the concentrations of each IL-22 fusion protein that induced 200 pg / mL IL-10 with and without uPA protease. [Figure 22D]Assessment of the activity of IL-22 fusion proteins with and without protease digestion. To assess whether amino acid modifications at the VH / VL interface promoted IL-22 release, IL-22 activity was assayed using the concentration of IL-10 secreted from cells in response to IL-22. A total of three assay plates were assayed, and Figure 22D corresponds to the evaluation results for plate 2. For each assay plate, an IL-22 fusion protein without any modifications at the VH / VL interface, i.e., a control fusion protein ("Ab4H / Ab4L FP14"), was included as a reference. To compare IL-22 activity among IL-22 fusion proteins, the IL-10 response curve was interpolated at a concentration of 200 pg / mL. The activity window was calculated as the ratio of the concentrations of each IL-22 fusion protein that induced 200 pg / mL IL-10 with and without uPA protease. [Figure 22E] Assessment of the activity of IL-22 fusion proteins with and without protease digestion. To assess whether amino acid modifications at the VH / VL interface promoted IL-22 release, IL-22 activity was assayed using the concentration of IL-10 secreted from cells in response to IL-22. A total of three assay plates were assayed, and Figure 22E corresponds to the evaluation results for plate 2. For each assay plate, an IL-22 fusion protein without any modifications at the VH / VL interface, i.e., a control fusion protein ("Ab4H / Ab4L FP14"), was included as a reference. To compare IL-22 activity among IL-22 fusion proteins, the IL-10 response curve was interpolated at a concentration of 200 pg / mL. The activity window was calculated as the ratio of the concentrations of each IL-22 fusion protein that induced 200 pg / mL IL-10 with and without uPA protease. [Figure 22F]Assessment of the activity of IL-22 fusion proteins with and without protease digestion. To assess whether amino acid modifications at the VH / VL interface promoted IL-22 release, IL-22 activity was assayed using the concentration of IL-10 secreted from cells in response to IL-22. A total of three assay plates were assayed, and Figure 22F corresponds to the evaluation results for plate 3. For each assay plate, an IL-22 fusion protein without any modifications at the VH / VL interface, i.e., a control fusion protein ("Ab4H / Ab4L FP14"), was included as a reference. To compare IL-22 activity among IL-22 fusion proteins, the IL-10 response curve was interpolated at a concentration of 200 pg / mL. The activity window was calculated as the ratio of the concentrations of each IL-22 fusion protein that induced 200 pg / mL IL-10 with and without uPA protease. [Figure 23A] Schematic diagram of IL-22 release from the fusion protein "FP15" from which VL-IL-22 is released. [Figure 23B] Assessment of the activity of IL-22 fusion proteins with and without protease digestion. To assess whether amino acid modifications at the VH / VL interface promoted IL-22 release, IL-22 activity was assayed using the concentration of IL-10 secreted from cells in response to IL-22. Two assay plates were assayed in total, and Figure 23B corresponds to the evaluation results of plate 1. On each assay plate, an IL-22 fusion protein without any modifications at the VH / VL interface, i.e., a control fusion protein ("Ab5H / Ab5L FP15"), was included as a reference. To compare IL-22 activity among IL-22 fusion proteins, the IL-10 response curve was interpolated at a concentration of 200 pg / mL. The activity window was calculated as the ratio of the concentrations of each IL-22 fusion protein that induced 200 pg / mL IL-10 with and without uPA protease. [Figure 23C]Assessment of the activity of IL-22 fusion proteins with and without protease digestion. To assess whether amino acid modifications at the VH / VL interface promoted IL-22 release, IL-22 activity was assayed using the concentration of IL-10 secreted from cells in response to IL-22. Two assay plates were assayed in total, and Figure 23C corresponds to the evaluation results for plate 1. For each assay plate, an IL-22 fusion protein without any modifications at the VH / VL interface, i.e., a control fusion protein ("Ab5H / Ab5L FP15"), was included as a reference. To compare IL-22 activity among IL-22 fusion proteins, the IL-10 response curve was interpolated at a concentration of 200 pg / mL. The activity window was calculated as the ratio of the concentrations of each IL-22 fusion protein that induced 200 pg / mL IL-10 with and without uPA protease. [Figure 23D] Assessment of the activity of IL-22 fusion proteins with and without protease digestion. To assess whether amino acid modifications at the VH / VL interface promoted IL-22 release, IL-22 activity was assayed using the concentration of IL-10 secreted from cells in response to IL-22. Two assay plates were assayed in total, and Figure 23D corresponds to the evaluation results of plate 2. For each assay plate, an IL-22 fusion protein without any modifications at the VH / VL interface, i.e., a control fusion protein ("Ab5H / Ab5L FP15"), was included as a reference. To compare IL-22 activity among IL-22 fusion proteins, the IL-10 response curve was interpolated at a concentration of 200 pg / mL. The activity window was calculated as the ratio of the concentrations of each IL-22 fusion protein that induced 200 pg / mL IL-10 with and without uPA protease. [Figure 23E]Assessment of the activity of IL-22 fusion proteins with and without protease digestion. To assess whether amino acid modifications at the VH / VL interface promoted IL-22 release, IL-22 activity was assayed using the concentration of IL-10 secreted from cells in response to IL-22. Two assay plates were assayed in total, and Figure 23E corresponds to the evaluation results of plate 2. For each assay plate, an IL-22 fusion protein without any modifications at the VH / VL interface, i.e., a control fusion protein ("Ab5H / Ab5L FP15"), was included as a reference. To compare IL-22 activity among IL-22 fusion proteins, the IL-10 response curve was interpolated at a concentration of 200 pg / mL. The activity window was calculated as the ratio of the concentrations of each IL-22 fusion protein that induced 200 pg / mL IL-10 with and without uPA protease. [Figure 24A] Schematic diagram of IL-22 release from the fusion protein "FP16" through VH release. [Figure 24B] Assessment of the activity of IL-22 fusion proteins with and without protease digestion. To assess whether amino acid modifications at the VH / VL interface promoted IL-22 release, IL-22 activity was assayed using the concentration of IL-10 secreted from cells in response to IL-22. Two assay plates were assayed in total, and Figure 24B corresponds to the evaluation results of plate 1. On each assay plate, an IL-22 fusion protein without any modifications at the VH / VL interface, i.e., a control fusion protein ("Ab5H / Ab5L FP16"), was included as a reference. To compare IL-22 activity among IL-22 fusion proteins, the IL-10 response curve was interpolated at a concentration of 300 pg / mL. The activity window was calculated as the ratio of the concentrations of each IL-22 fusion protein that induced 300 pg / mL IL-10 with and without uPA protease. [Figure 24C]Assessment of the activity of IL-22 fusion proteins with and without protease digestion. To assess whether amino acid modifications at the VH / VL interface promoted IL-22 release, IL-22 activity was assayed using the concentration of IL-10 secreted from cells in response to IL-22. Two assay plates were assayed in total, and Figure 24C corresponds to the evaluation results for plate 1. For each assay plate, an IL-22 fusion protein without any modifications at the VH / VL interface, i.e., a control fusion protein ("Ab5H / Ab5L FP16"), was included as a reference. To compare IL-22 activity among IL-22 fusion proteins, the IL-10 response curve was interpolated at a concentration of 300 pg / mL. The activity window was calculated as the ratio of the concentrations of each IL-22 fusion protein that induced 300 pg / mL IL-10 with and without uPA protease. [Figure 24D] Assessment of the activity of IL-22 fusion proteins with and without protease digestion. To assess whether amino acid modifications at the VH / VL interface promoted IL-22 release, IL-22 activity was assayed using the concentration of IL-10 secreted from cells in response to IL-22. Two assay plates were assayed in total, and Figure 24D corresponds to the evaluation results of plate 2. On each assay plate, an IL-22 fusion protein without any modifications at the VH / VL interface, i.e., a control fusion protein ("Ab5H / Ab5L FP16"), was included as a reference. To compare IL-22 activity among IL-22 fusion proteins, the IL-10 response curve was interpolated at a concentration of 300 pg / mL. The activity window was calculated as the ratio of the concentrations of each IL-22 fusion protein that induced 300 pg / mL IL-10 with and without uPA protease. [Figure 24E]Assessment of the activity of IL-22 fusion proteins with and without protease digestion. To assess whether amino acid modifications at the VH / VL interface promoted IL-22 release, IL-22 activity was assayed using the concentration of IL-10 secreted from cells in response to IL-22. Two assay plates were assayed in total, and Figure 24E corresponds to the evaluation results for plate 2. For each assay plate, an IL-22 fusion protein without any modifications at the VH / VL interface, i.e., a control fusion protein ("Ab5H / Ab5L FP16"), was included as a reference. To compare IL-22 activity among IL-22 fusion proteins, the IL-10 response curve was interpolated at a concentration of 300 pg / mL. The activity window was calculated as the ratio of the concentrations of each IL-22 fusion protein that induced 300 pg / mL IL-10 with and without uPA protease. [Figure 25A] Assessment of the activity of IL-22 fusion proteins with and without protease digestion and the activity of recombinant IL-22. IL-22 activity of IL-22 fusion proteins without mutations at the VH / VL interface and selected IL-22 fusion protein variants for FP14 was assessed in the presence or absence of uPA protease, along with recombinant IL-22 as a reference control. To compare IL-22 activity, an IL-10 response curve was interpolated at a concentration of 250 pg / mL for FP14. The activity window for each fusion protein was calculated as the ratio of the concentration of IL-22 fusion protein that induced the indicated amount of IL-10 with and without uPA protease. All three selected fusion protein variants showed lower IL-22 bioactivity than recombinant human IL-22 in the absence of uPA, and IL-22 bioactivity was restored to the same level as recombinant human IL-22 in the presence of uPA. [Figure 25B]Assessment of the activity of IL-22 fusion proteins with and without protease digestion and the activity of recombinant IL-22. IL-22 activity of IL-22 fusion proteins without mutations at the VH / VL interface and IL-22 fusion protein variants selected for FP15 was assessed in the presence or absence of uPA protease, along with recombinant IL-22 as a reference control. To compare IL-22 activity, an IL-10 response curve was interpolated at a concentration of 400 pg / mL for FP15. The activity window for each fusion protein was calculated as the ratio of the IL-22 fusion protein concentrations that induced the indicated amount of IL-10 with and without uPA protease. All three selected fusion protein variants showed lower IL-22 bioactivity than recombinant human IL-22 in the absence of uPA, and IL-22 bioactivity was restored to the same level as recombinant human IL-22 in the presence of uPA. [Figure 25C] Assessment of the activity of IL-22 fusion proteins with and without protease digestion and the activity of recombinant IL-22. The IL-22 activity of IL-22 fusion proteins without mutations at the VH / VL interface and selected IL-22 fusion protein variants for FP16 was assessed in the presence or absence of uPA protease, along with recombinant IL-22 as a reference control. To compare IL-22 activity, an IL-10 response curve was interpolated at a concentration of 400 pg / mL for FP16. The activity window for each fusion protein was calculated as the ratio of the IL-22 fusion protein concentrations that induced the indicated amount of IL-10 with and without uPA protease. All three selected fusion protein variants showed lower IL-22 bioactivity than recombinant human IL-22 in the absence of uPA, and IL-22 bioactivity was restored to the same level as recombinant human IL-22 in the presence of uPA. DETAILED DESCRIPTION OF THE INVENTION

[0013] Description of Aspects General Technology The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully explained in such references as "Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (FM Ausubel et al., eds. 1987, and regularly updated); PCR: The Polymerase Chain Reaction (Mullis et al., ed., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

[0014] The following definitions and detailed descriptions are provided to facilitate understanding of the disclosure set forth herein. All references mentioned herein are specifically incorporated by reference.

[0015] I. Definition Proteins / Polypeptides As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" typically refers to a peptide having a length of approximately four amino acids or more and does not refer to a specific length of the product. As used herein, the term also includes fragments of polypeptides. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides can be derived from natural biological sources or produced by recombinant technology, but are not necessarily translated from a designated nucleic acid sequence. Polypeptides may be generated in any manner, including chemical synthesis. Polypeptides as described herein may be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids in size. Polypeptides may have a defined three-dimensional structure, but do not necessarily have such a structure. Polypeptides that have a defined three-dimensional structure are said to be folded, and polypeptides that do not have a defined three-dimensional structure but rather can adopt a number of different conformations are said to be unfolded.

[0016] amino acid Herein, amino acids are represented by one-letter or three-letter codes, or both, such as Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, or Val / V. To represent an amino acid at a specific position, a number representing the specific position can be combined with the one-letter or three-letter code of the amino acid, as appropriate. For example, amino acid 37V, an amino acid contained in an antibody variable region, represents Val at position 37 according to the Kabat numbering system.

[0017] Amino acid modification The terms "amino acid modification," "amino acid alteration," or "amino acid mutation," as used interchangeably herein, refer to the modification of an amino acid in the amino acid sequence of a protein or polypeptide by 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, which may be used as appropriate. Several methods known in the art for replacing amino acids with amino acids other than natural amino acids may also be used (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a tRNA-containing cell-free translation system (Clover Direct (Protein Express)) having a non-naturally occurring amino acid bound to an amber suppressor tRNA complementary to the UAG stop codon (amber codon) is also preferably used. Herein, examples of amino acid modifications at designated positions include substitution or deletion of the designated residue, or insertion of at least one amino acid residue adjacent to the designated residue, or any combination of these substitutions, deletions, and insertions. An insertion "adjacent" to a designated residue means insertion within 1 to 2 residues of the designated residue. The insertion may be N-terminal or C-terminal to the designated residue. A preferred amino acid modification herein is substitution.

[0018] replacement An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence with another, different "replacement" amino acid residue. One or more replacement residues may be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and may be selected from the group consisting of alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine ​​(Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). Preferably, the replacement residue is not cysteine. Substitution with one or more non-naturally occurring amino acid residues is also encompassed by the definition of amino acid substitution herein. A "non-naturally occurring amino acid residue" refers to a residue other than the naturally occurring amino acid residues listed above that can be covalently linked to an adjacent amino acid residue in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs, such as those described in Ellman et al. Meth. Enzym. 202:301-336 (1991). To generate such non-naturally occurring amino acid residues, the procedures of Noren et al. Science 244:182 (1989) and Ellman et al., supra, can be used. Briefly, these procedures involve chemically activating a suppressor tRNA with the non-naturally occurring amino acid residue, followed by in vitro transcription and translation of the RNA.

[0019] Insert An "amino acid insertion" refers to the incorporation of at least one amino acid into a predetermined amino acid sequence. Insertions typically consist of one or two amino acid residues, although the present application contemplates larger "peptide insertions," e.g., from about three to about five, or even up to about ten amino acid residues. The inserted residues may be naturally occurring or non-naturally occurring, as disclosed above.

[0020] deletion An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0021] As used herein, the term "and / or" when referring to sites of amino acid modification includes all combinations appropriately represented by "and / or." Specifically, for example, the phrase "amino acids at positions 37, 45, and / or 47 are substituted" includes the following amino acid modification variations: (a) position 37, (b) position 45, (c) position 47, (d) positions 37 and 45, (e) positions 37 and 47, (f) positions 45 and 47, and (g) positions 37, 45, and 47.

[0022] Herein, the one-letter or three-letter code of the amino acid before and after the modification can be used as appropriate to represent an amino acid modification, with the one-letter or three-letter code of the amino acid before and after the modification preceded and followed by a number representing a specific position. For example, the modification F37V or Phe37Val used to replace an amino acid contained in an antibody variable region represents a substitution of Val for Phe at position 37 as defined by the Kabat numbering. Specifically, the number represents the amino acid position as defined by the Kabat numbering; the one-letter or three-letter code of the amino acid before the number represents the amino acid before substitution; and the one-letter or three-letter code of the amino acid after the number represents the amino acid after substitution. Similarly, the modification P238A or Pro238Ala used to replace an amino acid in the Fc region contained in an antibody constant region represents a substitution of Ala for Pro at position 238 as defined by the EU numbering. Specifically, the numbers represent amino acid positions defined by EU numbering; the one-letter or three-letter code of the amino acid preceding the number represents the amino acid before substitution; and the one-letter or three-letter code of the amino acid following the number represents the amino acid after substitution.

[0023] Percent (%) Amino Acid Identity "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0024] Ligand-binding moieties / molecules In some embodiments, the fusion protein is a polypeptide comprising a ligand-binding moiety or a ligand-binding molecule, which further comprises a ligand-binding domain. As used herein, the term "ligand-binding moiety" or "ligand-binding molecule" refers to a moiety or molecule capable of binding to a ligand, and particularly to a moiety or molecule that binds to a ligand when the moiety or molecule is in an uncleaved state. In this context, "binding" generally refers to binding through interactions primarily based on non-covalent bonds, such as electrostatic forces, van der Waals forces, or hydrogen bonds. Preferred examples of binding modes of ligand-binding moieties or molecules include, but are not limited to, antigen-antibody reactions, through which antigen-binding domains, antigen-binding molecules, antibodies, antibody fragments, etc. bind to antigens. In certain embodiments, ligand-binding moieties or molecules include, but are not limited to, antibody fragments, antibodies, and molecules formed from antibody fragments (e.g., diabodies, chimeric antigen receptors (CARs)), including multispecific binding molecules (e.g., bispecific diabodies and bispecific antibodies).

[0025] Ligand-binding domain As used herein, the term "ligand-binding domain" refers to a portion of a ligand-binding moiety or molecule that binds only to a portion (epitope) of a ligand when the ligand-binding moiety / molecule is bound to the ligand. In the present invention, a ligand-binding domain is limited only by the fact that the domain binds to a ligand when the ligand-binding moiety / molecule is in its uncleaved state, and can have any structure, so long as the domain is capable of binding to the ligand of interest when the ligand-binding moiety / molecule is in its uncleaved state.Examples of ligand-binding domains include, but are not limited to, antigen-binding domains, antibody heavy chain variable regions (VH), antibody light chain variable regions (VL), antibody Fv regions, single domain antibodies (sdAb), scaffold peptides, and peptide aptamers (Reverdatto S. et al., Curr Top Med Chem. 2015; 15(12): 1082-1101), IL-12 receptor, a module called an A domain of approximately 35 amino acids contained in an in vivo cell membrane protein avimer (WO2004 / 044011 and WO2005 / 040229), an adnectin containing a 10Fn3 domain that acts as a protein binding domain derived from the glycoprotein fibronectin expressed on the cell membrane (WO2002 / 032925), an affibody containing an IgG binding domain scaffold that constitutes a three-helix bundle composed of 58 amino acids of protein A (WO1995 / 001937), and a DARPin (designated ankyrin repeat), which is a molecular surface-exposed region of ankyrin repeat (AR), each of which has a structure of 33 amino acid residues folded into subunits of one turn, two antiparallel helices, and one loop. These include anticalin (WO2003 / 029462), which has four loop regions connecting eight antiparallel chains bent toward the central axis at one end of a barrel structure highly conserved in lipocalin molecules such as the neutrophil gelatinase-associated lipocalin (NGAL) and the anticalin (WO2003 / 029462), and a depressed region in the internal parallel sheet structure of a horseshoe-fold structure composed of repeated leucine-rich repeat (LRR) modules of variable lymphocyte receptors (VLRs) without immunoglobulin structure, as found in the adaptive immune systems of jawless vertebrates such as lampreys or hagfish (WO2008 / 016854).

[0026] antigen-binding domain As used herein, the term "antigen-binding domain" refers to a region that specifically binds to an antigen or partially complements an antigen. As used herein, an antigen-binding molecule comprises an antigen-binding domain. When the molecular weight of an antigen is large, the antigen-binding domain can bind only to a specific portion of the antigen. This specific portion is called an epitope. In one embodiment, the antigen-binding domain comprises an antibody fragment that binds to a specific antigen. The antigen-binding domain can be provided by one or more antibody variable domains. In a non-limiting embodiment, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen-binding domains include "scFv (single-chain Fv)," "single-chain antibody (single-chain antibody)," "Fv," "scFv2 (single-chain Fv2)," "Fab," or "F(ab')2." In another embodiment, the antigen-binding domain comprises a non-antibody protein or a fragment thereof that binds to a specific antigen. In a specific embodiment, the antigen-binding domain comprises a hinge region. In some embodiments, the antigen is a ligand. As used herein, when the antigen is a ligand, the terms "antigen-binding domain" and "ligand-binding domain" may be used interchangeably to refer to the region that specifically or partially binds to the ligand as an antigen.

[0027] As used herein, the term "binding to the same epitope" means that the epitopes bound by two antigen-binding domains at least partially overlap. The degree of overlap is not limited, but is at least 10% or more, preferably 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, and particularly preferably 90% or more. 100% overlap is most preferred.

[0028] In one embodiment, the fusion protein of the present invention comprises an antigen-binding domain capable of binding to an antigen, e.g., interleukin-12 (IL-12) or IL-22. The terms "fusion protein that binds IL-12," "polypeptide that binds IL-12," or "antibody that binds IL-12" or "anti-IL-12" antibody refer to a measurable and reproducible interaction between a protein or antibody and IL-12 that determines the presence of that antigen, e.g., IL-12, in the presence of a heterogeneous population of molecules, including biological molecules. The same is true for IL-22, etc. The fusion protein or antibody binds to its antigen with greater affinity, avidity, more readily, and / or with a longer duration than it binds to other antigens. In one embodiment, the extent of binding of the fusion protein or antibody to an unrelated antigen is less than about 10% of the binding of the fusion protein or antibody to the antigen, as measured, e.g., by radioimmunoassay (RIA). In certain embodiments, the fusion protein or antibody that specifically binds to an antigen / target is at a concentration of 1 micromolar (μM) or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 The fusion protein or antibody has a dissociation constant (Kd) of 1 M (M). In certain embodiments, the fusion protein or antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0029] Ligand / Antigen As used herein, the terms "ligand" (or "ligand moiety") and "antigen" may be used interchangeably and are limited only by the presence of an epitope to which the ligand-binding domain or antigen-binding domain binds. The terms "ligand" and "antigen" refer to any molecule to which a ligand-binding domain or antigen-binding domain can specifically bind. Preferred examples of ligands / antigens include, but are not limited to, peptides, polypeptides, and proteins of animal or human origin. Preferred examples of ligands / antigens for use in treating diseases caused by target tissues 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 the tissue containing the target cells, molecules expressed on the surface of cells that have an immunological role in the target cells and the tissue containing the target cells, macromolecules present in the stroma of the tissue containing the target cells, soluble molecules such as cytokines, chemokines, polypeptide hormones, growth factors, apoptosis inducers, PAMPs, DAMPs, nucleic acids, and fragments thereof, or other molecules involved in immunoregulatory and inflammatory processes. Examples of ligands or antigens include interleukins, interferons, hematopoietic factors, members of the TNF superfamily, chemokines, growth factors, members of the TGF-β family, myokines, adipokines, or neurotrophic factors. More specifically, examples include CXCL9, CXCL10, CXCL11, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-22, IFN-α, IFN-β, IFN-γ, MIG, I-TAC, RANTES, MIP-1a, MIP-1b, IL-1R1 (interleukin-1 receptor, type I), IL-1R2 (interleukin-1 receptor, type II), IL-1RAcP (interleukin-1 receptor accessory protein), or IL-1Ra (protein accession number NP_776214, mRNA accession number NM_173842.2).

[0030] specificity As used herein, the term "specificity" refers to the property of one of the specifically binding molecules not substantially binding to molecules 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 specific antigen. This term is also used when the antigen-binding domain has specificity for a particular epitope among multiple epitopes contained in an antigen. In this context, the term "does not substantially bind" means that the binding activity of a specific binding molecule for molecules other than its binding partner is 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less of its binding activity for the binding partner molecule, as determined according to the method described in the section on binding activity.

[0031] Affinity The term "affinity," as used herein, refers to the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., a ligand-binding molecule, a ligand, an antigen-binding molecule, or an antibody) and its binding partner (e.g., a ligand, a ligand receptor, or an antigen). Unless otherwise specified, "binding affinity," as used herein, refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., a ligand-binding molecule and a ligand, a ligand and a ligand receptor, an antigen-binding molecule and an antigen, or an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of the dissociation rate constant and the association rate constant (Koff and Kon, respectively). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0032] Antibodies and antibody fragments In one embodiment, the ligand-binding portion of the presently claimed fusion proteins comprises an antibody. As used herein, the term "antibody" is used in the broadest sense to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0033] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0034] Full-length antibody / natural antibody The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0035] As used herein, the term "native antibody" refers to immunoglobulin molecules with various structures found in nature. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain (VH), or antibody heavy chain variable domain (VH), followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain (VL), or light chain variable domain (VL), or antibody light chain variable domain (VH), followed by a constant light chain (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. As used herein, the terms "CL" or "CL region" or "CL domain" are used interchangeably, and the same is applicable to the other domains "VH," "VL," "CH1," "CH2," and "CH3," when each of these terms is paired with a "region" or "domain" in reference.

[0036] Monoclonal antibodies As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants (e.g., variants containing naturally occurring mutations or variants that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0037] Chimeric, humanized, and human antibodies As used herein, the term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0038] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0039] A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0040] Human Consensus Framework A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0041] Acceptor Human Framework An "acceptor human framework," for purposes of this specification, is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise those same amino acid sequences or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0042] Affinity matured antibodies As used herein, the term "affinity matured" antibody refers to an antibody with one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody for antigen compared to a parent antibody that does not possess such modifications.

[0043] Antibody Class The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0044] IgG antibody-like polypeptide As used herein, the term "IgG antibody-like polypeptide" or "IgG antibody-like molecule" is used to define a polypeptide having a portion substantially similar in structure to a constant domain or constant region as in an IgG antibody and a portion substantially similar in structure to a variable domain or variable region as in an IgG antibody, and having a conformation substantially similar to that of an IgG antibody. In an IgG antibody-like molecule, the antibody CH1-like domain and CL-like domain may be used interchangeably; that is, the domain linked to the portion similar to the antibody hinge region may be an antibody CH1 domain or an antibody CL domain, as long as an interaction similar to the interaction between the CH1 and CL of an IgG antibody exists between the domains. However, as used herein, an "IgG antibody-like molecule" may or may not exhibit antigen-binding activity while retaining a structure similar to that of an IgG antibody. As used herein, when the term "full length IgG antibody comprising a protease cleavage site" or a full length IgG antibody comprising a protease cleavage site is referred to herein, such term or phrase is used interchangeably to refer to the above-mentioned "IgG antibody-like polypeptide" or "IgG antibody-like molecule," so long as it serves the purpose for the proper function of the fusion protein.

[0045] Substantially similar As used herein, the terms "substantially similar" or "substantially the same" refer to a similarity between two numerical values ​​(e.g., between one relating to an antibody of the invention and one relating to a reference / comparator antibody) that is sufficiently high that one of skill in the art would consider the difference between the two numerical values ​​to have little or no biological and / or statistical significance in terms of the biological characteristic measured by the numerical value (e.g., Kd value).

[0046] Constant and Fc regions As used herein, the term "constant region" or "constant domain" refers to a region or domain in an antibody other than the variable region. For example, an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 Da, composed of two identical light chains and two identical heavy chains connected through disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy chain domain or heavy chain variable domain, followed by a heavy chain constant region (CH) containing a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chain of a natural antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain. As used herein, the terms "CH1," "CH1 domain," and "CH1 region" are used interchangeably. As used herein, the terms "CL," "CL domain," and "CL region" are used interchangeably.

[0047] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residues of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0048] Mutant Fc region A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution, e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native-sequence Fc region or in the Fc region of the parent polypeptide compared to the native-sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably has at least about 80% homology with the native-sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto. Mutant constant region A "variant constant region" comprises an amino acid sequence that differs from that of a native-sequence constant region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant constant region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native-sequence constant region or in the constant region of the parent polypeptide compared to the native-sequence constant region or the constant region of the parent polypeptide. The variant constant regions herein preferably have at least about 80% homology with the native-sequence constant region and / or the constant region of the parent polypeptide, most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto.

[0049] Fc receptors "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR binds to IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs are reviewed, e.g., in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126:330-41 (1995). Other FcRs, including those identified in the future, are also encompassed by the term "FcR" herein.

[0050] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for regulating maternal IgG transfer to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).

[0051] In vivo binding to human FcRn and plasma half-life of human FcRn high-affinity binding polypeptides can be measured, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides with mutant Fc regions are administered. WO2000 / 42072 (Presta) describes antibody mutants with increased or decreased binding to FcR. See, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0052] Fc region containing antibody The term "Fc region-containing antibody" refers to an antibody that contains an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or by recombinant engineering of a nucleic acid encoding the antibody. Thus, a composition containing an antibody with an Fc region according to the present invention can contain an antibody with G446-K447, an antibody with G446 but without K447, an antibody with G446-K447 completely removed, or a mixture of the above three types of antibodies.

[0053] Functional Fc region A "functional Fc region" comprises an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors (BCRs)); and the like. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays, for example, as disclosed within the definitions herein.

[0054] Human effector cells "Human effector cells" refer to leukocytes that express one or more FcRs and exert effector function. In certain embodiments, the cells express at least FcγRIII and exert ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, for example, from blood.

[0055] Antibody-dependent cell-mediated cytotoxicity "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcR) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill them with cytotoxins. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent Nos. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta), may be performed. Useful effector cells for such assays include PBMCs and NK cells. Alternatively, or additionally, the ADCC activity of a molecule of interest may be assessed in vivo in an animal model, such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0056] Complement-dependent cytotoxicity "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are bound to their corresponding antigens. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with mutant Fc regions) and increased or decreased C1q binding ability are described, for example, in U.S. Patent No. 6,194,551 B1 and WO 1999 / 51642. See also, for example, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0057] Variable region As used herein, the term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). As used herein, "heavy chain variable domain (VH)" is used interchangeably with "antibody heavy chain variable domain (VH)" or "antibody heavy chain variable region (VH)" or "VH" or "antibody VH" or "VH domain", and "light chain variable domain (VL)" is used interchangeably with "antibody light chain variable domain (VH)" or "antibody light chain variable region (VL)" or "VL" or "antibody VL" or "VL domain".

[0058] HVR or CDR As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) A combination of (a), (b), and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0059] Framework As used herein, the term "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0060] Isolated antibodies An "isolated" antibody is one that has been separated from a component of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0061] Isolated nucleic acids An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0062] An "isolated nucleic acid encoding an anti-IL-12 antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including nucleic acid molecules carried on a single vector or separate vectors, and nucleic acid molecules present in one or more locations in a host cell. The same is true for anti-IL-22 antibodies, etc.

[0063] An "isolated nucleic acid encoding a fusion polypeptide that binds IL-12" refers to one or more nucleic acid molecules that encode a polypeptide of Formula I (or fragments thereof), including nucleic acid molecules on a single vector or separate vectors, and nucleic acid molecules present in one or more locations in a host cell. The same is true for IL-22, etc.

[0064] Vectors and host cells As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of effecting the expression of nucleic acids to which they are operatively linked. Such vectors are also referred to herein as "expression vectors."

[0065] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0066] Individual / Subject An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0067] Pharmaceutical preparations The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. A "pharmaceutical formulation" may alternatively be referred to as a "pharmaceutical composition."

[0068] Pharmaceutically acceptable carrier A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation / composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0069] Effective dose An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount, at dosages and for periods of time necessary, effective to achieve a desired therapeutic or prophylactic result.

[0070] Attached document The term "package insert" is used to refer to instructions typically included in commercial packaging of therapeutic products that contain information about the indications, usage, dosage, method of administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0071] treatment As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) refers to a clinical intervention intended to alter the natural course of the individual being treated and can be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.

[0072] cancer As used herein, the terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0073] Cell proliferative disorders As used herein, the terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0074] B-cell neoplasms / Hodgkin's disease "B-cell neoplasms" includes Hodgkin's disease, including lymphocyte predominant Hodgkin's disease (LPHD); non-Hodgkin's lymphoma (NHL); follicular center cell (FCC) lymphoma; acute lymphocytic leukemia (ALL); chronic lymphocytic leukemia (CLL); and hairy cell leukemia. Non-Hodgkin lymphomas include low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, bulky disease NHL, plasmacytoid lymphocytic lymphoma, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström's macroglobulinemia. Treatment of recurrence of these cancers is also a consideration. LPHD is a type of Hodgkin's disease that tends to recur frequently, even after radiation or chemotherapy treatment. CLL is one of the four major types of leukemia. A cancer of mature B cells called lymphocytes, CLL develops due to the progressive accumulation of cells in the blood, bone marrow, and lymphatic tissues. Indolent lymphoma is a slow-growing, incurable disease in which the average patient survives 6 to 10 years after multiple periods of remission and relapse.

[0075] Breast tumors The term "breast tumor" or "breast cancer" refers to any tumor or cancer of the breast, including, for example, adenocarcinomas such as invasive or ductal carcinoma in situ, invasive or lobular carcinoma in situ, medullary carcinoma, colloid carcinoma, and papillary carcinoma; and less common forms such as cystosarcoma phyllodes, sarcoma, squamous cell carcinoma, and carcinosarcoma.

[0076] Colon tumors The term "colon tumor" or "colon cancer" refers to any tumor or cancer of the colon (large intestine from the cecum to the rectum).

[0077] Colorectal tumors The term "colorectal tumor" or "colorectal cancer" refers to any tumor or cancer of the large intestine, including the colon (the large intestine from the cecum to the rectum) and rectum, including, for example, adenocarcinoma and less common forms such as lymphoma and squamous cell carcinoma.

[0078] Non-Hodgkin lymphoma As used herein, the term "non-Hodgkin's lymphoma" or "NHL" (non-Hodgkin's lymphoma) refers to cancers of the lymphatic system other than Hodgkin's lymphoma. Hodgkin's lymphomas can generally be distinguished from non-Hodgkin's lymphomas by the presence of Reed-Sternberg cells in Hodgkin's lymphomas and their absence in non-Hodgkin's lymphomas. Examples of non-Hodgkin's lymphomas encompassed by this term as used herein include any that would be identified as such by a skilled artisan (e.g., an oncologist or pathologist) according to classification schemes known in the art, such as the Revised European-American Lymphoma (REAL) scheme described in Color Atlas of Clinical Hematology, Third Edition; A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Limited 2000) (see especially Figs. 11.57, 11.58, and / or 11.59).More specific examples include, but are not limited to, the following: relapsed or refractory NHL, newly diagnosed low-grade NHL, stage III / IV NHL, chemotherapy-resistant NHL, precursor B-lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and / or prolymphocytic leukemia and / or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and / or lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone MALT lymphoma, nodal marginal zone lymphoma, Marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or plasma cell myeloma, low-grade / follicular lymphoma, intermediate-grade / follicular NHL, mantle cell lymphoma, follicle center lymphoma (follicular), intermediate-grade diffuse NHL, diffuse large B-cell lymphoma, aggressive NHL (including aggressive newly diagnosed NHL and aggressive relapsed NHL), relapsed after autologous stem cell transplant or autologous stem cell transplant NHL not responding to autologous cell transplantation, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, bulky disease NHL, Burkitt lymphoma, precursor (peripheral) T-cell lymphoblastic leukemia and / or lymphoma, adult T-cell lymphoma and / or leukemia, T-cell chronic lymphocytic leukemia and / or precursor Lymphocytic leukemia, large granular lymphocytic leukemia, mycosis fungoides and / or Sézary syndrome, extranodal natural killer / T-cell (nasal type) lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, cutaneous lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, peripheral T-cell (not otherwise specified) lymphoma, and angioimmunoblastic T-cell lymphoma.

[0079] ovarian cancer "Ovarian cancer" refers to a heterogeneous group of malignant tumors originating from the ovary. Approximately 90% of malignant ovarian tumors are epithelial in origin; the remainder are germ cell and stromal tumors. Epithelial ovarian tumors are classified into the following histologic subtypes: serous adenocarcinoma (accounting for about 50% of epithelial ovarian tumors); endometrioid adenocarcinoma (about 20%); mucinous adenocarcinoma (about 10%); clear cell carcinoma (about 5-10%); and Brenner (transitional cell) tumor (less common). The prognosis for ovarian cancer, the sixth most common cancer in women, is generally poor, with 5-year survival rates ranging from 5% to 30%. For reviews of ovarian cancer, see Fox et al. (2002) "Pathology of epithelial ovarian cancer," in Ovarian Cancer, ch. 9 (Jacobs et al., eds., Oxford University Press, New York); Morin et al. (2001) "Ovarian Cancer," in Encyclopedic Reference of Cancer, pp. 654-656 (Schwab, ed., Springer-Verlag, New York). Methods for diagnosing or treating any of the above epithelial ovarian tumor subtypes, particularly the serous adenocarcinoma subtype, are contemplated by the present invention.

[0080] recurrence "Relapse" refers to the return of a patient's disease to its previous disease state, especially the return of symptoms after a clear or partial recovery. Unless otherwise indicated, relapse refers to the process of returning or returning to a disease state prior to a previous treatment (including, but not limited to, chemotherapy and stem cell transplant treatment).

[0081] refractory "Refractory" refers to the resistance or non-responsiveness of a disease or condition to treatment (e.g., the number of neoplastic plasma cells increases even when treatment is administered). Unless otherwise indicated, the term "refractory" refers to resistance or non-responsiveness to any prior treatment (including, but not limited to, chemotherapy and stem cell transplant treatment).

[0082] Gastric tumor As used herein, the term "gastric tumor" or "gastric cancer" refers to any tumor or cancer of the stomach, including, for example, adenocarcinoma (e.g., diffuse and intestinal types) and less common forms such as lymphoma, leiomyosarcoma, and squamous cell carcinoma.

[0083] tumor As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as used herein.

[0084] Inhibition of cell growth or proliferation / suppression of cell growth "Inhibiting cell growth or proliferation" or "suppressing cell growth" means reducing cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.

[0085] chemotherapy drugs "Chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylolmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including the synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®)), acetylcamptothecin, and scopolectin. (including scopolectin, and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictiin; spongistatin; chlorambucil, chlornaphazine, chlorophosphamide (chlorophosphamide), estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, fenesterine, prednimustine, trofosfamide, nitrogen mustards, such as uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine;Enediyne antibiotics {e.g., calicheamicin, particularly calicheamicin gamma 11 and calicheamicin omega 11 (e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)) See also); CDP323, an oral alpha-4 integrin inhibitor; dynemicins, including dynemicin A; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores}, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposomal for injection (DOXIL®), liposomal doxorubicin TCL D-99 (including MYOCET®), pegylated liposomal doxorubicin (including CAELYX®, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, and lodorubicin antibiotics, such as rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calucelone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane. (anti-adrenals); folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydrochloride; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verraculin A) A), Roridin A, and Anguidine; urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, such as paclitaxel (TAXOL®), albumin-modified nanoparticle formulations of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents, such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin;Vinces, which interfere with the formation of microtubules by tubulin polymerization, include vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylornithine; DMFO; retinoids, such as retinoic acids, including bexarotene (TARGRETIN®); bisphosphonates, such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways associated with abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGFR). EGF-R; vaccines such as THERATOPE® vaccine and gene therapy vaccines (e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine); topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341);Bcl-2 inhibitors such as bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); sorafenib, ABT510; oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); lonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, which is an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, which is an abbreviation for a treatment regimen with oxaliplatin (ELOXANTIN™) in combination with 5-FU and leucovorin;

[0086] Chemotherapeutic agents, as defined herein, include "antihormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block, or inhibit the action of hormones that can promote cancer growth. They may be hormones themselves, including, but are not limited to: antiestrogens with a mixed agonist / antagonist profile, including selective estrogen receptor modulators (SERMs) such as tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, ketoxifene, and SERM3; pure antiestrogens without agonist properties, such as fulvestrant (FASLODEX®) and EM800 (such agents inhibit the estrogen receptor). steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and non-steroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole (FEMARA®), and aminoglutethimide, and vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazole. aromatase inhibitors, including other aromatase inhibitors including dazole; luteinizing hormone-releasing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and triptorelin; sex steroids, including progestins such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and Premarin, and androgens / retinoids such as fluoxymesterone, all-trans retinoic acid, and fenretinide; onapristone; antiprogesterones;Estrogen receptor down-regulators (ERDs); antiandrogens, such as flutamide, nilutamide, and bicalutamide; and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

[0087] Cytostatics / agents that inhibit cell proliferation As used herein, the terms "cytostatic agent" or "agent inhibiting cytostasis" interchangeably refer to a compound or composition that arrests cell proliferation either in vitro or in vivo. Thus, a cytostatic agent may significantly reduce the proportion of cells in S phase. Further examples of cytostatic agents include agents that block cell cycle progression by inducing G0 / G1 arrest or M-phase arrest. The humanized anti-Her2 antibody trastuzumab (HERCEPTIN®) is an example of a cytostatic agent that induces G0 / G1 arrest. Classical M-phase blockers include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Certain agents that arrest G1, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C, also affect S-phase arrest. Further information can be found in Murakami et al., Mendelsohn and Israel (eds.), The Molecular Basis of Cancer (WB Saunders, Philadelphia, 1995), Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs," e.g., page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew tree, is a semisynthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, thereby inhibiting mitosis in cells.

[0088] autoimmune disease "Autoimmune disease" refers to a non-malignant disease or disorder arising from and directed against an individual's own tissues. As used herein, autoimmune disease specifically excludes malignant or cancerous diseases or conditions, and specifically excludes B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myeloblastic leukemia.Examples of autoimmune diseases or disorders include, but are not limited to, inflammatory responses such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); systemic sclerosis and sclerosis; responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome (ARDS)); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions, such as eczema and asthma and other conditions involving T-cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; and systemic lupus erythematosus (SLE). (including, but not limited to, lupus nephritis, cutaneous lupus); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile-onset diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, typically seen in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); diseases associated with leukocyte leakage; and the central nervous system (CNS). Inflammatory disorders; multiple organ injury syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs' positive anemia); myasthenia gravis; antigen-antibody complex-mediated disease; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; Reiter's disease; Stiffman syndrome; Behçet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

[0089] Immunosuppressants / anti-inflammatory agents The term "immunosuppressant," as used herein with respect to adjunctive therapy, refers to a substance that acts to suppress or mask the immune system of the mammal being treated. This includes substances that suppress cytokine production, downregulate or suppress the expression of self-antigens, or mask MHC antigens. Examples of such agents include: 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077); non-steroidal anti-inflammatory drugs (NSAIDs); NSAIDs; ganciclovir, tacrolimus, glucocorticoids (e.g., cortisol or aldosterone), anti-inflammatory agents (e.g., cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, or leukotriene receptor antagonists); purine antagonists, such as azathioprine or mycophenolate mofetil (MMF); alkylating agents, such as cyclophosphamide; bromocriptine; danazol; dapsone; glutaraldehyde (which masks MHC antigens as described in U.S. Pat. No. 4,120,649); anti-idiopathic drugs against MHC antigens and MHC fragments. steroids, such as corticosteroids or glucocorticosteroids or glucocorticoid analogs (e.g., prednisone, methylprednisolone, including SOLU-MEDROL® methylprednisolone sodium succinate, and dexamethasone); dihydrofolate reductase inhibitors, such as methotrexate (oral or subcutaneous); antimalarials, such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; anti-interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosis factor (TNF) antibodies, anti-tumor necrosis factor (TNF-α), ... cytokine or cytokine receptor antibodies, including anti-TNF (necrosis factor: TNF)-alpha antibodies (infliximab (REMICADE®) or adalimumab), anti-TNF-alpha immunoadhesin (etanercept), anti-TNF-beta antibodies, anti-interleukin-2 (IL-2) antibodies and anti-IL-2 receptor antibodies, and anti-interleukin-6 (IL-6) receptor antibodies and antagonists (e.g., ACTEMRA™ (tocilizumab));anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; xenogeneic antilymphocyte globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4 / CD4a antibodies; soluble peptides containing the LFA-3 binding domain (WO 90 / 08187 published July 26, 1990); streptokinase; transforming growth factor-beta (TGF-beta); streptodornase; host-derived RNA or DNA; FK506; RS-61443; chlorambucil; deoxyspergualin; rapamycin; T cell receptors (Cohen et al., U.S. Pat. No. 5,114,721); T cell receptor fragments (Offner et al., Science, 251: 430-432 (1991); WO 90 / 11294; Ianeway, Nature, 341: 482 (1989); and WO91 / 01133); BAFF antagonists, such as BAFF and BR3 antibodies and zTNF4 antagonists (for a review, see Mackay and Mackay, Trends Immunol., 23:113-5 (2002) and see also definitions below); blocking antibodies against CD40-CD40 ligand (e.g., Durie et al., Science, 261:1328-30 (1993); Mohan et al., J. Immunol., 154:1470-80 (1995)) and CTLA4-Ig (Finck et al., Science, 265:1225-7 (1994)); and T cell receptor antibodies such as T10B9 (EP340,109). Some preferred immunosuppressants herein include cyclophosphamide, chlorambucil, azathioprine, leflunomide, MMF, or methotrexate.

[0090] II. Illustrative Embodiments of the Invention In one aspect, the present invention relates to a fusion protein comprising a polypeptide comprising at least one ligand-binding moiety comprising a ligand-binding domain comprising an antibody variable region, a protease cleavage site, and at least one ligand connected to the C-terminal region of the ligand-binding moiety by a non-cleavable peptide linker, wherein (a) in a first state, the ligand binds to the ligand-binding domain and the biological activity of the ligand is attenuated, and in a second state, the biological activity of the ligand is restored; (b) the fusion protein in the first state has a longer serum half-life than in the second state; and (c) the switch from the first state to the second state is mediated by the presence of a protease.

[0091] The difference between the "first state" and the "second state" can be the absence / presence of protease cleavage. The phrase "in the first state" can be rephrased as "before the protease cleavage site is cleaved by the protease," or "when the protease cleavage site is uncleaved by the protease," or "uncleaved state." The phrase "in the second state" can be rephrased as "after the protease cleavage site is cleaved by the protease," or "when the protease cleavage site is cleaved by the protease," or "cleaved state." The same applies to other embodiments described herein.

[0092] In one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (i) a ligand-binding portion comprising a ligand-binding domain and a constant region; (ii) a first peptide linker that contains a protease cleavage site and connects the ligand-binding domain to the constant region; (iii) the constant region, comprising a second peptide linker and, optionally, one or more amino acid residues modified from or to cysteine; (iv) a ligand portion connected to the C-terminal region of the constant region by a third peptide linker; a bivalent homodimeric fusion protein comprising two polypeptides, comprising: (a) in a first state, the ligand moiety binds to the ligand-binding domain and the biological activity of the ligand is attenuated, and in a second state, the biological activity of the ligand is restored; (b) the fusion protein in the first state has a longer serum half-life than in the second state; and (c) the switch from the first state to the second state is mediated by the presence of a protease. The bivalent homodimeric fusion protein.

[0093] In one aspect, the present invention provides a bivalent homodimeric fusion protein comprising an IgG antibody-like polypeptide fused to a ligand moiety, (i) a first peptide linker comprising a protease cleavage site between the (ia) VH and CH1 or (ib) VL and CL interface; (ii) a second peptide linker introduced into the hinge region that connects the CH1 region of the antibody to the Fc region and optionally contains one or more amino acid residues that are modified from or to cysteine; and (iii) a third peptide linker connecting the ligand moiety to the C-terminus of the Fc region of the antibody. Including, (a) in a first state, the ligand moiety binds to the antibody variable region and the biological activity of the ligand is attenuated, and in a second state, the biological activity of the ligand is restored; (b) the fusion protein in the first state has a longer serum half-life than in the second state; and (c) the switch from the first state to the second state is mediated by the presence of a protease. The bivalent homodimeric fusion protein.

[0094] In one aspect, the present invention provides a method for producing a compound having the general formula (I), each of which, from N-terminus to C-terminus, comprises: [Ligand-binding domain]-[Lx]-[Cx]-[Ly]-[ligand moiety] (I) A bivalent homodimeric fusion protein comprising two polypeptides represented by During the ceremony, Lx represents a peptide linker containing a protease cleavage site; Cx represents a constant region comprising a second peptide linker and, optionally, one or more amino acid residues modified to or from cysteine; Ly represents a third peptide linker; and (a) in a first state, the ligand moiety binds to the ligand-binding domain and the biological activity of the ligand moiety is attenuated, and in a second state, the biological activity of the ligand is restored; and (b) the fusion protein in the first state has a longer serum half-life than in the second state; and (c) the switch from the first state to the second state is mediated by the presence of a protease. The bivalent homodimeric fusion protein.

[0095] In one embodiment, the present invention relates to a bivalent homodimeric fusion protein comprising a full-length IgG antibody comprising an antigen-binding domain, wherein the antigen-binding domain comprises a variable region, the variable region comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) that associate with each other, and (a) a protease cleavage site at the boundary between the VH and CH1 or the boundary between the VL and CL of the variable region, and (b) a ligand moiety that binds to the variable region, wherein (a) in a first state, the ligand moiety binds to the variable region and the biological activity of the ligand moiety is attenuated, and in a second state, the biological activity of the ligand moiety is restored, (b) the fusion protein in the first state has a longer serum half-life than in the second state, and (c) the switching from the first state to the second state is mediated by the presence of a protease. As used herein, a full-length IgG antibody includes an IgG antibody-like polypeptide as described herein. In one aspect, the present invention relates to a bivalent homodimeric fusion protein comprising an IgG antibody-like polypeptide comprising an antigen-binding domain, wherein the antigen-binding domain comprises a variable region, wherein the variable region comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) that associate with each other, and wherein the variable region comprises (a) a protease cleavage site at the boundary between the VH and CH1 or the boundary between the VL and CL of the variable region, and (b) a ligand that binds to the variable region, wherein upon protease cleavage, (i) either the VH or the VL dissociates from the fusion protein, and (ii) the ligand dissociates from the variable region, and wherein the dissociation described in (i) is promoted by at least one amino acid modification made at the interface between the VH and VL that reduces the association between the VH and VL in the cleaved state compared to the uncleaved state, and wherein the amino acid residue for the modification is in the framework region (FR). In one aspect, the present invention provides a method for producing a compound having the general formula (I), each of which, from N-terminus to C-terminus, comprises: [Ligand-binding domain]-[Lx]-[Cx]-[Ly]-[ligand moiety] (I) A bivalent homodimeric fusion protein comprising two polypeptides represented by During the ceremony, Lx represents a peptide linker containing a protease cleavage site; Cx represents a constant region comprising a second peptide linker and, optionally, one or more amino acid residues modified to or from cysteine; Ly represents a third peptide linker; and the ligand-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), and the ligand-binding domain comprises at least one amino acid modification that reduces association between VH and VL in the presence of a protease that catalyzes cleavage of the protease cleavage site (the "cleaved state") compared to the absence of the protease (the "uncleaved state"), and the amino acid residue for the modification is in a framework region (FR). The bivalent homodimeric fusion protein.

[0096] In one aspect, the present invention relates to a polypeptide or antibody comprising at least one antigen-binding domain comprising a protease cleavage site, wherein upon cleavage at the protease cleavage site, antibody domains adjacent to the protease cleavage site dissociate. As used herein, the term "antibody domain" refers to a molecule other than a complete antibody, i.e., a portion of an antibody, including, but not limited to, antibody fragments such as VH, VL, VHH, CH1, CH2, CH3, CL, Fv, Fab, Fab', Fab'-SH, F(ab')2, scFv, etc.

[0097] In one embodiment, upon protease cleavage at the protease cleavage site, a portion of the polypeptide or antibody or its antibody domain dissociates from the remaining portion of the polypeptide or antibody. Dissociation is promoted by at least one amino acid modification made at the interface between the portion or domain and the corresponding interacting portion or domain. For example, if the antibody domain is VH, its corresponding interacting domain is VL, and if the antibody domain is VL, its corresponding interacting domain is VH.

[0098] As used herein, several molecular formats are included. As used herein, the terms "ligand" and "antigen" are used interchangeably and broadly refer to all molecules to which a ligand-binding domain or an antigen-binding domain can specifically bind. The terms "ligand-binding domain" and "antigen-binding domain" refer to molecules that can bind to a ligand or an antigen, respectively. When the ligand-binding domain includes an antibody fragment thereof that can bind to the ligand and neutralize the biological activity of the ligand, the ligand-binding domain can be used interchangeably with the antigen-binding domain.

[0099] Protease cleavage site In the present invention, the ligand-binding domain / moiety / molecule comprises at least one protease cleavage site. The protease cleavage site can be located anywhere within the ligand-binding domain / moiety / molecule, so long as, upon protease cleavage, the ligand is released from or becomes unbound to the ligand-binding domain, and the biological activity of the ligand binding to its binding partner is restored. As used herein, the phrase "liberating / releasing a ligand moiety / molecule" or "the ligand moiety / molecule is released" means that the ligand moiety / molecule is able to exert and / or increase its biological activity through interaction with its binding partner, compared to the biological activity of the ligand moiety / molecule bound to the uncleaved ligand-binding domain / moiety / molecule, but does not refer to or imply any particular level of release or any particular mode of action by which the ligand moiety / molecule is released.

[0100] For example, the protease cleavage site can be located near or even within the ligand-binding domain of the ligand-binding moiety / molecule. Protease cleavage at the protease cleavage site can affect, e.g., restore, the biological activity of the ligand to which the moiety / molecule can bind. As used herein, for example, the phrase "biological activity is restored" refers to a transition from a (first) state in which the ligand is bound to the ligand-binding moiety / molecule in an uncleaved state and unable to interact with its binding partner (i.e., biological activity is attenuated due to the absence of interaction) to a (second) state in which the ligand is not bound to the ligand-binding domain and can interact with the binding partner to exert its biological activity. The biological activity of the ligand bound to its binding partner is attenuated in the first state in which the ligand-binding domain is bound, and is restored in the second state in which the ligand-binding domain is unbound in the presence of a protease.

[0101] In some embodiments, in the presence of a protease, a ligand moiety / molecule linked to or bound to a ligand-binding moiety / molecule can be released from the ligand-binding domain of the ligand-binding moiety / molecule due to cleavage at a protease cleavage site located within or near the ligand-binding domain of the ligand-binding moiety / molecule. In some embodiments, even after cleavage, the ligand moiety / molecule can still be linked to the C-terminal region (e.g., Fc region / domain) of the ligand-binding moiety / molecule. In some embodiments, in the presence of a protease, the ligand moiety / molecule can be completely released from the ligand-binding moiety / molecule due to cleavage at a protease cleavage site located between the ligand moiety / molecule and the C-terminal region (e.g., Fc region / domain) of the ligand-binding moiety / molecule. In some embodiments, after cleavage, the ligand moiety / molecule is no longer linked to the C-terminal region (e.g., Fc region / domain) of the ligand-binding moiety / molecule.

[0102] In one embodiment, the ligand-binding moiety / molecule binds more weakly to the ligand moiety / molecule in the cleaved state compared to the uncleaved state (i.e., ligand binding is attenuated). In another embodiment, the ligand-binding moiety / molecule does not bind to the ligand or ligand moiety in the cleaved state compared to the uncleaved state (i.e., ligand binding is abolished). In embodiments where the ligand-binding moiety / molecule binds to the ligand moiety / molecule via an antigen-antibody reaction, the attenuation or absence of ligand binding can be assessed based on the biological activity of the ligand-binding moiety / molecule.

[0103] In one embodiment, the antigen-binding domain binds weaker to the ligand moiety / molecule in the cleaved state compared to the uncleaved state (i.e., ligand binding is attenuated). In another embodiment, the antigen-binding domain does not bind to the ligand or ligand moiety in the cleaved state compared to the uncleaved state (i.e., ligand binding is abolished). In this case, the antigen-binding domain binds to the ligand moiety / molecule via an antigen-antibody reaction, and the attenuation or lack thereof of ligand binding can be assessed based on the biological activity of the ligand-binding moiety / molecule.

[0104] The phrase "ligand binding is attenuated" means that the amount of the test ligand-binding molecule bound to the ligand is, for example, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, of the amount of the control ligand-binding molecule bound to the ligand, based on the above-mentioned measurement method. Any desired index may be used as an index of binding activity. For example, the dissociation constant (KD) may be used. When the dissociation constant (KD) is used as an index for evaluating binding activity, a larger dissociation constant (KD) of the test ligand-binding molecule for the ligand than that of the control ligand-binding molecule means that the test ligand-binding molecule has a weaker binding activity for the ligand than that of the control ligand-binding molecule. The phrase "ligand binding function is attenuated" means that the dissociation constant (KD) of the test ligand-binding molecule for the ligand is, for example, at least 2-fold, preferably at least 5-fold or at least 10-fold, and particularly preferably at least 100-fold, the dissociation constant (KD) of the control ligand-binding molecule for the ligand. Examples of control ligand-binding molecules include the uncleaved ligand-binding moiety / molecule or the uncleaved antibody or antibody fragment.

[0105] In some embodiments of the present invention, the biological activity of a ligand moiety / molecule is attenuated by binding to the ligand-binding domain of an uncleaved ligand-binding moiety / molecule. Examples of embodiments in which the biological activity of a ligand is attenuated include, but are not limited to, embodiments in which binding of the ligand moiety / molecule to the ligand-binding domain of an uncleaved ligand-binding moiety / molecule substantially or significantly interferes with or competes with binding of the ligand to its binding partner. When an antibody or fragment thereof having ligand-neutralizing activity is used as the ligand-binding moiety / molecule, the ligand-binding moiety / molecule bound to the ligand can attenuate, and to a greater extent, attenuate, i.e., inhibit, the biological activity of the ligand by exerting its neutralizing activity.

[0106] In some embodiments of the present invention, the biological activity of a ligand moiety / molecule is attenuated by binding to the antigen-binding domain of an antibody. Examples of embodiments in which the biological activity of a ligand is attenuated include, but are not limited to, embodiments in which binding of the ligand to the antigen-binding domain of an uncleaved antibody substantially or significantly interferes with or competes with binding of the ligand to its binding partner. Binding of the ligand to the antigen-binding domain attenuates or inhibits the biological activity of the ligand by exerting neutralizing activity through an antigen-antibody binding interaction.

[0107] In one embodiment of the present invention, the uncleaved ligand-binding moiety / molecule can sufficiently neutralize the biological activity of the ligand moiety upon binding to the ligand moiety. Specifically, the biological activity of the ligand moiety / molecule when bound to the uncleaved ligand-binding moiety / molecule is preferably lower than that of the ligand moiety / molecule when not bound to the uncleaved ligand-binding moiety / molecule. The biological activity of the ligand when bound to the uncleaved ligand-binding molecule can be, but is not limited to, 90% or less, preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less, particularly preferably 20% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the biological activity of the ligand when not bound to the uncleaved ligand-binding molecule. Administration of a ligand-binding moiety / molecule that sufficiently neutralizes the biological activity of a ligand can be expected to prevent the ligand from exerting its biological activity before it reaches the target tissue.

[0108] In another embodiment of the present invention, the intact antigen-binding domain can preferably sufficiently neutralize the biological activity of the ligand moiety upon binding to the ligand moiety. Specifically, the biological activity of the ligand moiety / molecule when bound to the intact antigen-binding domain is preferably lower than that of the ligand moiety / molecule when not bound to the intact antigen-binding domain. The biological activity of the ligand when bound to the intact antigen-binding domain can be, but is not limited to, 90% or less, preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less, particularly preferably 20% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the biological activity of the ligand when not bound to the intact antigen-binding domain. Administration of an antigen-binding domain that sufficiently neutralizes the biological activity of the ligand can be expected to prevent the ligand from exerting its biological activity before it reaches the target tissue.

[0109] Alternatively, the present invention provides a method for neutralizing the biological activity of a ligand. The method comprises the steps of contacting a ligand-binding molecule of the present invention with a ligand whose biological activity is to be neutralized and collecting the binding product of the two molecules. The biological activity of the neutralized ligand can be restored by cleaving the ligand-binding molecule in the collected binding product. Therefore, the method for neutralizing the biological activity of a ligand according to the present invention may further comprise the step of restoring the biological activity of the ligand by cleaving the ligand-binding molecule in the binding product consisting of the ligand and the ligand-binding molecule (in other words, eliminating the neutralizing activity of the ligand-binding molecule).

[0110] In one embodiment of the present invention, the binding activity of the cleaved ligand-binding moiety or molecule to the ligand moiety or molecule is preferably lower than the binding activity of the natural ligand-binding partner (e.g., the natural receptor for the ligand) to the ligand in vivo. The binding activity of the cleaved ligand-binding moiety / molecule to the ligand moiety / molecule is, but is not limited to, for example, 90% or less, preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less, particularly preferably 20% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the amount of ligand bound to the natural binding partner in vivo (per unit binding partner). Any desired index may be used as an index of binding activity. For example, the dissociation constant (KD) may be used. When using the dissociation constant (KD) as an index for evaluating binding activity, a dissociation constant (KD) of the cleaved ligand-binding moiety / molecule for the ligand that is greater than that of the natural in vivo ligand-binding partner means that the cleaved ligand-binding molecule has weaker binding activity for the ligand than that of the natural in vivo binding partner. The dissociation constant (KD) of the cleaved ligand-binding molecule for the ligand is, for example, at least 1.1 times, preferably at least 1.5 times, at least 2 times, at least 5 times, or at least 10 times, particularly preferably at least 100 times, the dissociation constant (KD) of the natural in vivo ligand-binding partner for the ligand. A ligand-binding molecule that has only low or almost no binding activity for the ligand after cleavage can be expected to ensure that the ligand is released by cleavage of the ligand-binding molecule and prevent it from rebinding to another ligand molecule.

[0111] The ligand desirably restores the inhibited biological activity after cleavage of the ligand-binding molecule. Desirably, the ligand binding of the cleaved ligand-binding molecule is attenuated such that the function of the ligand-binding molecule to inhibit the ligand biological activity is also attenuated. Those skilled in the art can confirm the biological activity of the ligand by known methods, for example, methods for detecting binding of the ligand to its binding partner, as disclosed herein.

[0112] As used herein, the phrase "attenuated binding activity" when referring to the binding activity of a ligand to its binding partner refers to a reduced or diminished binding activity compared to the binding activity of the ligand in the uncleaved state of the fusion polypeptide, and the degree of reduction or decrease includes, but is not limited to, complete abolition of the activity. Similarly, the phrases "inhibited biological activity" and "neutralizing the biological activity of a ligand" can be used interchangeably herein to refer to a reduction, including, but not limited to, complete elimination, of the binding activity of a ligand to its binding partner when the ligand is bound to the ligand-binding domain of the fusion polypeptide prior to protease cleavage.

[0113] As used herein, the phrase "biological activity restored" refers to a transition from a (first) state, when the ligand is bound to the ligand-binding moiety in an uncleaved state and unable to interact with a binding partner, to a (second) state, when the ligand is uncleaved and unable to interact with a binding partner to exert its biological activity. The term "restored" refers to the restoration of the ligand's ability to interact with a binding partner and exert its biological activity, which was inhibited when the ligand was bound to the ligand-binding domain in an uncleaved state. It includes any degree of interaction or increased interaction with the binding partner sufficient to exert its biological activity upon binding. In some embodiments, the ligand-binding moiety / molecule contains a protease cleavage site located within or near the ligand-binding domain in the ligand-binding moiety. In the presence of a protease, the ligand becomes unbound to the ligand-binding moiety and is free to interact with the binding partner and exert its biological activity. In some embodiments, the ligand's interaction with the binding partner to exert its biological activity occurs while the ligand remains attached to the C-terminus of the Fc region of the ligand-binding moiety by a non-cleavable peptide linker. As used herein, the term "biological activity" includes, but is not limited to, the physiological activity of a ligand (eg, the interaction of the ligand with its natural binding partner, such as a ligand receptor).

[0114] The biological activity of a ligand that binds to its ligand-binding partner can be confirmed by well-known methods such as FACS, ELISA, ALPHA (Amplified Luminescence Proximity Homogeneous Assay) screen or BIACORE using surface plasmon resonance (SPR) phenomenon, or BLI (BioLayer Interferometry) (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). The ALPHA screen is performed using two beads, donor and acceptor, according to ALPHA technology, based on the following principle: A luminescent signal is detected only when the two beads are positioned in close proximity through the interaction between a molecule bound to the donor bead and a molecule bound to the acceptor bead. A photosensitizer in the donor bead, excited by a laser, converts ambient oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and reaches the nearby acceptor bead, thereby triggering a chemiluminescent reaction in the bead, which ultimately emits light. In the absence of an interaction between the molecules bound to the donor bead and the molecules bound to the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and therefore no chemiluminescent reaction occurs.

[0115] For example, a biotin-labeled ligand-binding partner is bound to donor beads, and a glutathione S-transferase (GST)-tagged ligand is bound to acceptor beads. In the absence of an untagged competing ligand-binding partner, the ligand-binding partner interacts with the ligand, generating a signal at 520-620 nm. The untagged ligand-binding partner competes with the tagged ligand-binding partner for interaction with the ligand. The resulting decrease in fluorescence can be quantified to determine relative binding affinity. Biotinylation of a ligand-binding partner, such as an antibody, using sulfo-NHS-biotin or similar is known in the art. A suitable method for tagging a ligand with GST includes, for example, fusing a polynucleotide encoding the ligand with a polynucleotide encoding GST in frame; expressing the resulting GST-fusion ligand from cells carrying a vector enabling expression of the fusion gene; and purifying the GST-fusion ligand using a glutathione column. The resulting signals are preferably analyzed by fitting to a one-site competition model based on nonlinear regression analysis, for example, using the software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego).

[0116] One of the substances whose interaction is to be monitored (the ligand) is immobilized on a thin gold film on a sensor chip. Light is shone on the back of the sensor chip to cause total internal reflection at the interface between the gold film and the glass. As a result, a region of reduced reflection intensity (the SPR signal) is formed in a portion of the reflected light. The other substance whose interaction is to be monitored (the analyte) is then flowed over the surface of the sensor chip and allowed to bind to the ligand. The mass of the immobilized ligand molecule increases, causing a change in the refractive index of the solvent on the sensor chip surface. This change in refractive index shifts the position of the SPR signal (conversely, dissociation of the bound molecule returns the signal to its original position). The Biacore system plots the amount of shift, i.e., the change in mass on the sensor chip surface, on the vertical axis, and displays the change in mass over time as assay data (a sensorgram). Kinetics: The association rate constant (ka) and dissociation rate constant (kd) are determined from the sensorgram curve, and the dissociation constant (KD) is determined from the ratio between these constants. Inhibition assays or equilibrium analysis are also preferably used in the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010, and an example of an equilibrium analysis is described in Methods Enzymol. 2000; 323: 325-40.

[0117] As used herein, the phrase "biological activity is restored" does not limit the degree of binding of a ligand to its binding partner, as long as the biological activity resulting from the binding is observed in any measurement method, such as those described above. It can include an increase of 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more in the interaction between a ligand and its binding partner when comparing the ligand in the cleaved and uncleaved states. Any desired index may be used as an index of binding activity. For example, the binding rate constant (Kon) may be used. When the binding constant (Kon) is used, a binding constant of the ligand-binding partner for the test ligand (i.e., the cleaved, restored ligand) that is greater than that of the control ligand (i.e., the uncleaved ligand) refers to a stronger ligand-binding activity of the test ligand for the ligand-binding partner than that of the control ligand. In some embodiments, the binding constant is at least 2-fold, preferably at least 5-fold, or at least 10-fold, and particularly preferably at least 100-fold, that of the control ligand for the ligand binding partner.

[0118] For example, when detecting the biological activity of a ligand using Octet, a ligand-detecting antibody that recognizes the ligand is biotinylated and contacted with a biosensor. Subsequently, binding to the ligand in a sample can be measured to detect restoration of ligand-binding activity. Specifically, the amount of ligand is measured using a ligand-detecting antibody in a sample containing a ligand-binding molecule and a ligand before or after protease treatment. The amount of ligand detected in the sample before and after protease treatment can be compared to detect ligand release. Alternatively, the amount of ligand is measured using a ligand-detecting antibody in a sample containing a protease, a ligand-binding molecule, and a ligand, and in a sample containing a ligand-binding molecule and a ligand without a protease. The amount of ligand detected in a sample with or without a protease can be compared to determine restoration of the ligand-binding ability of the ligand moiety. When the ligand-binding molecule is fused to a ligand to form a fusion protein, the amount of ligand is measured using a ligand-detecting antibody in a sample containing the fusion protein before or after protease treatment. The amount of ligand detected in a sample before and after protease treatment can be compared to determine whether the ligand-binding ability of the ligand has been restored. Alternatively, the amount of ligand is measured using a ligand-detecting antibody in a sample containing a protease and a fusion protein, and in a sample containing the fusion protein without a protease. The amount of ligand detected in a sample with or without a protease can be compared to determine whether the ligand-binding ability of the ligand has been restored. More specifically, the restoration of the ligand-binding activity of a ligand can be detected by the method described in the Examples of this application.

[0119] In some embodiments, the biological activity of the ligand (i.e., the interaction of the ligand with its natural binding partner, such as a ligand receptor) is attenuated upon binding to the ligand-binding domain, and restoration of this biological activity of the ligand can be detected by a method for measuring the biological activity of the ligand in a sample. Specifically, the biological activity of the ligand can be measured in a sample containing the ligand-binding molecule and the ligand before or after protease treatment, and the results before and after protease treatment can be compared to detect restoration of its binding ability. Alternatively, the biological activity of the ligand can be measured in a sample containing the protease, the ligand-binding molecule, and the ligand, and a sample containing the ligand-binding molecule and the ligand without the protease, and the results can be compared to detect restoration of the binding ability of the ligand. When the ligand-binding molecule is fused to the ligand to form a fusion protein, the biological activity of the ligand can be measured in a sample containing the fusion protein before or after protease treatment, and the results before and after protease treatment can be compared to detect restoration of its binding ability. Alternatively, the biological activity of the ligand can be measured in a sample containing the fusion protein with the protease and a sample containing the fusion protein without the protease, and compared between these samples to detect restoration of its binding ability.

[0120] In some embodiments of the present invention, the uncleaved ligand-binding molecule forms a complex with the ligand through antigen-antibody binding. In more specific embodiments, the complex between the ligand-binding molecule and the ligand is formed through a non-covalent bond, e.g., antigen-antibody binding, between the ligand-binding molecule and the ligand.

[0121] In some embodiments, the uncleaved ligand-binding molecule is fused with a ligand molecule to form a fusion protein. The ligand-binding domain of the ligand-binding moiety and the ligand moiety in the fusion protein further interact with each other through antigen-antibody binding. The ligand-binding molecule and the ligand can be fused via a peptide linker. Even when the ligand-binding molecule and the ligand in the fusion protein are fused via a peptide linker, a non-covalent bond still exists between the ligand-binding domain of the ligand-binding moiety and the ligand moiety. In other words, even in embodiments in which the ligand-binding molecule is fused with the ligand, the non-covalent bond between the ligand-binding domain of the ligand-binding moiety and the ligand moiety is similar to when the ligand-binding molecule is not fused with the ligand. The non-covalent bond is weakened by cleavage of the ligand-binding moiety / molecule. In other words, the ligand binding of the ligand-binding moiety / molecule is weakened.

[0122] In some embodiments, in the fusion proteins of the present invention, the ligand moiety or molecule is connected to the C-terminal region of the ligand-binding moiety or molecule via a peptide linker. As used herein, the term "C-terminal region" refers to a region of a polypeptide extending from an internal amino acid residue in the polypeptide to the C-terminal amino acid residue of the polypeptide. In certain embodiments, for example, when the ligand-binding moiety / molecule is in the form of an antibody or an antibody fragment containing an Fc region, the C-terminal region of the ligand-binding moiety / molecule typically refers to the region of amino acid residues 1 to 250 from the C-terminus of the ligand-binding moiety / molecule. In preferred embodiments, the ligand moiety / molecule is connected to the C-terminal amino acid residue of the ligand-binding moiety / molecule via a peptide linker. The peptide linker may be attached to the ligand moiety / molecule and the C-terminal region of the ligand-binding moiety / molecule by any covalent bond, such as a peptide bond. The length of the peptide linker is not particularly limited, as long as it connects the ligand moiety / molecule to the ligand-binding domain in the ligand-binding moiety / molecule. The above-mentioned peptide linker may or may not contain a protease cleavage site. In a preferred embodiment, the peptide linker described above does not contain a protease cleavage site.

[0123] In some aspects, the ligand moiety / molecule of the invention is IL-12. In one embodiment, the ligand moiety / molecule of the invention is IL-12, which is connected to the C-terminal amino acid residue of the ligand binding moiety / molecule via a peptide linker attached to the p35 subunit of IL-12 or the p40 subunit of IL-12. In one embodiment, the ligand moiety / molecule of the present invention is IL-12, which is connected to the C-terminal amino acid residue of the ligand-binding moiety / molecule via a peptide linker added to the N-terminus of the p35 subunit of IL-12 or the p40 subunit of IL-12. In some aspects, the ligand moiety / molecule of the present invention is IL-22. In one embodiment, the ligand moiety / molecule of the present invention is IL-22, which is connected to the C-terminal amino acid residue of the ligand-binding moiety / molecule via a peptide linker added to IL-22. In one embodiment, the ligand moiety / molecule of the present invention is IL-22, which is connected to the C-terminal amino acid residue of the ligand-binding moiety / molecule via a peptide linker added to the N-terminus of IL-22. In one embodiment, the ligand-binding domain of the present invention is connected to a hinge region included in the ligand-binding moiety via a peptide linker. In a preferred embodiment, the ...

Claims

**Claim 1**: A polypeptide comprising an antigen-binding domain containing a protease cleavage site, wherein: the antigen-binding domain comprises an antibody variable region containing a heavy-chain variable domain (VH) and a light-chain variable domain (VL) that associate with each other; VH associates with a heavy-chain constant region (CH1), and / or VL associates with a light-chain constant region (CL); the protease cleavage site is located at the boundary between the VH region and the CH1 region, or at the boundary between the VL region and the CL region; the protease cleavage site contains at least one amino acid modification at the interface between VH and VL that reduces the association between VH and VL upon cleavage at the protease cleavage site; the polypeptide. **Claim 2**: The polypeptide according to claim 1, wherein the at least one amino acid modification is a substitution or combination of substitutions of one or more amino acids present at the interface between VH and VL, and the one or more amino acids for the substitution are in a framework region (FR). **Claim 3**: The polypeptide according to claim 2, wherein the substitution or combination of substitutions of amino acids comprises a substitution of an amino acid to have the same charge as the corresponding interacting amino acid at the interface between VH and VL, or a substitution of an amino acid to have a neutral charge. **Claim 4**: The polypeptide according to claim 1, wherein at least one pair of amino acid modifications is made at the interface between VH and VL, and the pair of amino acid modifications comprises substitutions of both amino acids to have the same charge or a neutral charge. **Claim 5**: The polypeptide according to claim 3, wherein the substitution or combination of substitutions is selected from positions 37, 39, 44, 45, 47, 91, and 103 on VH, and / or positions 38, 43, 44, 46, 49, 87, and 98 on VL (all positions are according to Kabat numbering). **Claim 6**: The polypeptide according to claim 4, wherein the pair of amino acid modifications, or combination of substitutions, is selected from positions V37, Q39, G44, L45, W47, H91, Y91, and W103 on VH, and / or positions R38, A43, P44, L46, Y49, Y87, and F98 on VL (all positions are according to Kabat numbering).

7. The variable region is capable of binding to a ligand moiety, and upon protease cleavage, the ligand moiety dissociates from the variable region, the polypeptide according to claim 1.

8. The polypeptide according to claim 7, wherein the ligand moiety is fused to the C-terminal region or N-terminal region of the polypeptide.

9. The polypeptide according to claim 8, wherein the ligand moiety is an interleukin, chemokine, interferon, hematopoietic factor, member of the TNF superfamily, cell growth factor, member of the TGF-β family, myokine, adipokine, or neurotrophic factor.

10. The polypeptide according to claim 1, wherein the protease cleavage site comprises a protease cleavage sequence, or a combination of a protease cleavage sequence and a flexible linker.

11. The polypeptide according to claim 10, wherein the protease is selected from matriptase, urokinase (uPA), cathepsin, collagenase, furin, plasminogen, thrombin, serine protease, and metalloprotease.

12. The polypeptide according to claim 1, comprising an antibody or an antibody fragment thereof.

13. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 12.

14. The pharmaceutical composition according to claim 13, or the polypeptide according to any one of claims 1 to 12, for use in the treatment of a disease or disorder.

15. A method for producing the polypeptide according to any one of claims 1 to 12, comprising culturing a host cell containing a nucleic acid encoding the polypeptide, and recovering the polypeptide from the host cell.

Images