Cytokine prodrugs containing cleavable linkers

Abstract

We provide protease-cleavable cytokine prodrugs. [Solution] A protease-activated procytokine is provided, comprising a cytokine polypeptide sequence; an inhibitory polypeptide sequence capable of blocking the activity of the cytokine polypeptide sequence; a linker between the cytokine polypeptide sequence and the inhibitory polypeptide sequence, comprising a protease-cleavable polypeptide sequence; and a targeting sequence designed to bind to extracellular matrix components, integrins, or syndecans, or designed to bind to extracellular matrix components, IgB (CD79b), integrins, cadherins, heparan sulfate proteoglycans, syndecans, or fibronectin with pH-sensitive functionality; or a targeting sequence comprising a variant having one or two mismatches compared to a specific sequence.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority under U.S. Provisional Patent Application 62 / 961,537, filed on 15 January 2020, which is incorporated herein by reference in whole for all purposes.

[0002] Introduction and Overview This application relates to cytokine therapeutics, particularly cytokine prodrugs containing cleavable linkers. [Background technology]

[0003] Cytokines such as IL-2 are potent immunoproliferators that play a significant role in sustaining effective immune cell responses. IL-2 has been reported to induce complete and persistent regression in cancer patients, although immune-related adverse effects reduce its therapeutic potential. In any case, in some cases, systemic IL-2 administration can activate immune cells throughout the body. Systemic activation can lead to systemic toxicity and indiscriminate activation of immune cells, including those responsive to diverse epitopes, antigens, and stimuli. The therapeutic efficacy of IL-2 treatments may be affected by these severe toxicities.

[0004] IL-2 therapies can also suffer from a short serum half-life, which can be as short as a few minutes. Therefore, high doses of IL-2, which may be necessary to achieve optimal immunomodulatory effects, can also contribute to severe toxicity. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] As a result, there is a need for therapeutic agents that overcome systemic or untargeted functional impairments, severe toxicity, and poor pharmacokinetics. The present invention aims to satisfy one or more of these requirements, provide further benefits, and offer the public at least one useful option. [Means for solving the problem]

[0006] In one embodiment, a protease-activated procytokine (also referred to as a cytokine prodrug) is provided that can be administered to a subject in an inactive form. The inactive form may include a cytokine polypeptide sequence, a protease-cleavable polypeptide sequence, and an inhibitory polypeptide sequence that can block the activity of the cytokine polypeptide sequence. Such a prodrug may become active when the protease-cleavable polypeptide sequence is cleaved by a protease. Cleavage of the protease-cleavable polypeptide allows the inhibitory polypeptide sequence to dissociate from the cytokine polypeptide sequence.

[0007] Many tumors and tumor microenvironments exhibit abnormal protease expression. This invention provides cytokine prodrugs that are activated via proteolytic cleavage so as to become active upon contact with proteases in the tumor or tumor microenvironment. In some cases, this may result in an increase in active cytokines in and around the tumor or tumor microenvironment compared to other parts of the body or healthy tissue. One example of the potential benefits is the formation of a cytokine gradient. Such gradients may be formed when a cytokine prodrug is administered, selectively or preferentially activated in the tumor or tumor microenvironment, and then diffused from these areas to other parts of the body. These gradients may increase the transport of immune cells to the tumor and tumor microenvironment. Immune cells transported to the tumor can infiltrate the tumor. Infiltrating immune cells can carry out an immune response against cancer. Infiltrating immune cells can also secrete their own chemokines and cytokines. Cytokines may have either or both autocrine and paracrine effects in the tumor and tumor microenvironment. In some cases, immune cells include T cells or NK cells, such as T effector cells or cytotoxic T cells.

[0008] Also disclosed herein are methods of treatment and methods of administering the cytokine prodrugs described herein. Such administration may be systemic or local. In certain embodiments, the cytokine prodrugs described herein are administered systemically or locally for the treatment of cancer.

[0009] A further example of local administration is the administration of a cytokine prodrug, such as an IL-2 cytokine prodrug, to boost T regulatory cells. In some cases, the local administration of the IL-2 cytokine prodrug is in the area of inflammation. Such methods can also be used for the treatment of chronic autoimmune and / or inflammatory diseases.

[0010] The following embodiments are included.

[0011] Embodiment 1 is a cytokine polypeptide sequence; an inhibitory polypeptide sequence capable of blocking the activity of the cytokine polypeptide sequence; a linker between the cytokine polypeptide sequence and the inhibitory polypeptide sequence, comprising a protease-cleavable polypeptide sequence; and a targeting sequence designed to bind to an extracellular matrix component, integrin or syndecan or designed to bind to an extracellular matrix component, IgB (CD79b), integrin, cadherin, heparan sulfate proteoglycan, syndecan or fibronectin with a pH-sensitive function; or a targeting sequence comprising any one of SEQ ID NOs: 180-662 or a variant having one or two mismatches compared to any one of SEQ ID NOs: 180-662 a protease-activated pro-cytokine comprising.

[0012] Embodiment 2 is a protease-activated pro-cytokine of the immediately preceding () embodiment, further comprising a pharmacokinetic modulator.

[0013] Embodiment 3 is a protease-activated pro-cytokine of the immediately preceding embodiment, wherein the pharmacokinetic modulator comprises an immunoglobulin constant domain.

[0014] Embodiment 4 is a protease-activated pro-cytokine of Embodiment 2, wherein the pharmacokinetic modulator comprises an immunoglobulin Fc region.

[0015] Embodiment 5 is a protease-activated pro-cytokine of the immediately preceding embodiment, wherein the immunoglobulin is a human immunoglobulin.

[0016] Embodiment 6 is a protease-activated pro-cytokine of any one of Embodiments 4-5, wherein the immunoglobulin is IgG.

[0017] Embodiment 7 is a protease-activated pro-cytokine of the immediately preceding embodiment, wherein the IgG is IgG1, IgG2, IgG3 or IgG4.

[0018] Embodiment 8 is a protease-activated pro-cytokine of Embodiment 2, wherein the pharmacokinetic modulator comprises albumin.

[0019] Embodiment 9 is a protease-activated pro-cytokine of the immediately preceding embodiment, wherein the albumin is serum albumin.

[0020] Embodiment 10 is a protease-activated pro-cytokine of any one of Embodiments 8-9, wherein the albumin is human albumin.

[0021] Embodiment 11 is a protease-activated pro-cytokine of Embodiment 2, wherein the pharmacokinetic modulator comprises PEG.

[0022] Embodiment 12 is a protease-activated pro-cytokine of Embodiment 2, wherein the pharmacokinetic modulator comprises XTEN.

[0023] Embodiment 13 is the protease-activated procytokine of Embodiment 2, wherein the pharmacokinetic modulator includes CTP.

[0024] Embodiment 14 is a protease-activated procytokine of any of Embodiments 2 to 13, wherein the protease-cleavable polypeptide sequence is located between the cytokine polypeptide sequence and the pharmacokinetic modulator.

[0025] Embodiment 15 is a protease-activated procytokine of any of Embodiments 2 to 13, wherein the pharmacokinetic modulator is located between the cytokine polypeptide sequence and the protease-cleavable polypeptide sequence.

[0026] Embodiment 16 is a protease-activated procytokine of any of the previous embodiments, comprising a plurality of protease-cleavable polypeptide sequences.

[0027] Embodiment 17 is a protease-activated procytokine of the previous embodiment, in which the cytokine polypeptide sequence is sandwiched between protease-cleavable polypeptide sequences.

[0028] Embodiment 18 is a protease-activated procytokine of the immediately preceding embodiment having the structure PM-CL-CY-CL-IN (N-terminus → C-terminus or C-terminus → N-terminus) (where PM is a pharmacokinetic modulator, each CL is independently a protease-cleavable polypeptide sequence, CY is a cytokine polypeptide sequence, and IN is an inhibitory polypeptide sequence).

[0029] Embodiment 19 is a protease-activated procytokine of any of the previous embodiments, comprising a targeting sequence, wherein the targeting sequence is between a cytokine polypeptide sequence and a protease-cleavable polypeptide sequence or a protease-cleavable polypeptide sequence.

[0030] Embodiment 20 is a protease-activated procytokine of any of the previous embodiments, wherein the cytokine polypeptide sequence includes modifications to inhibit disulfide bond formation and optionally includes the rest of the wild-type sequence.

[0031] Embodiment 21 is a protease-activated procytokine of any of the previous embodiments, wherein the cytokine polypeptide sequence has at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with the wild-type cytokine polypeptide sequence or the cytokine polypeptide sequences in Table 1.

[0032] Embodiment 22 is a protease-activated procytokine of the immediately preceding embodiment, wherein the cytokine polypeptide sequence is a wild-type cytokine polypeptide sequence.

[0033] Embodiment 23 is a protease-activated procytokine of any of the previous embodiments, wherein the cytokine polypeptide sequence is a monomeric cytokine or a dimeric cytokine polypeptide sequence containing monomers linked covalently (preferably via a polypeptide linker) or non-covalently.

[0034] Embodiment 24 is a protease-activating procytokine of any of the previous embodiments, wherein the inhibitory polypeptide sequence includes a cytokine-binding domain.

[0035] Embodiment 25 is a protease-activated procytokine of the immediately preceding embodiment, wherein the cytokine-binding domain is the cytokine-binding domain of a cytokine receptor or the cytokine-binding domain of fibronectin.

[0036] Embodiment 26 is the protease-activated procytokine of Embodiment 24, wherein the cytokine-binding domain is an immunoglobulin cytokine-binding domain.

[0037] Embodiment 27 is a protease-activated procytokine of the previous embodiment, comprising a light chain variable domain and a heavy chain variable domain to which the immunoglobulin cytokine-binding domain binds to a cytokine.

[0038] Embodiment 28 is a protease-activated procytokine of any of Embodiments 26-27, wherein the immunoglobulin cytokine binding domain is scFv, Fab, or VHH.

[0039] Embodiment 29 is a protease-cleavable polypeptide sequence containing metalloprotease, serine protease, cysteine ​​protease, aspartate protease, threonine protease, glutamate protease, gelatinase, asparagine peptide lyase, cathepsin, kallikrein, plasmin, collagenase, hKl, hK10, hK15, stromelysin, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidine, bromelain, calpain, caspase, and Mir 1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, regmine, plasmmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, ADAM10, ADAM17, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), inter -A protease-activated procytokine of any of the preceding embodiments, recognized by leukin-1b-converting enzyme, thrombin, FAP (FAP-a), dipeptidyl peptidase or dipeptidyl peptidase IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, proteinase 3, mast cell chymase, mast cell tryptase or dipeptidyl peptidase.

[0040] Embodiment 30 is a protease-activated procytokine of any of the previous embodiments, wherein the protease-cleavable polypeptide sequence includes any sequence of SEQ ID NOs. 700 to 741 or a variant having one or two mismatches compared to any sequence of SEQ ID NOs. 700 to 741.

[0041] Embodiment 31 is a protease-activated procytokine of any of the previous embodiments, wherein the protease-cleavable polypeptide sequence is recognized by a matrix metalloproteinase.

[0042] Embodiment 32 is a protease-activated procytokine from any of the previous embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-1.

[0043] Embodiment 33 is a protease-activated procytokine from any of the previous embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-2.

[0044] Embodiment 34 is a protease-activated procytokine from any of the previous embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-3.

[0045] Embodiment 35 is a protease-activated procytokine from any of the previous embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-7.

[0046] Embodiment 36 is a protease-activated procytokine from any of the previous embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-8.

[0047] Embodiment 37 is a protease-activated procytokine from any of the previous embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-9.

[0048] Embodiment 38 is a protease-activated procytokine of any of the previous embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-12.

[0049] Embodiment 39 is a protease-activated procytokine from any of the previous embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-13.

[0050] Embodiment 40 is a protease-activated procytokine of any of the previous embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-14.

[0051] Embodiment 41 is a protease-activated procytokine of any of the previous embodiments, which is recognized by an MMP containing one or more protease-cleavable polypeptide sequences.

[0052] Embodiment 42 is a protease-activated procytokine of any of the previous embodiments, wherein the protease-cleavable polypeptide sequence is recognized by 2, 3, 4, 5, 6, or 7 copies of MMP-2, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13, and MMP-14.

[0053] Embodiment 43 is a protease-activated procytokine of any of the previous embodiments, wherein the protease-cleavable polypeptide sequence includes a variant sequence having one or two mismatches compared to any of the sequences of SEQ ID NOs. 80 to 94 or any of the sequences of SEQ ID NOs. 80 to 90.

[0054] Embodiment 44 is a protease-activated procytokine of the preceding embodiment, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 80 or a variant sequence having one or two mismatches compared thereto.

[0055] Embodiment 45 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 81 or a variant sequence having one or two mismatches compared thereto.

[0056] Embodiment 46 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 82 or a variant sequence having one or two mismatches compared thereto.

[0057] Embodiment 47 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 83 or a variant sequence having one or two mismatches compared thereto.

[0058] Embodiment 48 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 84 or a variant sequence having one or two mismatches compared thereto.

[0059] Embodiment 49 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 85 or a variant sequence having one or two mismatches compared thereto.

[0060] Embodiment 50 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 86 or a variant sequence having one or two mismatches compared thereto.

[0061] Embodiment 51 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 87 or a variant sequence having one or two mismatches compared thereto.

[0062] Embodiment 52 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 88 or a variant sequence having one or two mismatches compared thereto.

[0063] Embodiment 53 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 89 or a variant sequence having one or two mismatches compared thereto.

[0064] Embodiment 54 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 90 or a variant sequence having one or two mismatches compared thereto.

[0065] Embodiment 55 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence includes the sequence of SEQ ID NOs. 80 to 89 or 90.

[0066] Embodiment 56 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence includes the sequence of SEQ ID NO: 91.

[0067] Embodiment 57 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence includes the sequence of SEQ ID NO: 92.

[0068] Embodiment 58 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence includes the sequence of SEQ ID NO: 93.

[0069] Embodiment 59 is a protease-activated procytokine of any of Embodiments 1 to 43, wherein the protease-cleavable polypeptide sequence includes the sequence of SEQ ID NO: 94.

[0070] Embodiment 60 is a protease-activated procytokine of any of the previous embodiments, wherein the targeting sequence includes any sequence of SEQ ID NOs. 180 to 662 or a variant having one or two mismatches compared to any sequence of SEQ ID NOs. 180 to 662.

[0071] Embodiment 61 is a protease-activated procytokine of the preceding embodiment, wherein the targeting sequence includes any sequence from sequence numbers 180 to 662.

[0072] Embodiment 62 is a protease-activated procytokine from any of the previous embodiments, wherein the targeting sequence binds to denatured collagen.

[0073] Embodiment 63 is a protease-activated procytokine from any of Embodiments 1 to 61, wherein the targeting sequence binds to collagen.

[0074] Embodiment 64 is a protease-activated procytokine of any of Embodiments 62-63, wherein the collagen is collagen I.

[0075] Embodiment 65 is a protease-activated procytokine of any of Embodiments 62-63, wherein the collagen is collagen II.

[0076] Embodiment 66 is a protease-activated procytokine of any of Embodiments 62-63, wherein the collagen is collagen III.

[0077] Embodiment 67 is a protease-activated procytokine of any of Embodiments 62-63, wherein the collagen is collagen IV.

[0078] Embodiment 68 is a protease-activating procytokine of any of Embodiments 1 to 61, wherein the targeting sequence binds to an integrin.

[0079] Embodiment 69 is a protease-activating procytokine of the previous embodiment, wherein the integrin is one or more of α1β1 integrin, α2β1 integrin, α3β1 integrin, α4β1 integrin, α5β1 integrin, α6β1 integrin, α7β1 integrin, α9β1 integrin, α4β7 integrin, αvβ3 integrin, αvβ5 integrin, αIIbβ3 integrin, αIIIbβ3 integrin, αMβ2 integrin, or αIIbβ3 integrin.

[0080] Embodiment 70 is a protease-activated procytokine of any of Embodiments 1 to 61, wherein the targeting sequence binds to von Willebrand factor.

[0081] Embodiment 71 is a protease-activated procytokine from any of Embodiments 1 to 61, wherein the targeting sequence binds to IgB.

[0082] Embodiment 72 is a protease-activated procytokine from any of Embodiments 1 to 61, wherein the targeting sequence binds to heparin.

[0083] Embodiment 73 is a protease-activating procytokine of the immediately preceding embodiment, wherein the targeting sequence is bound to heparin and syndecane, heparan sulfate proteoglycan, or integrin, and optionally the integrin is one or more of α1β1 integrin, α2β1 integrin, α3β1 integrin, α4β1 integrin, α5β1 integrin, α6β1 integrin, α7β1 integrin, α9β1 integrin, α4β7 integrin, αvβ3 integrin, αvβ5 integrin, αIIbβ3 integrin, αIIIbβ3 integrin, αMβ2 integrin, or αIIbβ3 integrin.

[0084] Embodiment 74 is a protease-activated procytokine of any of Embodiments 72 to 73, wherein syndecane is one or more of syndecane-1, syndecane-4, and syndecane-2(w).

[0085] Embodiment 75 is a protease-activated procytokine from any of Embodiments 1 to 61, wherein the targeting sequence is bound to a heparan sulfate proteoglycan.

[0086] Embodiment 76 is a protease-activating procytokine from any of Embodiments 1 to 61, wherein the targeting sequence binds to a sulfated glycoprotein.

[0087] Embodiment 77 is a protease-activated procytokine from any of Embodiments 1 to 61, wherein the targeting sequence binds to hyaluronic acid.

[0088] Embodiment 78 is a protease-activated procytokine from any of Embodiments 1 to 61, wherein the targeting sequence binds to fibronectin.

[0089] Embodiment 79 is a protease-activating procytokine of any of Embodiments 1 to 61, wherein the targeting sequence binds to a cadherin.

[0090] Embodiment 80 is a protease-activated procytokine of any of the previous embodiments, wherein the targeting sequence is designed to bind to its target in a pH-sensitive manner.

[0091] Embodiment 81 is a protease-activated procytokine of the preceding embodiment, wherein the targeting sequence has a higher affinity for its target at a pH lower than normal physiological pH, and optionally the pH lower than normal physiological pH is less than 7 or less than 6.

[0092] Embodiment 82 is a protease-activated procytokine from the previous embodiment, wherein the targeting sequence has a higher affinity for its target at pH levels in the range of 5-7, for example, 5-5.5, 5.5-6, 6-6.5, or 6.5-7, than the normal physiological pH.

[0093] Embodiment 83 is a protease-activating procytokine of any of the previous embodiments, comprising one or more histidines in the targeting sequence, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 histidines.

[0094] Embodiment 84 is a protease-activated procytokine of any of the previous embodiments, wherein the targeting sequence includes a variant having one or two mismatches compared to any of the sequences of SEQ ID NOs. 641 to 662 or any of the sequences of SEQ ID NOs. 641 to 662.

[0095] Embodiment 85 is a protease-activating procytokine of the preceding embodiment, wherein the targeting sequence includes any sequence of sequence numbers 641 to 662.

[0096] Embodiment 86 is a protease-activated procytokine of any of Embodiments 80-86, in which the targeting sequence is designed to bind to an extracellular matrix component, IgB(CD79b), integrin, cadherin, heparan sulfate proteoglycan, syndecan, or fibronectin in a pH-sensitive manner.

[0097] Embodiment 87 is a protease-activated procytokine of the immediately preceding embodiment, wherein the extracellular matrix component is hyaluronic acid, heparin, heparan sulfate, or sulfated glycoprotein.

[0098] Embodiment 88 is a protease-activated procytokine of Embodiment 86, in which the targeting sequence is designed to bind to fibronectin in a pH-sensitive manner.

[0099] Embodiment 89 is a protease-activated procytokine of any of the previous embodiments, wherein the cytokine polypeptide sequence is an interleukin polypeptide sequence.

[0100] Embodiment 90 is a protease-activated procytokine from any of the previous embodiments, wherein the cytokine polypeptide sequence can bind to a receptor containing CD132.

[0101] Embodiment 91 is a protease-activated procytokine from any of the previous embodiments, wherein the cytokine polypeptide sequence can bind to a receptor containing CD122.

[0102] Embodiment 92 is a protease-activated procytokine from any of the previous embodiments, wherein the cytokine polypeptide sequence can bind to a CD25-containing receptor.

[0103] Embodiment 93 is a protease-activated procytokine of any of the previous embodiments, wherein the cytokine polypeptide sequence is an IL-2 polypeptide sequence.

[0104] Embodiment 94 is a protease-activated procytokine of an embodiment in which the IL-2 polypeptide sequence has at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with any of the sequences of SEQ ID NOs: 1 to 4.

[0105] Embodiment 95 is a protease-activated procytokine of the immediately preceding embodiment, wherein the IL-2 polypeptide sequence contains any of the sequences of SEQ ID NOs: 1 to 4.

[0106] Embodiment 96 is an IL-2 polypeptide sequence, which is either a human IL-2 polypeptide sequence or a protease-activated procytokine from any of Embodiments 93 to 95.

[0107] Embodiment 97 is a protease-activated procytokine of the previous embodiment, wherein the IL-2 polypeptide sequence includes the sequence of SEQ ID NO: 1.

[0108] Embodiment 98 is a protease-activated procytokine of any of Embodiments 93 to 95, wherein the IL-2 polypeptide sequence includes the sequence of SEQ ID NO: 2.

[0109] Embodiment 99 is a protease-activated procytokine of any of Embodiments 93 to 98, wherein the inhibitory polypeptide sequence includes the IL-2 binding domain of the IL-2 receptor (IL-2R).

[0110] Embodiment 100 is a protease-activating procytokine of the immediately preceding embodiment, wherein the inhibitory polypeptide sequence comprises an amino acid sequence having at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with any of the sequences of SEQ ID NOs. 10 to 19.

[0111] Embodiment 101 is a protease-activated procytokine of the immediately preceding embodiment, wherein IL-2R is human IL-2R.

[0112] Embodiment 102 is a protease-activating procytokine of any of Embodiments 93 to 98, wherein the inhibitory polypeptide sequence includes an IL-2-binding immunoglobulin domain.

[0113] Embodiment 103 is a protease-activated procytokine of any of Embodiments 93 to 98, wherein the IL-2-binding immunoglobulin domain is a human IL-2-binding immunoglobulin domain.

[0114] Embodiment 104 is a protease-activated procytokine of the immediately preceding embodiment, comprising a VL region containing hypervariable regions (HVRs) HVR-1, HVR-2, and HVR-3 having sequences of SEQ ID NOs. 33, 34, and 35, respectively, and a VH region containing HVR-1, HVR-2, and HVR-3 having sequences of SEQ ID NOs. 36, 37, and 38, respectively.

[0115] Embodiment 105 is a protease-activated procytokine of any of Embodiments 102 to 104, wherein the IL-2-binding immunoglobulin domain comprises a VL region containing an amino acid sequence having at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent or 99 percent identity with the sequence of SEQ ID NO: 32, and a VH region containing an amino acid sequence having at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent or 99 percent identity with the sequence of SEQ ID NO: 33.

[0116] Embodiment 106 is a protease-activated procytokine of the previous embodiment, wherein the IL-2-binding immunoglobulin domain comprises a VL region containing the sequence of SEQ ID NO: 32 and a VH region containing the sequence of SEQ ID NO: 33.

[0117] Embodiment 107 is a protease-activated procytokine of any of Embodiments 102 to 104, wherein the IL-2-binding immunoglobulin domain is scFv.

[0118] Embodiment 108 is a protease-activated procytokine of the preceding embodiment, wherein the IL-2-binding immunoglobulin domain comprises an amino acid sequence having at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with the sequence of SEQ ID NO: 30 or 31.

[0119] Embodiment 109 is a protease-activated procytokine of the preceding embodiment, wherein the IL-2-binding immunoglobulin domain comprises the sequence of SEQ ID NO: 30 or 31.

[0120] Embodiment 110 is the protease-activated procytokine of Embodiment 1, comprising any sequence of sequence numbers 803 to 852.

[0121] Embodiment 111 is a pharmaceutical composition comprising a protease-activated procytokine of any of the previous embodiments.

[0122] Embodiment 112 is a protease-activated procytokine or pharmaceutical composition of any of the previous embodiments for use in therapeutic purposes.

[0123] Embodiment 113 is a protease-activated procytokine or pharmaceutical composition of any of the previous embodiments for use in the treatment of cancer.

[0124] Embodiment 114 is a method for treating cancer, comprising administering any of the protease-activated procytokines or pharmaceutical compositions of the preceding embodiments to a subject in need thereof.

[0125] Embodiment 115 is the use of any of the protease-activated procytokines or pharmaceutical compositions of Embodiments 1 to 110 in the manufacture of a pharmaceutical for the treatment of cancer.

[0126] Embodiment 116 is a protease-activated procytokine for use or use in any of Embodiments 113 to 115, wherein the cancer is a solid tumor.

[0127] Embodiment 117 is a protease-activated procytokine for use or application of the method of the preceding embodiment for solid tumors that are metastatic and / or unresectable.

[0128] Embodiment 118 is a protease-activated procytokine for use or use in any of Embodiments 113 to 117, wherein the cancer is a PD-L1-expressing cancer.

[0129] Embodiment 119 is a protease-activated procytokine used in any way, use or for use of any of Embodiments 113 to 118, wherein the cancer is melanoma, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, gastric or gastrointestinal cancer, lymphoma, colon or colorectal cancer, endometrial cancer, thyroid cancer, or bladder cancer.

[0130] Embodiment 120 is a protease-activated procytokine for use or use in any of Embodiments 113-119, wherein the cancer is a high-frequency microsatellite-instability cancer.

[0131] Embodiment 121 is a protease-activated procytokine for use or application in any of Embodiments 113-120, wherein the cancer is a mismatch repair deficiency.

[0132] Embodiment 122 is a nucleic acid encoding a protease-activated procytokine of any of Embodiments 1 to 110.

[0133] Embodiment 123 is an expression vector containing the nucleic acid of Embodiment 121.

[0134] Embodiment 124 is a host cell containing the nucleic acid of Embodiment 121 or the vector of Embodiment 122.

[0135] Embodiment 125 is a method for producing protease-activated procytokines, comprising culturing the host cells of Embodiment 124 under conditions that produce protease-activated procytokines.

[0136] Embodiment 126 is the method of the preceding embodiment, further comprising the isolation of a protease-activated procytokine.

[0137] Embodiment 127 is a method for boosting T regulatory cells and / or reducing inflammatory or autoimmune activity, comprising administering a protease-activated procytokine from any of Embodiments 1 to 110 to a target area of ​​interest, for example, a target area of ​​inflammation.

[0138] Embodiment 128 is a method for treating an inflammatory or autoimmune disease or disorder in a subject, comprising administering a protease-activated procytokine from any of Embodiments 1 to 110 to a target area of ​​the subject, for example, an area of ​​inflammation or autoimmune activity of the subject. [Brief explanation of the drawing]

[0139] [Figure 1A] Figure 1A shows a description of the SDS-PAGE gel characterizing the exemplary cytokine prodrug structure and the purified cytokine prodrug (construct B). Abbreviations: PM, pharmacokinetic modulator; HMW, high molecular weight.

[0140] [Figure 1B] Figure 1B shows a description of an exemplary cytokine prodrug structure containing human IL-2 and IL-2Rα sequences and an MMP-cleavable linker, as well as SDS-PAGE gel and Western blots characterizing the purified cytokine prodrug (construct E). Abbreviations: Hu, human; MMP, matrix metalloproteinase; other abbreviations are as above.

[0141] [Figure 1C] Figure 1C shows a description of an exemplary cytokine prodrug structure containing mouse IL-2 and IL-2Rα sequences, an MMP-cleavable linker, and a further linker ("RET linker") containing a targeting sequence, as well as an SDS-PAGE gel characterizing the purified cytokine prodrug shown.

[0142] [Figure 1D] Figure 1D shows a description of an SDS-PAGE gel characterizing an exemplary cytokine prodrug structure and a purified cytokine prodrug, including a human IL-2 sequence and IL-2Rα sequence, an MMP-cleavable linker, and a further linker ("RET linker") containing a targeting sequence.

[0143] [Figure 2A]Figure 2A illustrates the protease-mediated cleavage of cytokine prodrugs and shows Western blot evidence of cleavage of construct A by MMP-9 at 1 hour, 2 hours, 4 hours, and overnight. Each Western blot includes a +MMP digestion lane and a -MMP simulated-digestion lane. The cleavage product was detectable at 1 hour, and the full-length cytokine prodrug was substantially undetectable at overnight +MMP.

[0144] [Figure 2B] Figure 2B illustrates the cleavage reaction of a cytokine prodrug containing a pharmacokinetic modulator by a protease, and shows Western blot evidence of cleavage of construct B by MMP-9 at 1 hour, 4 hours, and 20 hours. Each Western blot includes a +MMP digestion lane and a -MMP simulated-digestion lane. The cleavage product was detectable at 1 hour, and the full-length cytokine prodrug was only a faint band at 20 hours +MMP.

[0145] [Figure 2C] Figures 2C–E illustrate the cleavage reaction of cytokine prodrugs containing pharmacokinetic modulators by proteases, showing Western blot evidence of cleavage of construct E by MMP-9 at 1 hour, 4 hours, and 22 hours (2C); and cleavage of the construct at 18 hours (2D and 2E). Constructs BBB, CCC, and FFF in Figure 2E, which showed no substantial cleavage, had scrambled MMP sites. Each Western blot includes a +MMP9 digestion lane and a -MMP9 simulated-digestion lane. Cleavage products were detectable at 1 hour, and the full-length cytokine prodrug was essentially band-less at 22 hours +MMP. [Figure 2D]Figures 2C–E illustrate the cleavage reaction of cytokine prodrugs containing pharmacokinetic modulators by proteases, showing Western blot evidence of cleavage of construct E by MMP-9 at 1 hour, 4 hours, and 22 hours (2C); and cleavage of the construct at 18 hours (2D and 2E). Constructs BBB, CCC, and FFF in Figure 2E, which showed no substantial cleavage, had scrambled MMP sites. Each Western blot includes a +MMP9 digestion lane and a -MMP9 simulated-digestion lane. Cleavage products were detectable at 1 hour, and the full-length cytokine prodrug was essentially band-less at 22 hours +MMP. [Figure 2E] Figures 2C–E illustrate the cleavage reaction of cytokine prodrugs containing pharmacokinetic modulators by proteases, showing Western blot evidence of cleavage of construct E by MMP-9 at 1 hour, 4 hours, and 22 hours (2C); and cleavage of the construct at 18 hours (2D and 2E). Constructs BBB, CCC, and FFF in Figure 2E, which showed no substantial cleavage, had scrambled MMP sites. Each Western blot includes a +MMP9 digestion lane and a -MMP9 simulated-digestion lane. Cleavage products were detectable at 1 hour, and the full-length cytokine prodrug was essentially band-less at 22 hours +MMP.

[0146] [Figure 3A] Figure 3A shows the results of a CTLL-2 proliferation assay using construct A or its cleavage products. Construct A was cleaved with MMP-9, and the resulting products were incubated with CTLL-2 cells. The data show that MMP-9-treated construct A dose-dependently stimulated CTLL-2 cell proliferation and exhibited 10-fold greater activity than untreated construct A (EC50 comparison). EC50 values ​​are shown in nM.

[0147] [Figure 3B]Figure 3B shows the results of a CTLL-2 proliferation assay using construct B or its cleavage product. Construct B was cleaved with MMP-9, and the resulting product was incubated with CTLL-2 cells. For comparison, mRNA was also incubated with CTLL-2 cells. The data show that MMP-9 treated construct B dose-dependently stimulates CTLL-2 cell proliferation. Stimulation was minimal with the uncleaved construct B. EC50 values ​​are shown in nM.

[0148] [Figure 3C] Figures 3C–3J show the results of the HEK-Blue™ IL2 assay. Cells were treated with various concentrations of uncleaved or mMMP9-cleaved construct E for 22 hours (Figure 3C); human IL2 (Figure 3D); uncleaved or mMMP9-cleaved construct B for 19 hours; and uncleaved or mMMP9-cleaved constructs J, K, F, L, or I for 22 hours each (Figures 3E–J); and EC50 was determined based on OD630 as the readout for mML-2 stimulation. [Figure 3D] Figures 3C–3J show the results of the HEK-Blue™ IL2 assay. Cells were treated with various concentrations of uncleaved or mMMP9-cleaved construct E for 22 hours (Figure 3C); human IL2 (Figure 3D); uncleaved or mMMP9-cleaved construct B for 19 hours; and uncleaved or mMMP9-cleaved constructs J, K, F, L, or I for 22 hours each (Figures 3E–J); and EC50 was determined based on OD630 as the readout for mML-2 stimulation. [Figure 3E] Figures 3C–3J show the results of the HEK-Blue™ IL2 assay. Cells were treated with various concentrations of uncleaved or mMMP9-cleaved construct E for 22 hours (Figure 3C); human IL2 (Figure 3D); uncleaved or mMMP9-cleaved construct B for 19 hours; and uncleaved or mMMP9-cleaved constructs J, K, F, L, or I for 22 hours each (Figures 3E–J); and EC50 was determined based on OD630 as the readout for mML-2 stimulation. [Figure 3F]Figures 3C–3J show the results of the HEK-Blue™ IL2 assay. Cells were treated with various concentrations of uncleaved or mMMP9-cleaved construct E for 22 hours (Figure 3C); human IL2 (Figure 3D); uncleaved or mMMP9-cleaved construct B for 19 hours; and uncleaved or mMMP9-cleaved constructs J, K, F, L, or I for 22 hours each (Figures 3E–J); and EC50 was determined based on OD630 as the readout for mML-2 stimulation. [Figure 3G] Figures 3C–3J show the results of the HEK-Blue™ IL2 assay. Cells were treated with various concentrations of uncleaved or mMMP9-cleaved construct E for 22 hours (Figure 3C); human IL2 (Figure 3D); uncleaved or mMMP9-cleaved construct B for 19 hours; and uncleaved or mMMP9-cleaved constructs J, K, F, L, or I for 22 hours each (Figures 3E–J); and EC50 was determined based on OD630 as the readout for mML-2 stimulation. [Figure 3H] Figures 3C–3J show the results of the HEK-Blue™ IL2 assay. Cells were treated with various concentrations of uncleaved or mMMP9-cleaved construct E for 22 hours (Figure 3C); human IL2 (Figure 3D); uncleaved or mMMP9-cleaved construct B for 19 hours; and uncleaved or mMMP9-cleaved constructs J, K, F, L, or I for 22 hours each (Figures 3E–J); and EC50 was determined based on OD630 as the readout for mML-2 stimulation. [Figure 3I] Figures 3C–3J show the results of the HEK-Blue™ IL2 assay. Cells were treated with various concentrations of uncleaved or mMMP9-cleaved construct E for 22 hours (Figure 3C); human IL2 (Figure 3D); uncleaved or mMMP9-cleaved construct B for 19 hours; and uncleaved or mMMP9-cleaved constructs J, K, F, L, or I for 22 hours each (Figures 3E–J); and EC50 was determined based on OD630 as the readout for mML-2 stimulation. [Figure 3J]Figures 3C–3J show the results of the HEK-Blue™ IL2 assay. Cells were treated with various concentrations of uncleaved or mMMP9-cleaved construct E for 22 hours (Figure 3C); human IL2 (Figure 3D); uncleaved or mMMP9-cleaved construct B for 19 hours; and uncleaved or mMMP9-cleaved constructs J, K, F, L, or I for 22 hours each (Figures 3E–J); and EC50 was determined based on OD630 as the readout for mML-2 stimulation.

[0149] [Figure 3K] Figures 3K–3L show the results of CTLL-2 proliferation assays using construct M, construct N, or their cleavage products. Cleavage was performed with MMP-2 for 2 hours, and the resulting products were incubated with CTLL-2 cells. The data indicate that MMP-2-treated constructs M and N dose-dependently stimulate CTLL-2 cell proliferation. EC50 values ​​are shown in nM. [Figure 3L] Figures 3K–3L show the results of CTLL-2 proliferation assays using construct M, construct N, or their cleavage products. Cleavage was performed with MMP-2 for 2 hours, and the resulting products were incubated with CTLL-2 cells. The data indicate that MMP-2-treated constructs M and N dose-dependently stimulate CTLL-2 cell proliferation. EC50 values ​​are shown in nM.

[0150] [Figure 3M] Figure 3M shows the Coomassie-stained SDS-PAGE results comparing constructs E, M, and N. Constructs M and N showed reduced aggregation as well as greater stability and homogeneity.

[0151] [Figure 3N] Figures 3N to 3P show the results of CTLL-2 proliferation assays using constructs O, P, Q, or their cleavage products. Cleavage was performed with MMP2 for 2 hours, and the resulting products were incubated with CTLL-2 cells. The data indicate that MMP2-treated constructs O, P, and Q dose-dependently stimulate CTLL-2 cell proliferation. EC50 values ​​are shown in nM. [Figure 3O] Figures 3N to 3P show the results of CTLL-2 proliferation assays using constructs O, P, Q, or their cleavage products. Cleavage was performed with MMP2 for 2 hours, and the resulting products were incubated with CTLL-2 cells. The data indicate that MMP2-treated constructs O, P, and Q dose-dependently stimulate CTLL-2 cell proliferation. EC50 values ​​are shown in nM. [Figure 3P] Figures 3N to 3P show the results of CTLL-2 proliferation assays using constructs O, P, Q, or their cleavage products. Cleavage was performed with MMP2 for 2 hours, and the resulting products were incubated with CTLL-2 cells. The data indicate that MMP2-treated constructs O, P, and Q dose-dependently stimulate CTLL-2 cell proliferation. EC50 values ​​are shown in nM.

[0152] [Figure 3Q] Figures 3Q–3Y show the results of HEK-Blue™ IL2 assays using the construct or its cleavage products. Cleavage was performed with MMP9 for 18 or 22 hours, and the resulting products were incubated with HEK-Blue™ IL2 cells. EC50 was determined based on OD630 as the readout for mIL-2 stimulation. The data indicate that the MMP9-treated construct stimulates IL-2 in a dose-dependent manner. EC50 values ​​are shown in nM. [Figure 3R] Figures 3Q–3Y show the results of HEK-Blue™ IL2 assays using the construct or its cleavage products. Cleavage was performed with MMP9 for 18 or 22 hours, and the resulting products were incubated with HEK-Blue™ IL2 cells. EC50 was determined based on OD630 as the readout for mIL-2 stimulation. The data indicate that the MMP9-treated construct stimulates IL-2 in a dose-dependent manner. EC50 values ​​are shown in nM. [Figure 3S]Figures 3Q–3Y show the results of HEK-Blue™ IL2 assays using the construct or its cleavage products. Cleavage was performed with MMP9 for 18 or 22 hours, and the resulting products were incubated with HEK-Blue™ IL2 cells. EC50 was determined based on OD630 as the readout for mIL-2 stimulation. The data indicate that the MMP9-treated construct stimulates IL-2 in a dose-dependent manner. EC50 values ​​are shown in nM. [Figure 3T] Figures 3Q–3Y show the results of HEK-Blue™ IL2 assays using the construct or its cleavage products. Cleavage was performed with MMP9 for 18 or 22 hours, and the resulting products were incubated with HEK-Blue™ IL2 cells. EC50 was determined based on OD630 as the readout for mIL-2 stimulation. The data indicate that the MMP9-treated construct stimulates IL-2 in a dose-dependent manner. EC50 values ​​are shown in nM. [Figure 3U] Figures 3Q–3Y show the results of HEK-Blue™ IL2 assays using the construct or its cleavage products. Cleavage was performed with MMP9 for 18 or 22 hours, and the resulting products were incubated with HEK-Blue™ IL2 cells. EC50 was determined based on OD630 as the readout for mIL-2 stimulation. The data indicate that the MMP9-treated construct stimulates IL-2 in a dose-dependent manner. EC50 values ​​are shown in nM. [Figure 3V] Figures 3Q–3Y show the results of HEK-Blue™ IL2 assays using the construct or its cleavage products. Cleavage was performed with MMP9 for 18 or 22 hours, and the resulting products were incubated with HEK-Blue™ IL2 cells. EC50 was determined based on OD630 as the readout for mIL-2 stimulation. The data indicate that the MMP9-treated construct stimulates IL-2 in a dose-dependent manner. EC50 values ​​are shown in nM. [Figure 3W]Figures 3Q–3Y show the results of HEK-Blue™ IL2 assays using the construct or its cleavage products. Cleavage was performed with MMP9 for 18 or 22 hours, and the resulting products were incubated with HEK-Blue™ IL2 cells. EC50 was determined based on OD630 as the readout for mIL-2 stimulation. The data indicate that the MMP9-treated construct stimulates IL-2 in a dose-dependent manner. EC50 values ​​are shown in nM. [Figure 3X] Figures 3Q–3Y show the results of HEK-Blue™ IL2 assays using the construct or its cleavage products. Cleavage was performed with MMP9 for 18 or 22 hours, and the resulting products were incubated with HEK-Blue™ IL2 cells. EC50 was determined based on OD630 as the readout for mIL-2 stimulation. The data indicate that the MMP9-treated construct stimulates IL-2 in a dose-dependent manner. EC50 values ​​are shown in nM. [Figure 3Y] Figures 3Q–3Y show the results of HEK-Blue™ IL2 assays using the construct or its cleavage products. Cleavage was performed with MMP9 for 18 or 22 hours, and the resulting products were incubated with HEK-Blue™ IL2 cells. EC50 was determined based on OD630 as the readout for mIL-2 stimulation. The data indicate that the MMP9-treated construct stimulates IL-2 in a dose-dependent manner. EC50 values ​​are shown in nM.

[0153] [Figure 4] Figure 4 illustrates a serum stability assay using construct B and provides results showing that construct B was stable for 72 hours when incubated with serum collected from control or tumor-borne materials. Concentrations were measured by quantitative sandwich ELISA using mRNA capture antibody and mRNA Rα detection antibody.

[0154] [Figure 5] Figure 5 shows the study design, graphs, and pharmacokinetic (PK) parameters of construct B in mice. PK parameters were calculated using WinNonlin 7.0 (non-compartment model).

[0155] [Figure 6A] Figure 6A shows the design and results of an intratumor administration study in mice in which MC38 cells were subcutaneously injected on days 0–4 and treated with construct A, the medium, or human IL-2 on days 0–4 and 7–11, respectively. Construct A substantially inhibited tumor growth. In contrast, human IL-2 had an adverse effect on tumor control compared to the medium. Necrosis due to tumor growth was observed in the control and human IL-2 groups.

[0156] [Figure 6B] Figure 6B shows the experimental design in which mice treated as in Figure 6A were re-attacked with 2 × 10⁶ MC38 cells on day 40. Tumor growth was repelled, indicating that the treatment induced a sustained response, including anti-tumor immunological memory.

[0157] [Figure 7A] Figure 7A shows the study design in mice, in which MC38 cells were subcutaneously injected on day -10, and either construct B or the medium was administered intravenously every three days (Q3D) for a period of three weeks (a total of eight doses). No systemic toxicity was observed in essence. Construct B-treated mice showed virtually no tumor growth after the start of treatment, in contrast to medium-treated mice, whose tumors continued to grow until day 21. After day 21, several medium-treated mice were sacrificed because their tumor volume exceeded 3000 mm3. Therefore, subsequent tumor volume data for medium-treated mice are not shown because they tend to be biased towards mice with smaller tumor volumes compared to the population mean up to day 21.

[0158] [Figure 7B] Figure 7B shows the same mouse body weight data as in Figure 7A. The mouse body weight remained substantially constant during treatment with construct B, which is consistent with the absence of any apparent toxicity.

[0159] [Figure 8]Figure 8 shows the immunohistochemical results of tumor-infiltrating immune cells at day 21 of the media group tissue and at day 25 of the construct B-treated tumors in the experiment described above for Figure 7A. Significantly higher levels of all tested immune cell types were observed in construct B-treated mice compared to media-treated mice. Furthermore, the proportion of cells with markers consistent with the effector T cell phenotype was significantly higher than the proportion of CD4+Foxp3+(regulatory T) cells. Statistical analysis was performed using unpaired t-tests with Prism 5.0 software. P-values ​​between groups were calculated, and differences of p-value < 0.05 were considered statistically significant. * p<0.05, ** p<0.01, *** p<0.001.

[0160] [Figure 9] Figure 9 shows the quantitative analysis of MMP activity in a tumor-bearing mouse model based on fluorescence intensity over time after injection of MMPSense 680™.

[0161] [Figure 10] Figures 10A to 10D show the tumor volume over time in mice treated with the medium or construct B, as shown for each cancer model.

[0162] [Figure 11] Figures 11A–11D show the tumor volume over time (11A) and the levels of enzymes shown (11B–D) in mice treated with the medium or construct B, as shown in the B16F10 melanoma model.

[0163] [Figure 12] Figures 12A–12D show the tumor volume over time (12A) and the levels of enzymes shown (12B–D) in mice treated with the medium or construct B, as shown in the RM-1 prostate cancer model.

[0164] [Figure 13A] Figure 13A shows the MMP activity in each group, as measured as described in Figure 9.

[0165] [Figure 13B]Figures 13B to 13C show the tumor volume over time in mice treated with the medium or construct B, as shown for each cancer model. [Figure 13C] Figures 13B to 13C show the tumor volume over time in mice treated with the medium or construct B, as shown for each cancer model.

[0166] [Figure 14A] Figures 14A and 14B show the schematic structure of the linker and the binding of the MMP linker peptide, which includes the heparin-binding motif, to heparin-agarose beads. [Figure 14B] Figures 14A and 14B show the schematic structure of the linker and the binding of the MMP linker peptide, which includes the heparin-binding motif, to heparin-agarose beads.

[0167] [Figure 14C] Figure 14C shows the structural diagrams of the constructs and the heparin binding assay results for each construct. The assays were performed at pH 7.5, except where indicated as being performed at pH 6.

[0168] [Figure 14D] Figure 14D shows the schematic structure of the linker and the pH values ​​for the binding of each peptide to fibronectin.

[0169] [Figure 14E] Figure 14E shows the results of the fibronectin binding assay of the constructs. Unless otherwise stated, the assays were performed at pH 7.5.

[0170] [Figure 14F] Figure 14F shows the schematic structure of the linker and the binding of the MMP linker peptide containing the collagen-binding motif to the beads bound to collagen IV.

[0171] [Figure 14G]Figure 14G shows anti-mIL2 Western blots of the input (I), supernatant (S), collagen-bound (C), and control agarose-bound (A) fractions from a pull-down assay performed on the construct.

[0172] [Figure 15] Figure 15A shows fluorescence images of mice treated with each construct as described in Example 15. Figure 15B shows tumor-related fluorescence measured in mice treated with each construct as described in Example 15. Figures 15C-H show the amount of each construct present in tumor lysates prepared as described in Example 16. Here, mpk means mg / kg. Figures 15I-K show the amount of each construct present in serum samples prepared as described in Example 16.

[0173] [Figure 16A] Figures 16A and 16B show the tumor volume over time for the groups treated with each construct, as described in Example 17. [Figure 16B] Figures 16A and 16B show the tumor volume over time for the groups treated with each construct, as described in Example 17.

[0174] [Figure 17A] Figures 17A and 17B show the IFN-γ levels in the tumors after treatment with each construct, as described in Example 18. [Figure 17B] Figures 17A and 17B show the IFN-γ levels in the tumors after treatment with each construct, as described in Example 18.

[0175] [Figure 18A]Figures 18A-E show examples of element arrangements in cytokine prodrugs, including various combinations of cytokine polypeptide sequences (cytokines), pharmacokinetic modulators (PK EXT), protease-cleavable polypeptide sequences (PRO-LNK) in the linker, inhibitory polypeptide sequences (inhibitors), and, in some cases, one or more targeting sequences (RET LNK) or further linkers (LNK). Targeting sequences are shown in white text on a dark background. In Figures 18A and 18C, the pharmacokinetic modulator is on the same side as the inhibitory polypeptide sequence of the protease-cleavable sequence and therefore does not affect the pharmacokinetics of the cytokine polypeptide sequence. In Figures 18B and 18D, the pharmacokinetic modulator is on the same side as the cytokine polypeptide sequence of the protease-cleavable sequence and therefore affects the pharmacokinetics of the cytokine polypeptide sequence. In Figure 18E, the protease-cleavable sequence is present on each side of the pharmacokinetic modulator. This arrangement results in an intermediate outcome because the pharmacokinetic modulator is separated from the cytokine polypeptide sequence more slowly than the inhibitory polypeptide sequence. [Figure 18B]Figures 18A-E show examples of element arrangements in cytokine prodrugs, including various combinations of cytokine polypeptide sequences (cytokines), pharmacokinetic modulators (PK EXT), protease-cleavable polypeptide sequences (PRO-LNK) in the linker, inhibitory polypeptide sequences (inhibitors), and, in some cases, one or more targeting sequences (RET LNK) or further linkers (LNK). Targeting sequences are shown in white text on a dark background. In Figures 18A and 18C, the pharmacokinetic modulator is on the same side as the inhibitory polypeptide sequence of the protease-cleavable sequence and therefore does not affect the pharmacokinetics of the cytokine polypeptide sequence. In Figures 18B and 18D, the pharmacokinetic modulator is on the same side as the cytokine polypeptide sequence of the protease-cleavable sequence and therefore affects the pharmacokinetics of the cytokine polypeptide sequence. In Figure 18E, the protease-cleavable sequence is present on each side of the pharmacokinetic modulator. This arrangement results in an intermediate outcome because the pharmacokinetic modulator is separated from the cytokine polypeptide sequence more slowly than the inhibitory polypeptide sequence.

[0176] [Figure 18C]Figures 18A-E show examples of element arrangements in cytokine prodrugs, including various combinations of cytokine polypeptide sequences (cytokines), pharmacokinetic modulators (PK EXT), protease-cleavable polypeptide sequences (PRO-LNK) in the linker, inhibitory polypeptide sequences (inhibitors), and, in some cases, one or more targeting sequences (RET LNK) or further linkers (LNK). Targeting sequences are shown in white text on a dark background. In Figures 18A and 18C, the pharmacokinetic modulator is on the same side as the inhibitory polypeptide sequence of the protease-cleavable sequence and therefore does not affect the pharmacokinetics of the cytokine polypeptide sequence. In Figures 18B and 18D, the pharmacokinetic modulator is on the same side as the cytokine polypeptide sequence of the protease-cleavable sequence and therefore affects the pharmacokinetics of the cytokine polypeptide sequence. In Figure 18E, the protease-cleavable sequence is present on each side of the pharmacokinetic modulator. This arrangement results in an intermediate outcome because the pharmacokinetic modulator is separated from the cytokine polypeptide sequence more slowly than the inhibitory polypeptide sequence.

[0177] [Figure 18D]Figures 18A-E show examples of element arrangements in cytokine prodrugs, including various combinations of cytokine polypeptide sequences (cytokines), pharmacokinetic modulators (PK EXT), protease-cleavable polypeptide sequences (PRO-LNK) in the linker, inhibitory polypeptide sequences (inhibitors), and, in some cases, one or more targeting sequences (RET LNK) or further linkers (LNK). Targeting sequences are shown in white text on a dark background. In Figures 18A and 18C, the pharmacokinetic modulator is on the same side as the inhibitory polypeptide sequence of the protease-cleavable sequence and therefore does not affect the pharmacokinetics of the cytokine polypeptide sequence. In Figures 18B and 18D, the pharmacokinetic modulator is on the same side as the cytokine polypeptide sequence of the protease-cleavable sequence and therefore affects the pharmacokinetics of the cytokine polypeptide sequence. In Figure 18E, the protease-cleavable sequence is present on each side of the pharmacokinetic modulator. This arrangement results in an intermediate outcome because the pharmacokinetic modulator is separated from the cytokine polypeptide sequence more slowly than the inhibitory polypeptide sequence.

[0178] [Figure 18E]Figures 18A-E show examples of element arrangements in cytokine prodrugs, including various combinations of cytokine polypeptide sequences (cytokines), pharmacokinetic modulators (PK EXT), protease-cleavable polypeptide sequences (PRO-LNK) in the linker, inhibitory polypeptide sequences (inhibitors), and, in some cases, one or more targeting sequences (RET LNK) or further linkers (LNK). Targeting sequences are shown in white text on a dark background. In Figures 18A and 18C, the pharmacokinetic modulator is on the same side as the inhibitory polypeptide sequence of the protease-cleavable sequence and therefore does not affect the pharmacokinetics of the cytokine polypeptide sequence. In Figures 18B and 18D, the pharmacokinetic modulator is on the same side as the cytokine polypeptide sequence of the protease-cleavable sequence and therefore affects the pharmacokinetics of the cytokine polypeptide sequence. In Figure 18E, the protease-cleavable sequence is present on each side of the pharmacokinetic modulator. This arrangement results in an intermediate outcome because the pharmacokinetic modulator is separated from the cytokine polypeptide sequence more slowly than the inhibitory polypeptide sequence. [Modes for carrying out the invention]

[0179] Detailed description of one embodiment This specification describes exemplary embodiments and applications of the present invention. However, the present invention is not limited to these exemplary embodiments and applications or the manner in which such exemplary embodiments and applications operate or are described. The term “or” is used in an inclusive sense, i.e., equivalent to “and / or,” unless the context indicates otherwise. It should be noted that any singular expression and use of terminology used herein and in the appended claims includes multiple subjects unless explicitly and obviously limited to one subject. The terms “include,” “contain,” and their grammatical variations as used herein are intended to be non-limiting so as not to exclude other similar items that may be substituted for or added to the listed items. Chapter divisions in this specification are provided solely for the convenience of the reader and do not limit any combination of elements described herein. In the event of any inconsistency or contradiction between what is included by reference and what is explicitly stated herein, the explicitly stated content shall prevail.

[0180] overview Provided herein are protease-activated procytokines (also referred to herein as cytokine prodrugs) comprising a linker containing a protease-cleavable linker and a targeting sequence described herein, for example, designed to bind to an extracellular matrix component, integrin, or syndecan; or a targeting sequence designed to bind to an extracellular matrix component, IgB(CD79b), integrin, cadherin, heparan sulfate proteoglycan, syndecan, or fibronectin with pH-sensitive function; or a targeting sequence containing any of the sequences of SEQ ID NOs. 180-662. The cleavable linker may be present between the cytokine polypeptide sequence and the inhibitory polypeptide sequence such that the ability of the cytokine polypeptide sequence to activate immune cells is reduced or removed compared to the free cytokine polypeptide sequence. Proteolysis of the linker can release the cytokine so that it can activate immune cells.

[0181] In one embodiment, a protease-cleavable linker is cleavable by a protease expressed at higher levels in the tumor microenvironment (TME) than in healthy tissue of the same type. In one embodiment, the protease-cleavable linker is an MMP-cleavable linker, such as any matrix metalloproteinase (MMP)-cleavable linker described herein. While not intended to be bound by any particular theory, increased expression of proteases, including but not limited to MMPs, in the tumor microenvironment (TME) can provide a mechanism for achieving selective or preferential activation of cytokine prodrugs at or near the tumor site. Some of the protease-cleavable linkers described herein are considered particularly suitable for achieving such selective or preferential activation.

[0182] In other embodiments, the cytokine prodrug includes a targeting sequence, for example, a targeting sequence designed to bind to an extracellular matrix component, such as an integrin or syndecan, or to fibronectin in a pH-sensitive function. The targeting sequence can promote the accumulation and / or extension of the lifetime of the cytokine prodrug and / or active cytokine in the ECM. In one embodiment, the targeting sequence is combined with a protease-cleavable linker that is cleavable by a protease highly expressed in the TME and / or by an MMP.

[0183] In any of the embodiments described above, the cytokine prodrug may further include a pharmacokinetic modulator that, for example, extends the half-life of the prodrug and, optionally, the half-life of the active cytokine.

[0184] Examples of cytokine prodrugs and the sequences of their components are shown in Tables 1 and 2. In Table 1, "X Hy" (Pip)" represents a hydrophobic amino acid residue. In one embodiment, the hydrophobic amino acid residue is any of glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). In one embodiment, the hydrophobic amino acid residue is any of Ala, Leu, Val, Ile, Pro, Phe, Met, and Trp. In one embodiment, the hydrophobic amino acid residue is any of Leu, Val, Ile, Pro, Phe, Met, and Trp. In one embodiment, the hydrophobic amino acid residue is any of Ala, Leu, Val, Ile, Phe, Met, and Trp. In one embodiment, the hydrophobic amino acid residue is any of Leu, Val, Ile, Phe, Met, and Trp. "(Pip)" represents piperidine. "(Hof)" represents homophenylalanine. "(Cit)" represents citrulline. "(Et)" represents ethionine. "C(me)" represents methylcysteine. Underlining is used to indicate a variant in a given sequence.

[0185] The present invention further provides the use of these cytokine prodrugs, for example, for the treatment of cancer. In some embodiments, cytokine prodrugs are selectively or preferentially cleaved in the tumor microenvironment, which may result in beneficial effects, such as improved recruitment and / or activation of immune cells near the tumor and / or reduced systemic exposure to active cytokines. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 Table 1-23 Table 1-24 Table 1-25 Table 1-26 Table 1-27 Table 1-28 Table 1-29 Table 1-30 Table 1-31 Table 1-32 Table 1-33 Table 1-34 Table 1-35 Table 1-36 Table 1-37 Table 1-38 Table 1-39 Table 1-40 Table 1-41 Table 1-42 Table 1-43 Table 1-44 Table 1-45 Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6 Table 2-7 Table 2-8 Table 2-9 Table 2-10 Table 2-11 Table 2-12 Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21

[0186] Definition As used herein, “cytokine polypeptide sequence” refers to a polypeptide sequence (may be a large sequence, e.g., part of a fusion polypeptide) that has significant sequence identity with wild-type cytokines and, when isolated from an inhibitory polypeptide sequence, can bind to and activate cytokine receptors. In some embodiments, the cytokine polypeptide sequence has at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with the sequence of a wild-type cytokine, e.g., a wild-type human cytokine. In some embodiments, the cytokine polypeptide sequence differs from a wild-type cytokine, e.g., a wild-type human cytokine, by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. Cytokines include, but are not limited to, chemokines. Exemplary cytokine polypeptide sequences are provided in Table 1. This definition applies to IL-2 polypeptide sequences, replacing “cytokine” with “IL-2”.

[0187] The “inhibitory polypeptide sequence” used herein is a sequence in a cytokine prodrug that inhibits the activity of a cytokine polypeptide sequence in the prodrug. The inhibitory polypeptide sequence binds to the cytokine polypeptide sequence, and such binding is reduced or removed by the action of an appropriate protease on the protease-cleavable polypeptide sequence. Exemplary inhibitory polypeptide sequences are provided in Table 1.

[0188] The "protease-cleavable polypeptide sequence" used here is a sequence that serves as a substrate for cleavage by a protease. The protease-cleavable polypeptide sequence is located in a cytokine prodrug such that its cleavage reduces or removes the binding of the inhibitory polypeptide sequence to the cytokine polypeptide sequence.

[0189] A polypeptide containing a protease-cleavable polypeptide sequence used herein is recognized by a protease or a group of proteases if, when exposed to a protease under conditions that enable protease cleavage, it results in significantly greater cleavage than that seen in a control polypeptide having an unrelated sequence, and / or if the protease-cleavable polypeptide sequence corresponds to a known recognition sequence of a protease (e.g., as described elsewhere in this specification for various exemplary proteases).

[0190] As used herein, a “pharmacokinetic modulator” is a portion of a cytokine prodrug that extends its in vivo half-life. The pharmacokinetic modulator may be a fusion domain in the cytokine prodrug or a post-translationally linked chemical component. The linkage may, but is not necessarily, be covalent. Exemplary pharmacokinetic modulator polypeptide sequences are provided in Table 1. Exemplary non-polypeptide pharmacokinetic modulators are described elsewhere in this specification.

[0191] The “targeting sequence” used herein is a sequence that localizes the majority of a cytokine prodrug to a target region, such as the tumor microenvironment. The targeting sequence may bind to extracellular matrix components or other components found in the target region, such as integrins or syndecans. Exemplary targeting sequences are provided in Table 2.

[0192] As used herein, "extracellular matrix components" refer to extracellular proteins or polysaccharides found in vivo. Endogenous and superficial membrane proteins in cells, including fibronectin, cadherins, integrins, and syndecans, are not considered extracellular matrix components.

[0193] As used herein, "immunoglobulin constant domain" refers to a domain that exists in or has significant sequence identity with the domain of the constant region of immunoglobulins such as IgG. An example of a constant domain is C H2 and C H are 3 domains. Unless otherwise specified, a polypeptide or prodrug comprising an immunoglobulin constant domain may comprise more than one immunoglobulin constant domain. In certain embodiments, the immunoglobulin constant domain has at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity to the sequence of a wild-type immunoglobulin constant domain, such as a wild-type human immunoglobulin constant domain. In certain embodiments, the immunoglobulin constant domain differs from a wild-type immunoglobulin constant domain, such as a wild-type human immunoglobulin constant domain, by 1, 2, 3, 4, 5, 6, 7, 8, 9, or more than 10 amino acids. In certain embodiments, the immunoglobulin constant domain has the same sequence as a wild-type immunoglobulin constant domain, such as a wild-type human immunoglobulin constant domain. Exemplary immunoglobulin constant domains are included within the sequences provided in Table 1. This definition applies with each “immunoglobulin constant” replaced by “C H 2” or “C H 3” to the C H 2 and C H 3 domains, provided that the C H 2 domain sequence does not have a higher percent identity to the C H 2 domain wild-type sequence than to the non-C H 2 immunoglobulin constant domain wild-type sequence, and that the C H 3 domain sequence does not have a higher percent identity to the C H 3 domain wild-type sequence than to the non-C H 3 immunoglobulin constant domain wild-type sequence. These definitions include domains having minor truncations compared to the wild-type sequence, provided that the truncation does not inhibit the substantially normal folding of the domain.

[0194] As used herein, “immunoglobulin Fc region” refers to the region of an immunoglobulin heavy chain comprising the C H 2 and C H 3 domains defined above. The Fc region may be a variable domain or C HDoes not include 1 domain.

[0195] In the context of the primary sequence of a polypeptide used here, if the first component is on one side of another component and the second component is on the other, then that component is "between" the first and second components. This term does not require them to be immediately adjacent. Therefore, in structure 1-2-3-4, 2 is between 1 and 4, and also between 1 and 3.

[0196] As used here, "domain" can refer, depending on the context, to a structural domain of a polypeptide or a functional collection of at least one domain (but possibly more structural domains). For example, C H Two domains refer to a portion of a sequence that is recognized as such. The immunoglobulin cytokine binding domain may include VH and VL structural domains.

[0197] As used herein, "denatured collagen" refers to cleavage products resulting from the action of MMPs on gelatin and collagen, and more generally to collagen or its fragments in forms that do not exist in the natural structure of full-length collagen.

[0198] In this context, "designed to bind to... with pH-sensitive function" means that the polypeptide sequence (e.g., the targeting sequence) exhibits pH-dependent differential binding affinity to its binding partner. For example, a polypeptide sequence may have a higher affinity at relatively acidic pH than the normal physiological pH (approximately 7.4). High affinity may occur at pH levels lower than 7, e.g., in the range of pH 5.5–7, 6–7, or 5.5–6.5, or below pH 6.

[0199] As used herein, the “cytokine-binding domain of a cytokine receptor” refers to the extracellular portion or fragment or truncation of the cytokine receptor that can bind to a cytokine polypeptide sequence. In some embodiments, the sequence of the cytokine-binding domain of a cytokine receptor has at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with the cytokine-binding domain of a wild-type cytokine receptor, e.g., the cytokine-binding domain of a wild-type human cytokine receptor. Exemplary sequences of cytokine-binding domains of cytokine receptors are provided in Table 1. This definition also applies to the IL-2-binding domain of an IL-2 receptor, by replacing “cytokine” with “IL-2”.

[0200] As used herein, a "cytokine-binding immunoglobulin domain" refers to one or more immunoglobulin variable domains (e.g., VH and VL domains) capable of binding to a cytokine polypeptide sequence. Exemplary sequences of cytokine-binding immunoglobulin domains are provided in Table 1. This definition also applies to IL-2-binding immunoglobulin domains, by replacing "cytokine" with "IL-2".

[0201] As used herein, “substantially” and its other grammatical forms mean sufficient to function for the intended purpose. The term “substantially” therefore allows for minor, slight variations from absolute or perfect conditions, dimensions, measurements, results, etc., as anticipated by those skilled in the art, but without apparent impact on the overall performance. When used in relation to numerical values ​​or parameters or features that can be expressed numerically, “substantially” means within 10 percent.

[0202] The term "plural" as used here can refer to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0203] In this context, if the alignment of the first sequence to the second sequence shows a match with the first sequence at a position of X% or more of the second sequence as a whole, then the first sequence is considered to "contain a sequence that is at least X% identical" to the second sequence. For example, the sequence QLYV contains the sequence QLY and a sequence that is 100% identical because the alignment results in 100% identity with all three locations of the second sequence. Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. Those skilled in the art will understand what choice of algorithm and parameter setting is appropriate for a given pair of sequences to be aligned; generally, for sequences of similar length with predicted identity >50% at the amino acid level or >75% at the nucleotide level, the Needleman-Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by EBI on the www.ebi.ac.uk web server is generally appropriate.

[0204] As used herein, “subject” refers to any member of the animal kingdom. In some embodiments, “subject” refers to a human being. In some embodiments, “subject” refers to a non-human animal. In some embodiments, “subject” refers to a primate. In some embodiments, “subject” includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, insects and / or worms. In some embodiments, a non-human subject is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates and / or pigs). In some embodiments, a subject may be a transgenic animal, a genetically modified animal and / or a clone. In some embodiments of the present invention, a subject is an adult, adolescent or infant. In some embodiments, the terms “individual” or “patient” are used interchangeably with “subject” and are intended to be so.

[0205] Cytokine polypeptide sequences The cytokine polypeptide sequence may be a wild-type cytokine polypeptide sequence or a sequence having one or more differences from a wild-type cytokine polypeptide sequence. In one embodiment, the cytokine polypeptide sequence is a human cytokine polypeptide sequence (which may be wild-type or have one or more differences). In one embodiment, the cytokine includes modifications to inhibit disulfide bond formation and optionally otherwise includes a wild-type sequence. In one embodiment, the cytokine polypeptide sequence has at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with a wild-type cytokine polypeptide sequence or the cytokine polypeptide sequences in Table 1. In one embodiment, the cytokine is a dimeric cytokine, e.g., a heterodimeric cytokine. In one embodiment, the cytokine is a homodimeric cytokine. Monomers can be linked together as a fusion protein, for example, by a linker or covalent (e.g., a disulfide bond) or non-covalent interaction. In one embodiment, the cytokine polypeptide sequence is an interleukin polypeptide sequence. In one embodiment, the cytokine polypeptide sequence can bind to a receptor containing CD132. In one embodiment, the cytokine polypeptide sequence can bind to a receptor containing CD122. In another embodiment, the cytokine polypeptide sequence can bind to a receptor containing CD25. IL-2

[0206] In one embodiment, the cytokine polypeptide sequence is an IL-2 polypeptide sequence. The IL-2 polypeptide sequence is a wild-type IL-2 polypeptide sequence or a sequence having one or more differences from a wild-type IL-2 polypeptide sequence. In one embodiment, the IL-2 polypeptide sequence is a human IL-2 polypeptide sequence (which may be wild-type or have one or more differences). In one embodiment, IL-2 includes a sequence of modification to inhibit disulfide bond formation (e.g., aldethleukin (commercially available as Proleukin®)) and optionally otherwise includes a wild-type sequence. In one embodiment, the IL-2 polypeptide sequence is at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identical to a wild-type IL-2 polypeptide sequence or the IL-2 polypeptide sequences in Table 1.

[0207] Inhibitory polypeptide sequence Various types of inhibitory polypeptide sequences can be used in the cytokine prodrug according to the present invention. In one embodiment, the inhibitory polypeptide sequence includes a cytokine-binding domain.

[0208] The cytokine-binding domain can be the cytokine-binding domain of a cytokine receptor. The cytokine-binding domain of a cytokine receptor may be provided as a sufficient portion of the extracellular component of the cytokine receptor or of the cytokine polypeptide sequence of a cytokine prodrug. In one embodiment, the cytokine-binding domain of a cytokine receptor has at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with the sequence of the cytokine-binding domain of a wild-type cytokine receptor, for example, the wild-type cytokine-binding domain of a human cytokine receptor.

[0209] The cytokine-binding domain may be a fibronectin cytokine-binding domain. In one embodiment, the fibronectin cytokine-binding domain has at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with the sequence of the cytokine-binding domain of the wild-type fibronectin cytokine receptor, for example, the wild-type human fibronectin cytokine-binding domain.

[0210] The cytokine-binding domain may be an immunoglobulin cytokine-binding domain. The immunoglobulin cytokine-binding domain may be an Fv, scFv, Fab, VHH, or other immunoglobulin sequence having antigen-binding activity against a cytokine polypeptide sequence. A VHH antibody (or nanobody) is an antigen-binding fragment of a heavy-chain-only antibody.

[0211] Further examples of inhibitory polypeptide sequences that may be provided to inhibit the cytokine polypeptide sequence of a cytokine prodrug include anticarin, affilin, affibody molecule, affimer, affitin, alphabody, avimer, DARPins, finomer, Knitz domain peptide, monobody, and binding domains based on other manipulated scaffolds such as SpA, GroEL, lipocalin, and CTLA4 scaffolds.

[0212] IL-2 inhibitory polypeptide sequence In a cytokine prodrug containing an IL-2 polypeptide sequence, the inhibitory polypeptide sequence is any of the above-described types of IL-2 inhibitory polypeptide sequences. In one embodiment, the IL-2 inhibitory polypeptide sequence is an immunoglobulin IL-2 inhibitory polypeptide sequence. In one embodiment, the IL-2 inhibitory polypeptide sequence includes an anti-IL-2 antibody or a functional fragment thereof. In one embodiment, the immunoglobulin IL-2 inhibitory polypeptide sequence includes a set of six anti-IL2 hypervariable regions (HVRs) as shown in Table 1 (e.g., SEQ ID NOs. 34-39 or 750-755). In one embodiment, the IL-2 inhibitory polypeptide sequence includes a set of anti-IL2 VH and VL sequences having at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with the anti-IL2 VH and VL sequences shown in Table 1, either as separate sequences or as part of an scFv. In one embodiment, the IL-2 inhibitory polypeptide sequence comprises a set of anti-IL2 VH and VL sequences, having sequences of the set of anti-IL2 VH and VL sequences shown in Table 1, either as individual sequences or as part of an scFv. Exemplary IL-2 inhibitory polypeptide sequences include SEQ ID NOs: 10-31, 40-51 and 747 and combinations of SEQ ID NOs: 32 and 33 or combinations of SEQ ID NOs: 748 and 749.

[0213] Protease-cleavable sequences Protease-cleavable sequences include various types of proteases, such as metalloproteases, serine proteases, cysteine ​​proteases, aspartate proteases, threonine proteases, glutamate proteases, gelatinases, asparagine peptide lyases, cathepsins, kallikreins, plasmins, collagenases, hKl, hK10, hK15, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidine, bromelain, calpain, caspases, and Mir Sequences that can be cleaved by 1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, regmine, plasmmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, ADAM10, ADAM17, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAP-a), dipeptidyl peptidase or dipeptidyl peptidase IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, proteinase 3, mast cell chymase, mast cell tryptase or dipeptidyl peptidase may be selected. In one embodiment, the protease-cleavable sequence includes any sequence in Table 1 (e.g., SEQ ID NOs. 80-90 or 700-741) or a variant having one or two mismatches compared to any sequence in Table 1 (e.g., SEQ ID NOs. 80-90 or 700-741). The protease does not generally need to be an exact copy of the recognition sequence; as such, the exemplary sequences may vary in some amino acid positions. In one embodiment, the protease-cleavable sequence includes a sequence that matches an MMP consensus sequence, such as any of SEQ ID NOs. 91-94. Those skilled in the art will be familiar with further sequences recognized by these types of proteases.

[0214] Matrix metalloproteinase-cleavable sequences In one embodiment, the protease-cleavable sequence is a matrix metalloprotease (MMP)-cleavable sequence. Exemplary MMP-cleavable sequences are provided in Table 1. In one embodiment, the MMP-cleavable sequence is cleavable by a plurality of MMPs and / or one or more MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13 and / or MMP-14. Table 1, for example, sequence numbers 80-90, provides exemplary MMP-cleavable sequences.

[0215] Targeting array In one embodiment, the targeting sequence promotes the localization, accumulation, and / or retention of cytokine prodrugs and / or cytokine polypeptide sequences (e.g., after proteolysis of protease-cleavable sequences) in a region of interest, e.g., the tumor microenvironment (TME). The targeting sequence may be a sequence that binds to an extracellular matrix component. Exemplary extracellular matrix components are collagen or denatured collagen (in any case, collagen may be collagen I, II, III, or IV), poly(I), von Willebrand factor, IgB (CD79b), heparin, sulfated glycoprotein, or hyaluronic acid.

[0216] In other embodiments, the targeting sequence binds to a target other than an extracellular matrix component. In one embodiment, the targeting sequence binds to IgB(CD79b), fibronectin, integrin, cadherin, heparan sulfate proteoglycan, or syndecane. In one embodiment, the targeting sequence binds to at least one integrin, such as one or more of α1β1 integrin, α2β1 integrin, α3β1 integrin, α4β1 integrin, α5β1 integrin, α6β1 integrin, α7β1 integrin, α9β1 integrin, α4β7 integrin, αvβ3 integrin, αvβ5 integrin, αIIbβ3 integrin, αIIIbβ3 integrin, αMβ2 integrin, or αIIbβ3 integrin. In one embodiment, the targeting sequence binds to at least one syndecane, such as one or more of syndecane-1, syndecane-4, and syndecane-2(w). Cytokine prodrugs containing such targeting sequences may also include MMP cleavable linkers as described elsewhere in this specification, such as MMP cleavable linkers containing any of sequence numbers 80-90 or variants having one or two mismatches compared to any of sequence numbers 80-90.

[0217] In one embodiment, the targeting sequence includes any of the sequences shown in Table 2 (e.g., any of sequence numbers 180 to 640) or variants having one or two mismatches compared to such sequences.

[0218] pH-sensitive targeting sequence In one embodiment, the targeting sequence is designed to bind to its target with pH-sensitive function. In one embodiment, the targeting sequence may have high affinity at relatively acidic pH levels compared to normal physiological pH (approximately 7.4). High affinity may occur at pH levels lower than 7, e.g., in the range of pH 5.5–7, 6–7, or 5.5–6.5, or below pH 6. The presence of histidine in the targeting sequence may confer pH-sensitive binding. While not intended to be bound by any particular theory, it is thought that histidine is more likely to be protonated at lower pH levels, which may make binding to the load-electrode target more energetically likely. Therefore, in one embodiment, the targeting sequence contains one or more histidines, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 histidines. The inclusion of pH-sensitive targeting sequences can enhance the differentiation of cytokine prodrugs between tumor and normal tissue, so that cytokine prodrugs are preferentially retained in the tumor microenvironment compared to the normal extracellular matrix. Therefore, pH-sensitive targeting elements can further promote tumor-specific delivery of cytokine prodrugs, and thus further reduce or eliminate toxicity caused by cytokine activity in the normal extracellular matrix.

[0219] pH-sensitive binding to a target can be useful when it is desirable to localize or retain a cytokine prodrug or its cytokine polypeptide sequence in a region with a pH different from normal physiological pH. For example, the tumor microenvironment may be more acidic than blood and / or healthy tissue. That is, pH-sensitive binding to a target can improve the retention of the cytokine prodrug or its cytokine polypeptide sequence in the region of interest, which can enable lower doses than would otherwise be required and / or reduce systemic exposure and / or adverse effects.

[0220] In one embodiment, the targeting sequence is designed to bind to any of the targets described herein with pH sensitivity. In a specific embodiment, the target is an extracellular matrix component such as hyaluronic acid, heparin, heparan sulfate, or sulfated glycoprotein. In another specific embodiment, the target is fibronectin.

[0221] Exemplary targeting sequences for conferring target binding in pH-sensitive function are provided in Table 2 (e.g., SEQ ID NOs. 641-662). In one embodiment, the targeting sequence includes any of the sequences SEQ ID NOs. 641-662 or a variant having one or two mismatches compared to any of the sequences SEQ ID NOs. 641-662.

[0222] Pharmacokinetic modulator In one embodiment, the cytokine prodrug comprises a pharmacokinetic modulator. The pharmacokinetic modulator may be conjugated to the cytokine prodrug covalently or non-covalently. The pharmacokinetic modulator can extend the half-life of the cytokine prodrug and, optionally, the cytokine polypeptide sequence, for example, so that a smaller dose is required and less prodrug administration is needed over a longer period to achieve the desired result. Various forms of pharmacokinetic modulators are known in the art and may be used in the cytokine prodrugs of the present invention. In one embodiment, the pharmacokinetic modulator comprises a polypeptide (see Examples below). In one embodiment, the pharmacokinetic modulator comprises a non-polypeptide moiety (e.g., polyethylene glycol, polysaccharide, or hyaluronic acid). The non-polypeptide moiety may be conjugated to the prodrug using known approaches, e.g., conjugation to the prodrug; for example, reactive amino acid residues may be used to facilitate conjugation or added to the prodrug.

[0223] In one embodiment, the pharmacokinetic modulator alters the size, shape, and / or charge of the prodrug, for example, in a manner that reduces clearance. For example, a negatively charged pharmacokinetic modulator may inhibit renal clearance. In one embodiment, the pharmacokinetic modulator increases the hydrodynamic volume of the prodrug. In one embodiment, the pharmacokinetic modulator reduces renal clearance, for example, by increasing the hydrodynamic volume of the prodrug.

[0224] In one embodiment, the cytokine prodrug comprising a pharmacokinetic modulator (e.g., any of the pharmacokinetic modulators described herein) has a molecular weight of at least 70 kDa, for example, at least 75 or 80 kDa.

[0225] For further descriptions of various approaches to providing pharmacokinetic modulators, see, for example, Strohl, BioDrugs 29:215-19 (2015) and Podust et al., J. Controlled Release 240:52-66 (2016).

[0226] Polypeptide pharmacokinetic modulator In one embodiment, the pharmacokinetic modulator comprises polypeptides, for example, immunoglobulin sequences (see exemplary embodiments below), albumin, CTP (a load-electronically charged carboxy-terminal peptide of chorionic gonadotropin β chains that undergoes sialylation in vivo and in suitable host cells), inactive polypeptides (e.g., structurally indeterminate polypeptides such as XTEN, polypeptides containing residues Ala, Glu, Gly, Pro, Ser, and Thr), transferrin, homo-amino acid polypeptides, or elastin-like polypeptides.

[0227] Exemplary polypeptide sequences suitable for use as pharmacokinetic modulators are provided in Table 1 (e.g., any of SEQ ID NOs. 70-74). In one embodiment, the pharmacokinetic modulator has at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with the pharmacokinetic modulator sequences in Table 1 (e.g., any of SEQ ID NOs. 70-74).

[0228] In any embodiment in which the pharmacokinetic modulator includes a polypeptide sequence from an organism, the polypeptide sequence may be a human polypeptide sequence.

[0229] Immunoglobulin pharmacokinetic modulators In one embodiment, the pharmacokinetic modulator includes an immunoglobulin sequence, e.g., one or more immunoglobulin constant domains. In another embodiment, the pharmacokinetic modulator includes an Fc region. The immunoglobulin sequence (e.g., one or more immunoglobulin constant domains or Fc regions) may be a human immunoglobulin sequence. The immunoglobulin sequence (e.g., one or more immunoglobulin constant domains or Fc regions) may have at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with the sequence of a wild-type immunoglobulin sequence (e.g., one or more immunoglobulin constant domains or Fc regions), such as a wild-type human immunoglobulin sequence. In any of these embodiments, the immunoglobulin sequence may be an IgG sequence (e.g., IgG1, IgG2, IgG3, or IgG4). Exemplary immunoglobulin pharmacokinetic modulator sequences include the combination of SEQ ID NOs. 70-74 and SEQ ID NOs. 756 and 757.

[0230] Arrangement of components The listing of cytokine prodrug components herein does not imply any specific order other than those explicitly stated (for example, it may be specified that the protease-cleavable sequence lies between the cytokine polypeptide sequence and the inhibitory polypeptide sequence). The components of a cytokine prodrug may be arranged in various ways to provide properties suitable for a particular application. The components of a cytokine prodrug may all be on a single polypeptide chain or on multiple polypeptide chains crosslinked by covalent bonds such as disulfide bonds. For example, when a pharmacokinetic modulator contains Fc, one or more components may be bound to one chain, while one or more other components may be bound to other chains. Fc may be a heterodimer Fc, such as a knob-into-hole Fc (where one chain of Fc contains a knob mutation and the other chain contains a hole mutation). For an exemplary general description of knob and hole mutations, see, for example, Xu et al., mAbs 7:1, 231-242 (2015). An exemplary knob mutation (e.g., for human IgG1 Fc) is K360E / K409W. An exemplary hole mutation (e.g., for human IgG1 Fc) is Q347R / D399V / F405T. See SEQ ID NOs. 756 and 757.

[0231] For example, the pharmacokinetic modulator may be located on the same side as the cytokine polypeptide sequence of the protease-cleavable sequence, meaning that cleavage of the protease-cleavable sequence does not separate the pharmacokinetic modulator from the cytokine polypeptide sequence. Examples of such structures include CY-PM-CL-IN, IN-CL-CY-PM, and any other rearrangements (or variations in which further elements are included between, before or after, the listed components), where CL is not between CY and PM, and CY is the cytokine polypeptide sequence, PM is the pharmacokinetic modulator, CL is the protease-cleavable sequence, and IN is the inhibitory polypeptide sequence. In such embodiments, the pharmacokinetic modulator modulates the pharmacokinetics of both the prodrug and the active cytokine polypeptide sequence. In one embodiment, the pharmacokinetic modulator is Fc, in which case the components before and after PM in the above exemplary structures may bind to the same or different chains of Fc as described above.

[0232] In one embodiment, the pharmacokinetic modulator may be located on the same side as the inhibitory polypeptide sequence of the protease-cleavable sequence, meaning that cleavage of the protease-cleavable sequence separates the pharmacokinetic modulator from the cytokine polypeptide sequence. Such embodiments may be useful in providing the prodrug with a longer half-life than that of the active form.

[0233] In one embodiment, the targeting sequence may be located on the same side as the cytokine polypeptide sequence of the protease-cleavable sequence, meaning that cleavage of the protease-cleavable sequence does not separate the targeting sequence from the cytokine polypeptide sequence. Such embodiments may be useful for promoting the localization or retention of both the prodrug and the active form in a region of interest, e.g., the tumor microenvironment. When a pharmacokinetic modulator is used, depending on the desired effect, the targeting sequence may be on the same side as the protease-cleavable linker (e.g., to promote low-dose and / or low-frequency dosing) or on the other side (e.g., to avoid prolonged immune stimulation).

[0234] In one embodiment, the targeting sequence may be located on the same side as the inhibitory polypeptide sequence of the protease-cleavable sequence, meaning that cleavage of the protease-cleavable sequence separates the targeting sequence from the cytokine polypeptide sequence. Such embodiments may be useful for providing a cytokine gradient emanating from a region of interest or for providing such a gradient more rapidly than would occur with a targeting sequence located on the same side as the protease-cleavable sequence. When a pharmacokinetic modulator is used, depending on the desired effect, the targeting sequence may be on the same side as the protease-cleavable linker (e.g., to minimize systemic exposure to the active form of cytokine and / or to avoid prolonged immunostimulation) or on the other side (e.g., to facilitate low-dose and / or low-frequency dosing).

[0235] Several exemplary arrangements are shown in Figures 9 and 10A-E. In one embodiment, the cytokine prodrug comprises components arranged in an N-terminus → C-terminus or C-terminus → N-terminus order, according to any of the examples in Figures 9 and 10A-E, along with optionally additional components inserted between any of the described components.

[0236] Exemplary prodrugs IL-2 The following table shows exemplary combinations of components according to one embodiment of the disclosed cytokine prodrug. The numbers indicate the sequence number of a component. CY is a cytokine polypeptide sequence, CL is a protease-cleavable sequence, IN is an inhibitory polypeptide sequence, and PM, when present, is a pharmacokinetic modulator. When a range is indicated, any of the listed sequence numbers may be selected. When two sequence numbers are cited contiguously (using "and"), both sequence numbers exist and can function together (they may be fused to each other or not, as desired, for example, by covalent linking, intervening linkers, or crosslinking). For example, sequence numbers 32 and 33 are VL and VH domains that can function together to form cytokine-binding immunoglobulin domains such as sequence numbers 748 and 749. Sequence numbers 256 and 257 are Fc polypeptide chains that form heterodimeric knob-into-hole Fc that can act as pharmacokinetic modulators. The components may be arranged in any manner consistent with this disclosure, for example, as shown elsewhere in this specification. In one embodiment, the cytokine prodrug comprises the sequence combination shown in Table 3A. [Table 3A-1] [Table 3A-2] TIFF2026086601000069.tif242170 [Table 3A-3] [Table 3A-4] [Table 3A-5] [Table 3A-6] [Table 3A-7] [Table 3A-8] [Table 3A-9] [Table 3A-10]

[0237] Furthermore, as can be seen in Table 3A or elsewhere, any cytokine prodrug described herein may further include a targeting sequence, such as any of the targeting sequences described herein. In one embodiment, the targeting sequence is any of SEQ ID NOs. 180-662.

[0238] Furthermore, any of the cytokine prodrugs listed in Table 3A may contain a consensus sequence, such as sequence numbers 91-94, instead of the listed protease-cleavable sequences.

[0239] Also included in the present invention are cytokine prodrugs comprising a sequence having at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with any of the sequences of the above cytokine prodrugs.

[0240] In one embodiment, the cytokine prodrug contains a sequence that is at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identical to any of the sequences of SEQ ID NOs. 100 to 111. In one embodiment, the cytokine prodrug contains any of the sequences of SEQ ID NOs. 100 to 111. In one embodiment, the cytokine prodrug contains any of the sequences of SEQ ID NOs. 803 to 852.

[0241] Combination of protease-cleavable sequences and targeting sequences As shown in Table 3A or elsewhere, any embodiment of the suitability of the cytokine prodrugs described herein may include combinations of protease-cleavable sequences and targeting sequences shown in Table 4. When a range is indicated, any of the listed sequence numbers may be selected. The components may be arranged in any manner consistent with this disclosure, for example, as shown elsewhere in this specification (e.g., Figures 9 and 10A-E and the section on component arrangement). [Table 4-1] [Table 4-2]

[0242] Also included in the present invention are cytokine prodrugs comprising a sequence having at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with any of the sequences of the above cytokine prodrugs.

[0243] Pharmaceutical preparations The cytokine prodrug pharmaceutical formulations described herein may be prepared by mixing such cytokine prodrugs of desired purity in the form of lyophilized formulations or aqueous solutions with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are nontoxic to the recipient at commonly used doses and concentrations and include buffers such as phosphoric acid, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than approximately 10 residues) polypeptides; and blood Proteins such as clear albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0244] Preparations used for in vivo administration are generally sterile. For example, sterility can be easily achieved by filtration through a sterile filtration membrane.

[0245] use In one embodiment, one or more of the cytokine prodrugs, compositions, or pharmaceutical formulations described herein are for use in the manufacture of a medicament for the treatment or prevention of a disease or disorder in a subject. In one embodiment, one or more of the cytokine prodrugs, compositions, or pharmaceutical formulations described herein are for use in a method of creating a cytokine gradient in a subject, comprising administering the subject a protease-activated procytokine or pharmaceutical composition, wherein the subject comprises a site in which a protease that cleaves a protease-cleavable polypeptide sequence is abnormally high, and optionally the site comprises cancer. In one embodiment, abnormally high level means that the level of protease is higher than the level of protease in healthy tissue of the same type as the site in which the abnormally high level is located (e.g., in the subject being treated or in a healthy subject). In one embodiment, abnormally high level means that the level of protease is higher than the average level of protease in soft tissue.

[0246] In one embodiment, a method is provided for treating or preventing a disease or disorder in a subject, comprising administering to the subject one of the cytokine prodrugs or pharmaceutical compositions described herein. In one embodiment, the disease or disorder is cancer, for example, a solid tumor. In one embodiment, the cancer is melanoma, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, gastric or gastrointestinal cancer, lymphoma, colon or colorectal cancer, endometrial cancer, thyroid cancer, or bladder cancer. The cancer (for example, any of the aforementioned cancers) may have one or more of the following characteristics: PD-L1 positive; metastatic; unresectable; mismatch repair abnormality (MMRd); and / or high microsatellite instability (MSI-H).

[0247] In one embodiment, a method is provided for boosting T regulatory cells and / or reducing inflammatory or autoimmune activity, comprising administering a cytokine prodrug to a target area, e.g., an area of ​​inflammation. The cytokine prodrug for use in such a method may include an IL-2 polypeptide sequence. In another embodiment, a method is provided for treating autoimmune and / or inflammatory diseases, comprising administering a cytokine prodrug to a target area, e.g., an area of ​​inflammation or autoimmune activity. The cytokine prodrug for use in such a method may include an IL-2 polypeptide sequence. These methods utilize the ability of certain cytokines to stimulate T regulatory cells at relatively low levels, which can exert anti-inflammatory effects and reduce or suppress autoimmune activity.

[0248] The cytokine prodrug in any of the above methods and uses may be delivered to the subject using any suitable route of administration. In one embodiment, the cytokine prodrug is delivered non-enterally. In another embodiment, the cytokine prodrug is delivered intravenously.

[0249] The cytokine prodrugs provided herein may be used alone or in combination with other agents in treatment. For example, the cytokine prodrugs provided herein may be co-administered with at least one further therapeutic agent.

[0250] Such combination therapies described above include combination administrations (two or more therapeutic agents contained in the same or separate formulations) and separate administrations in which the cytokine prodrugs provided herein may be administered before, concurrently with, and / or after the administration of further therapeutic agents and / or adjuvants.

[0251] Cytokine prodrugs are formulated, dose-determined, and administered in a manner consistent with the context of advanced medical treatment. Factors to consider in this context include the specific disorder being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to the healthcare professional. Cytokine prodrugs are formulated with one or more drugs, if desired, to prevent or treat the disorder in question. The effective dose of such other drugs depends on the amount of cytokine prodrug present in the formulation, the type of disorder or treatment, and the other factors mentioned above. These are generally used in the same doses and routes of administration as described herein, or in approximately 1–99% of the doses described herein, or in any dose and route determined empirically / clinically appropriate.

[0252] For the prevention or treatment of a disease, the appropriate dose of a cytokine prodrug (when used alone or in combination with one or more other further therapeutic agents) depends on the type of disease being treated, the type of cytokine prodrug, the severity and course of the disease, whether the cytokine prodrug is administered for preventive or therapeutic purposes, prior treatment, the patient's medical history and response to antibodies or immunoconjugates, and the discretion of the treating physician. Cytokine prodrugs are preferably administered to the patient as a single dose or as part of a series of treatments.

[0253] Nucleic acids, host cells, and production methods Cytokine prodrugs or their precursors can be produced using recombinant methods and compositions. In one embodiment, an isolated nucleic acid encoding the cytokine prodrug described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising a cytokine polypeptide sequence, a linker and an inhibitory polypeptide sequence and any other polypeptide cytokine prodrug components that may be present. Exemplary nucleic acid sequences are provided in Table 1. In a further embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. In a further embodiment, host cells containing such nucleic acids are provided. In one such embodiment, the host cells contain a vector (e.g., transformed) containing the nucleic acid encoding the cytokine prodrug of the present invention. In one embodiment, the host cells are eukaryotes, e.g., Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method for producing the cytokine prodrug disclosed herein is provided, wherein the method comprises culturing host cells containing the nucleic acid encoding the cytokine prodrug as described above under conditions suitable for cytokine prodrug expression and optionally recovering antibodies from the host cells (or host cell culture medium).

[0254] For recombinant production of cytokine prodrugs, for example, the nucleic acids encoding the cytokine prodrug described above are prepared and / or isolated (e.g., after construction using synthesis and / or molecular cloning techniques) and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily prepared and / or isolated using known techniques.

[0255] Suitable host cells for cloning or expressing cytokine prodrug encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, cytokine prodrugs can be produced in bacteria, especially when glucosylation is not required. For example, see U.S. Patents 5,648,237, 5,789,199 and 5,840,523 regarding polypeptide expression in bacteria. After expression, cytokine prodrugs may be isolated from bacterial cell paste in soluble fractions and further purified.

[0256] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for cytokine prodrug encoding vectors, including fungal and yeast strains in which the glucosylation pathway has been "humanized," resulting in the production of polypeptides with a partial or complete human glucosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0257] Suitable host cells for cytokine prodrug expression can also be derived from multicellular organisms (plants, invertebrates, and vertebrates). Examples of invertebrate cells include insect cells. In particular, numerous baculovirus strains have been identified that can be used together with insect cells for transfection of armyworm cells.

[0258] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429.

[0259] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293 cells) as described in, e.g., Graham et al., J. Gen Virol. 36:59 (1977); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); e.g., TRI cells as described in, Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include DHFR - Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0.

[0260] This description and exemplary embodiments should not be construed as limiting. For the purposes of this specification and the appended claims, unless otherwise specified, all figures representing quantities, percentages or ratios and other numerical values ​​used herein and in the claims should be understood in all examples as being modified by the term “about,” even if not already modified by it. “About” indicates a degree of variation that does not substantially affect the nature of the subject matter described, for example, within 10%, 5%, 2%, or 1%. Thus, unless otherwise indicated, the numerical parameters shown in the following specification and the appended claims are approximations that may vary depending on the desired nature. At a minimum, and rather than attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be interpreted in light of at least the number of significant figures stated and by applying the usual rounding. [Examples]

[0261] The following embodiments are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the invention in any way.

[0262] Example 1: Construction of a mammalian expression vector encoding a fusion protein. The coding sequence for the entire protein domain, including the linker sequence, was synthesized as a whole gene (Genscript, NJ). The total synthesized gene was designed to include the coding sequences for the N-terminal signal peptide (to promote protein secretion), the 5' Kozak sequence, and specific restriction sites at the 5' and 3' ends. These genes were then cloned in a targeted manner into the mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA). Examples of fusion protein constructs are listed in Table 5A. Site-directed mutagenesis was performed using standard molecular biology techniques and appropriate kits (GeneArt, Regensburg).

[0263] [Table 5A-1] [Table 5A-2] [Table 5A-3] [Table 5A-4]

[0264] Example 2: Expression and purification of the fusion protein. Transient expression of fusion proteins Fusion proteins were produced using various mammalian cell expression methods (ExpiCHO-S TM Expi293F TM and Freestyle CHO-S TM (Life Technologies). Briefly, the expression construct was transiently transfected into cells using the reagents provided in each expression kit, according to the manufacturer's protocol. The fusion protein was then expressed and secreted into the cell culture supernatant. Samples were collected daily from the productive culture, and cell density and viability were assessed. The optimal collection time was determined by analyzing the protein expression titer and product integrity in the cell culture supernatant by SDS-PAGE. The cell culture supernatant was collected generally between 4 and 12 days, when the culture viability was generally >75%. On the day of collection, the cell culture supernatant was purified by centrifugation and vacuum filtration for further use.

[0265] Purification of fusion proteins The fusion protein was purified from the cell culture supernatant using a one-step or two-step method. Briefly, the Fc domain containing the protein was purified by protein A affinity chromatography (HiTrap MabSelect SuRe, GE Healthcare). The His-tagged protein was first purified using a nickel-agarose column (Ni-NTA Agarose, Qiagen), followed by anion exchange chromatography (HiTrap Capto Q ImpRes, Sigma). The entire purified sample was concentrated to a typical concentration of >1 mg / mL by ultrafiltration with buffer exchange. The purity and homogeneity of the final sample (generally >90%) were evaluated by SDS-PAGE under reducing and non-reducing conditions, followed by immunoblotting using anti-His or anti-Fc antibodies. The purified protein was divided into equal portions and stored at -80°C until use. Figure 1 shows a successful example of purified fusion protein.

[0266] Example 3: Cleavage of fusion protein by MMP protease Recombinant MMP9 and / or MMP2 (R&D Systems) were first activated with p-aminophenylmercury acetate, and the fusion proteins were digested or simulated digested at 37°C for 1 hour, 2 hours, 4 hours, and overnight (18-22 hours) using this activated protease or an equivalent volume of the unactivated solution. The cleavage assay was set up in TCNB buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35 (w / v), pH 7.5. The digested proteins were divided into equal parts and stored at -80°C before testing. Equal volumes of digests were then analyzed by SDS-PAGE, followed by Western blotting, to assess the degree of cleavage. The digests were also evaluated using functional assays such as CTLL-2 proliferation and HEK-Blue interleukin reporter assays. As shown in Figures 2A-E, essentially complete cleavage of fusion proteins with functional sites by the MMP9 protease is observed after overnight incubation. In contrast, proteins containing scrambled MMP cleavage sites are not cleaved (Figure 2E).

[0267] Example 4: Detection of mouse IL-2 / IL-2Ra fusion protein and mouse IL-2 by ELISA An ELISA assay was developed to detect and quantify fusion proteins containing IL-2 and IL-2Ra moieties. Wells of a 96-well plate were coated overnight with 100 μL of rat anti-mouse IL-2 monoclonal antibody (JES6-1A12; ThermoFisher) at a concentration of 1 mg / ml in PBS. After washing, the wells were blocked with TBS / 0.05% Tween 20 / 1% BSA, and then the fusion protein and / or unknown biological sample was added at room temperature for 1 hour. After washing, anti-mouse IL-2Ra biotin-labeled detection antibody (BAF2438, R&D systems) was added, and binding was detected using Ultra Strepavidin HRP (ThermoFisher). The ELISA plate was developed by adding chromogenic tetramethylbenzidine substrate (Ultra TMB, ThermoFisher). The reaction was stopped by adding 0.5 M H2SO4, and the absorbance was read at 450–650 nm.

[0268] A second ELISA assay was developed to detect and quantify mouse IL-2 and / or fusion proteins containing the IL-2 moiety. Wells of a 96-well plate were coated overnight with 100 μL of rat anti-mouse IL-2 monoclonal antibody (JES6-1A12; ThermoFisher) at a concentration of 1 mg / ml in PBS. After washing, the wells were blocked with TBS / 0.05% Tween 20 / 1% BSA, and then the fusion protein and / or unknown biological sample was added at room temperature for 1 hour. After washing, anti-mouse IL-2 biotin-labeled detection antibody (JES6-5H4, ThermoFisher) was added, and binding was detected using Ultra Strepavidin HRP (ThermoFisher). The ELISA plate was developed by adding chromogenic tetramethylbenzidine substrate (Ultra TMB, ThermoFisher). The reaction is stopped by adding 0.5 M H2SO4, and the absorbance is read at 450-650 nm. This assay allows for the simultaneous detection of both free mouse IL-2 and mouse IL-2 in the state of the prodrug fusion protein.

[0269] Example 5: Immunoblot analysis of IL-2, IL-2Ra, 6x histidine, and Fc Untreated and digested fusion proteins were evaluated for cleavage products by Western blotting. The following monoclonal antibodies were used: rat anti-mouse IL-2 antibody (JES6-1A12; ThermoFisher), goat anti-mouse IL-2 polyclonal antibody (AF-402-NA; R&D systems), mouse anti-6xHis monoclonal antibody (MA1-21315, ThermoFisher), anti-mIgG Fc HRP conjugate (ThermoFisher cat# A16084), and anti-human IL2 antibody (Invitrogen, cat# MA5-17097, mouse IgG1). Detection was performed using goat anti-rat HRP conjugate antibody, donkey anti-goat HRP conjugate antibody, or goat anti-mouse HRP conjugate (Jackson Immuno Research, West Grove, PA), and developed using SuperSignal West Femto Maximum sensitive detection reagent (ThermoFisher) as recommended by the manufacturer.

[0270] Example 6: IL-2 functional cell-based assay IL-2 activity was measured using CTLL-2 cells (ATCC) or the reporter cell line HEK Blue IL2 (Invivogen, San Diego). Briefly, for the CTLL-2 assay, untreated and digested samples of a certain titer were added to a 96-well plate at a ratio of 40,000 CTLL-2 cells per well in 100 μl of medium and incubated at 37°C in 5% CO2 for 18–22 hours. At the end of this time, 50 μg / well of Thiazolyl Blue Tetrazolium Bromide (MTT) (Sigma-Aldrich) was added, and the plate was incubated at 37°C in 5% CO2 for 5 hours. Cells were lysed with 10% SDS (Sigma) acidified with 100 μl / well of HCl, incubated at 37°C for 4 hours, and absorbance was read at 570 nm. Recombinant human or mouse IL-2 (Peprotech and R&D systems, respectively) were used as positive controls. Figures 3A-B, 3K-L, and 3N-P show examples of untreated and digested fusion proteins evaluated by the CTLL-2 proliferation assay.

[0271] HEK-Blue TM IL-2 cells were specifically designed to monitor IL-2-induced activation of the JAK-STAT pathway. Indeed, stimulation with human or mouse IL-2 initiates the JAK / STAT5 pathway and induces secreted embryonic alkaline phosphatase (SEAP) production. SEAP is quanti-blue TM These cells can be readily detected when using a SEAP detection medium. These cells respond to human and mouse IL-2. For the HEK Blue assay, untreated and digested samples were titrated and added to a 96-well plate in 200 μl of medium with 50,000 HEK Blue cells per well, incubated at 37°C in 5% CO2 for 20–24 hours. The following day, SEAP levels were measured by adding 20 μL of cell supernatant to QuantiBlue reagent, followed by incubation at 37°C for 1–3 hours and reading the absorbance at 630 nm. Figures 3C–J, 3Q–Y, and Tables 5B–5C show the results obtained from IL2 fusion proteins tested with the HEK Blue IL2 assay.

[0272] [Table 5B]

[0273] [Table 5C]

[0274] The aggregation, stability, and homogeneity of constructs E, M, and N were compared using Coomassie-stained SDS-PAGE analysis (Figure 3M). Constructs M and N showed reduced aggregation and greater stability and homogeneity, consistent with improvements due to the deletion of O-glucosylation sites.

[0275] Example 7: In vitro serum stability of fusion protein Construct B was incubated with serum collected from 8-week-old female C57BL / 6 naive and MC38 tumor-bearing mice at 37°C for up to 72 hours, respectively, to test both nonspecific and MMP-specific off-target cleavage (n=2 per serum type, tumor volume >3000 mm³ at collection). 3 Samples were collected at 0, 4, 8, 24, 48, and 72 hours, and intact non-MMP cleaved fusion proteins were quantified using an in-house developed sandwich ELISA. The results (see Figure 4) show that fusion protein levels were stable in both serum types, indicating 1) the absence of off-target protein cleavage up to 72 hours and 2) the absence of active MMPs in circulation.

[0276] Example 8: Pharmacokinetic evaluation of fusion protein in non-tumor-carrying mice For this study, 8-10 week old female C57BL / 6 mice (Jackson Labs) were assigned to different groups (3 mice / treatment group). Mice received a single dose of the fusion protein by IV injection (3.5 mg / kg). Blood samples were collected from 3 mice / group / time point at the following times: before administration (0 hours), and 10, 30, 1, 4, 12, 24, 48, 72, 96, and 120 hours after administration. Blood samples were collected in Eppendorf tubes, treated with serum, and stored at -80°C until the study. The samples were then evaluated by ELISA to quantify intact fusion protein levels. As shown in Figure 5, the mean serum concentration of the fusion protein was plotted over time, and the PK parameter was calculated using WinNonlin 7.0 (non-compartment model).

[0277] Example 9: In vivo efficacy of fusion protein in a syngeneic MC38 colorectal cancer model a. Intratumor injection of construct A Pilot PK data showed that construct A was rapidly removed from circulation (approximately 30-fold decrease in serum levels within 30 minutes of IV injection). This is common to small therapeutic proteins with molecular weights below the renal glomerular filtration cutoff of approximately 60-70 kDa. Therefore, this fusion protein was deemed unsuitable for systemic IV administration for POC in vivo efficacy studies. Instead, a 3-arm direct intratumor delivery design was chosen: medium, recombinant human IL-2 (r hIL2), and construct A (n=3 mice / arm). IL-2 has previously demonstrated antitumor activity by direct tumor injection in various syngeneic models, and based on this data, a dose of 5 μg / day (corresponding to 50,000 U / day) of r hIL2 was selected. Construct A was then subjected to EC observed in the CTLL-2 assay. 50 To compensate for the difference, the drug was administered at a concentration of 70 μg / day, which is 5 molars higher than recombinant IL-2. All drugs and media were administered daily to subcutaneous MC38 tumor masses (approximately 200 mm at the start of administration) growing in the flanks of C57BL / 6 mice. 3The tumor was injected for 12 days, with a 2-day rest period after the first 5 injections (total of 10 injections). Tumor and body weight were measured twice a week during the study period. Tumor volume was calculated using the following formula: (longest diameter × shortest diameter) 2 ) / 2. As shown in Figure 6A, significant antitumor activity was observed with construct A. In fact, complete tumor removal was observed in the construct A-treated group, while no tumor regression was observed in the medium or r hIL2-treated group. When 'cured', MC38 tumor cells (10 in the opposite flank) were placed in construct A-treated mice. 6 The mice were re-inoculated with the cells on day 40, and no tumor mass was established one month after the re-inoculation, suggesting the existence of a 'memory' immune response in these mice (Figure 6B).

[0278] b. Systemic IV injection of construct B The purpose of this study is to evaluate the efficacy of construct B in MC38-carrying female C57BL / 6 mice. For this study, 6-8 week old female C57BL / 6 mice (Jackson Labs) were fed MC38 cells (10 6 Cells / animals were subcutaneously inoculated, and the average tumor volume was approximately 80 mm². 3 When this was reached, the animals were randomized into two groups based on tumor volume (8 mice / treatment group). The animals were administered according to the following study design. TIFF2026086601000086.tif28155

[0279] Mice were administered the drug for 21 days, followed by a further week of observation. Tumors and body weight were measured twice a week during the study period. Tumor volume was calculated using the following formula: (longest diameter × shortest diameter) 2 ) / 2. Figure 7 shows the mean tumor volume over time for both groups (Figure 7a) and the individual body weight of the media and treated animals (Figure 7B).

[0280] The results demonstrated superior efficacy in the treatment group, with 92% inhibition of tumor growth at day 21, while no adverse events were observed. Notably, in the colorectal cancer syngeneic setting, complete tumor regression ('cure') occurred in 3 out of 8 patients.

[0281] Example 10: Evaluation of immune cell populations by immunohistochemistry (IHC) in MC38 colorectal cancer samples The purpose of this study is to evaluate immune targets in tumor samples using IHC. See details below. • CD4+Foxp3 double immunofluorescence staining • Single IHC staining of CD8, CD25, CD3, CD4, and CD335

[0282] It should be noted that before performing IHC, H&E staining was performed on all controls and tumors treated with construct B to confirm tissue quality.

[0283] Seven tumor samples were selected from a systemic in vivo efficacy study, and formalin-fixed paraffin-embedded (FFPE) blocks were prepared according to standard embedding procedures. TIFF2026086601000087.tif28156

[0284] The following antibody was used. TIFF2026086601000088.tif168160

[0285] FFPE blocks were sectioned using a manual rotary microtome (4 μm thick / section), and the optimized IHC assay protocol was used for all antibodies. All stained sections were manipulated at 40x magnification using a NanoZoomer-S60 Image System. High-resolution images of the entire section were created and further analyzed.

[0286] Scoring method: All images are HALO TM The images were analyzed using an Image Analysis platform. All slide images were analyzed, and necrotic areas were excluded. Total cells and IHC-positive cells were counted. The IHC score is shown as the ratio of positive cells to the total number of cells in the entire section, as shown in Figure 8. The results indicate a significant increase in tumor-infiltrating immune cells after construct B treatment.

[0287] Example 11: Evaluation of in vivo MMP activity in various syngeneic tumor models. MMP-activating fluorescent probe, MMPSense 680 TM The degree of MMP activity was evaluated using an in vivo model utilizing this probe. This probe is optically silent in its intact state and becomes highly fluorescent after MMP-mediated cleavage, and is designed to be used as a real-time in vivo imaging tool (Perkin Elmer). After a single IV injection of the probe into tumor-bearing mice, fluorescence images were captured for 6 days, and the fluorescence intensity in the tumor area, which is directly proportional to the present MMP activity, was quantified (Figure 9). All models showed essentially different levels of MMP activity.

[0288] Example 12: In vivo efficacy of construct B in various syngeneic tumor models. In the efficacy test, malignant cells were subcutaneously inoculated into C57BL / 6 or BALB / c mice, and the average tumor volume was 90 mm². 3 When tumor volume was reached, animals were randomized into two groups based on tumor volume (n=10 mice / treatment group). Mice were administered 20 mg / kg intravenously every 3 days (Q3D). Tumors, body weight, and clinical findings were measured / acquired twice weekly during the study period. Tumor volumes are shown in Figures 10A-D, 11A, 12A, and 13B-C. Robust antitumor activity was observed in several models, with notable 49% tumor growth inhibition (TGI) observed at D12 in the B16F10 melanoma model and 58% tumor TGI observed at day 10 in the high-grade Ras / Myc transformed RM-1 prostate cancer model (Figures 10C-D and Table 6). Notably, these models showed no signs of toxicity, including weight loss and elevated liver and / or kidney enzyme levels, and clinical findings were normal. Liver and kidney enzyme results corresponding to Figures 11A and 12A are shown in Figures 11B-D and 12B-D, respectively.

[0289] [Table 6] The p-value represents the unpaired t-test (graphpad prism) between the medium and construct group B for max TGI.

[0290] The difference in efficacy between the MC38 and B16F10 models may be partly due to lower MMP activity measured in B16F10 tumors compared to the MC38 setting, resulting in less functional IL-2 being released to TME (Figure 13A).

[0291] Example 13: Next-generation retained linker peptide binding assay A series of peptides containing MMP cleavable sites, with or without tumor-retaining sequences, were synthesized and conjugated to the fluorophore EDANS(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid) (custom synthesis, ThermoFisher). Table 7 lists the peptides. These peptides were then tested for their ability to bind to ECM proteins such as heparin, fibronectin, and collagen, which are abundant in tumor stroma.

[0292] [Table 7] Underlined text indicates MMP cleavage sites. Bold italics indicate retained motifs when present. * This represents the Edans fluorophore conjugated to the peptide.

[0293] The full-binding assay was set up with 10 mM TrisHCl pH 7.5 and / or 10 mM TrisHCl pH 6. Peptides (20 μM) were incubated in a shaker with agarose crosslinked to heparin or control agarose beads (Sigma and Pierce, respectively) for 2 hours at room temperature. The beads were then washed four times and resuspended in 100 μL of binding buffer in a black 96-well plate. Peptide binding was quantified by measuring the fluorescence of the samples using the EDANS excitation / emission spectrum (Ex340 / Em490). Figure 14A shows that MMP linker peptides containing several next-generation heparin-binding motifs bound to heparin-agarose beads, while first-generation MMP linkers lacking the retention sequence did not. One such peptide showed enhanced binding to heparin at pH 6 (tumor pH) compared to pH 7.5 (normal tissue pH) (Figure 14B).

[0294] For fibronectin and collagen binding assays, streptavidin-conjugated magnetic beads (Mag Sepharose, Cytiva, and Dynabead, and ThermoFisher, respectively) were first incubated with biotin-labeled fibronectin (Cytoskeleton) or biotin-labeled collagen IV (Prospec) for 1 hour with gentle shaking. After multiple washes, the ECM-coated beads were then incubated with Edans peptide (20 μM) for 2 hours at room temperature in neutral or acidic binding buffer. The beads were then washed and resuspended in 100 μL of binding buffer in a black 96-well plate. Peptide binding was quantified by measuring the fluorescence of the sample using the excitation / emission spectrum (Ex340 / Em490) of EDANS. Figure 14D shows that peptide 13 can bind to fibronectin and shows enhanced binding at pH 6 (tumor pH) compared to pH 7.5 (normal tissue pH). Figure 14F shows that peptide 14 binds strongly to collagen IV, while peptide 15 binds only to a lesser extent.

[0295] Example 14: Next-generation tumor-retaining IL-2 fusion protein binding assay We designed and successfully fabricated a series of IL-2 fusion proteins containing tumor-retaining sequences in the linker region (Table 3 and Figures 1C-D). These proteins were then tested for their ability to bind to ECM proteins such as heparin, fibronectin, and collagen, which are abundant in tumor stroma.

[0296] 96-well plates were coated with 25 μg / mL heparin-BSA conjugate (donated by Dr. Mueller of Boerhinger Ingelheim) or control BSA for 18–22 hours on a shaker (350 rpm) at room temperature. After washing, the wells were blocked with PBS-0.05% Tween 20 / 1% BSA for 90 minutes, and then the fusion protein was added at room temperature for 2 hours with shaking, after titration in 1% BSA / PBS-0.05% Tween 20 pH 7.5 and / or pH 6. After washing, anti-mouse IL-2 biotin-labeled detection antibody (JES6-5H4, ThermoFisher) was added, and binding was detected using Ultra Strepavidin HRP (ThermoFisher). The plates were developed by adding chromogenic tetramethylbenzidine substrate (Ultra TMB, ThermoFisher). The reaction was stopped by adding 0.5M H2SO4, and the absorbance was read at 450-650nm. IL-2 fusion variant constructs Y and CC bound to heparin in a dose-dependent manner at acidic pH, with higher affinity than construct B (Figure 14C). Notably, construct CC preferentially bound to heparin at acidic pH, and EC 50 The strongest bond was approximately 10 nM, while the bond in structure B was much weaker, EC 50 The value is >100 times higher.

[0297] A similar plate-based assay was developed to investigate the binding of IL-2 fusion variants to fibronectin. 96-well plates were coated with 4 μg / mL fibronectin (Sigma) or control BSA for 18–22 hours at room temperature on a shaker (350 rpm). After washing, wells were blocked with protein-free barrier buffer (Pierce) for 90 minutes, and the fusion protein was then titrated in barrier buffer-0.1% Tween 20 pH 7.5 and / or pH 6 and added at room temperature for 1 hour with shaking. After washing, anti-mouse IL-2 biotin-labeled detection antibody (JES6-5H4, ThermoFisher) was added, and binding was detected using Ultra Streptavidin HRP (ThermoFisher). The plates were developed by adding chromogenic tetramethylbenzidine substrate (Ultra TMB, ThermoFisher). The reaction was stopped by adding 0.5 M H2SO4, and the absorbance was read at 450-650 nm. Construct EE preferentially bound to fibronectin at acidic pH, showing dose-dependent binding, while no binding was observed at pH 7.5 (Figure 14E). No significant binding of construct B was observed under neutral or acidic conditions.

[0298] To demonstrate binding to collagen, a pull-down assay was performed using collagen-crosslinked agarose (Sigma). IL-2 fusion proteins were incubated with collagen-agarose or control agarose beads in 1% BSA / PBS-0.05% Tween 20 for 18–22 hours at 4°C, with gentle rotation. After washing, protein binding to the beads was evaluated by suspending the beads in SDS sample buffer (Life Technologies). The bound proteins were then isolated by SDS-PAGE on a 4–12% BisTris gradient gel, followed by immunoblotting using a goat anti-mouse IL-2 polyclonal antibody (AF-402-NA; R&D systems). A donkey anti-goat HRP conjugate antibody was used for detection (Jackson Immuno Research, West Grove, PA), and the blots were developed using SuperSignal West Femto Maximum sensitivity detection reagent (ThermoFisher) as recommended by the manufacturer. The blot image is shown in Figure 14G. Constructs GG and II bound particularly well to collagen-agarose beads but not to IL-2 fusion protein-bound control agarose beads. Quantification of the blot using the iBright imaging system (Invitrogen) showed that the fractions of bound constructs GG and II were low (<1% of input), but 2.5-fold and 1.4-fold higher than the fraction of bound construct B, respectively (Table 8).

[0299] [Table 8]

[0300] Example 15: The next-generation retention linker IL-2 fusion protein shows significant retention in tumors in vivo. The level of IL-2 fusion protein present in tumors was assessed in vivo using fluorescently labeled proteins and real-time whole-body imaging. Non-cleavable constructs GGG and DD were conjugated to the Dylight 650 probe according to the manufacturer's protocol (Dylight 650 antibody-labeled kit, ThermoFisher). It was confirmed that conjugation did not significantly alter the protein's binding to heparin. BALB / c mice were subcutaneously inoculated with an EMT6 breast cancer syngeneic model, with an average tumor volume of 240 mm². 3 When the tumor volume reached a certain point, the animals were randomized into three groups based on tumor volume (n=2 mice / treatment group). The table below shows the study design. TIFF2026086601000092.tif44160

[0301] After a single administration of labeled IL-2 fusion protein to tumor-bearing mice, fluorescence images (excitation 640 / emission 680 matching the Dylight 650 probe ex / em spectrum) were collected over 96 hours using an IVIS system (PerkinElmer, IVIS Lumina Series III), as shown in Figure 15A. Fluorescence intensity in the tumor region was quantified across groups, and the mean background tumor fluorescence (group 1) was subtracted from group 2 and 3 values ​​at each time point to normalize the data against the initial fluorescence intensity of the same amount of each labeled protein. Figure 15B shows tumor-associated fluorescence, with group 3 being approximately twice as high as group 2 at each time point tested. This indicates accumulation of the next-generation retained linker construct DD, which is retained in the tumor at twice the level compared to the first-generation IL-2 fusion protein construct GGG.

[0302] Example 16: Next-generation retaining MMP-linkers increase drug and IL2 levels in tumors and serum in vivo. In tumor samples collected during a preclinical efficacy study comparing construct B and the retained linker IL-2 fusion drug, the levels of full-length IL2-IL2Ra fusion protein and IL-2 were quantified (see Example 17).

[0303] Tumors (n=3 / group) were collected 24 hours after the last injection, flash-frozen, and stored at -80°C until further processing. Tumor lysates were produced using tissue extraction reagents (ThermoFisher) and standard techniques supplemented with protease and phosphatase inhibitors, and protein concentrations were determined using a BCA assay (Pierce).

[0304] Lysates were tested using in-house ELISA to measure full-length IL-2 fusion protein (IL-2 capture / IL-2Ra detection) and IL-2 fusion protein + free IL-2 (IL-2 capture / IL-2 detection). Tumor free IL-2 levels were calculated by subtracting drug levels from drug + IL-2 datasets. Results were normalized to 1 mg of tumor lysate, and mean values ​​are shown in Figures 15C-H. The level of construct CC (20 mg / kg dose) in tumors was approximately three times higher than that of construct B, despite construct B being administered at 40 mg / kg (Figure C). A comparison of drug levels in samples from the 10 mg / kg dosing cohort shows that the highest levels were present in tumors treated with collagen-binding construct GG (Figure 15F). This indicates that retention linker technology can lead to a robust increase in drug levels in tumors in vivo. Similarly, IL-2 levels in tumors treated with construct CC, construct GG, and construct II are increased compared to tumors treated with construct B (Figure 15E / H). This implies that next-generation retention linker technology can retain both full-length drug and post-cleavage-released IL-2 in the TME.

[0305] Equivalent serum samples (n=3 / group) were also tested using in-house ELISA to quantify the full-length IL-2 fusion drug, and the results are shown in Figures 15I-K. 24 hours after administration, circulating drug levels for construct B (40 mg / kg) and construct CC (20 mg / kg) were approximately equivalent despite the dose difference. However, in the 10 mg / kg cohort, serum drug levels for construct GG and construct II were approximately 5 times and 3 times higher than those for construct B (Figure 15J). Further serum samples collected on days 17 (construct B 20 mg / kg), 21 (construct Y 20 mg / kg), 4, and 8 after the final IV injection were assayed for the full-length IL-2 fusion drug. Figure 15K shows that circulating construct Y drug levels were significantly more than 10 times higher than construct B, despite serum being collected 4 days later. In summary, these data indicate that retained linker technology increases circulating drug levels.

[0306] Example 17: In vivo efficacy of retained linker IL-2 drug in the B16F10 analogue model In the initial efficacy trial, C57BL / 6 mice were subcutaneously inoculated with B16F10 melanoma cells, resulting in an average tumor volume of 70-90 mm². 3 When the tumor volume was reached, the animals were randomized into 5 groups based on tumor volume (n=6 mice / treatment group). The mice were administered intravenously every 3 days (Q3D) for a total of 5 times, according to the design described below. TIFF2026086601000093.tif71160

[0307] Tumor volume was measured twice a week during the study period. The mean tumor volume is shown in Figure 16A. Antitumor activity was observed in all treatment groups, but the most potent tumor growth inhibition (TGI) was observed in constructs Y and CC (77% and 78%, respectively) with retained linker drugs, compared to approximately 60% TGI in the construct B treatment group (dose-independent, Table 9).

[0308] [Table 9] The p-value represents the unpaired t-test (graphpad prism) between the media and test groups on day 13.

[0309] In a second efficacy study using the same model, C57BL / 6 mice were subcutaneously inoculated with B16F10 melanoma cells, resulting in an average tumor volume of 70-90 mm². 3 When the tumor volume was reached, the animals were randomized into 5 groups based on tumor volume (n=6 mice / treatment group). The mice were administered intravenously every 3 days (Q3D) for a total of 5 times, according to the design described below. TIFF2026086601000095.tif71160

[0310] Tumor volume was measured twice weekly during the study period until day 20, seven days after the fifth dose. On day 20, mice were administered the drug again, and the animals were sacrificed 24 hours later. Tissue and blood (treated with serum) were collected and stored at -80°C for further testing. The mean tumor volume is shown in Figure 16B. In this aggressive model, only mild antitumor activity was observed with construct B at 10 mg / kg (27% TGI day 15, Table 10). Notably, at equivalent doses, the total-retaining linker IL-2 fusion drug showed superior TGI (Table 10). In particular, collagen-binding drug constructs GG and II (10 mg / kg dose) showed robust tumor control similar to that observed with twice the dose of construct B (comparing 57% TGI day 15 in Table 10 and 61% TGI day 13 in Table 9, respectively). Furthermore, after a 7-day drug-free period, construct B showed reduced efficacy, while the total-retaining linker drug maintained a similar level of tumor control until day 20. In summary, Figures 16A-B demonstrate that retaining linker IL-2 drugs exhibit superior antitumor efficacy in a preclinical melanoma model. This is likely due to the high levels of both circulating drug in the serum and resident drug in the TME, which allow for long-term antitumor activity even after extended drug-free periods.

[0311] [Table 10]

[0312] Example 18: IFN-γ levels in tumor samples IFN-γ cytokine levels in tumor lysates (n=3 / group) were measured using the Luminex kit according to the manufacturer's (Invitrogen) protocol. Results were normalized to 1 mg lysate, and the mean values ​​are shown in Figure 17A / B. Higher levels of IFN-γ were observed in tumors treated with the whole-retaining linker IL-2 compared to tumors treated with construct B. IFN-γ was undetectable in tumors treated with the medium.

Claims

1. Cytokine polypeptide sequence; Inhibitory polypeptide sequences that can block the activity of cytokine polypeptide sequences; A linker between a cytokine polypeptide sequence and an inhibitory polypeptide sequence, comprising a protease-cleavable polypeptide sequence; and Targeting sequences designed to bind to extracellular matrix components, integrins, or syndecans, or designed to bind to extracellular matrix components, IgB (CD79b), integrins, cadherins, heparan sulfate proteoglycans, syndecans, or fibronectin with pH-sensitive functionality; or targeting sequences comprising any sequence of SEQ ID NOs. 180-662 or variants having one or two mismatches compared to any sequence of SEQ ID NOs. 180-662. Protease-activated procytokines, including

2. The protease-activated procytokine according to claim 1, further comprising a pharmacokinetic modulator.

3. The protease-activated procytokine according to claim 2, wherein the pharmacokinetic modulator comprises an immunoglobulin constant domain.

4. The protease-activated procytokine according to claim 2, wherein the pharmacokinetic modulator comprises an immunoglobulin Fc region.

5. The protease-activated procytokine according to claim 4, wherein the immunoglobulin is human immunoglobulin.

6. A protease-activated procytokine according to any one of claims 4 to 5, wherein the immunoglobulin is IgG.

7. The protease-activated procytokine according to claim 6, wherein IgG is IgG1, IgG2, IgG3, or IgG4.

8. The protease-activated procytokine according to claim 2, wherein the pharmacokinetic modulator comprises albumin.

9. The protease-activated procytokine according to claim 8, wherein albumin is serum albumin.

10. A protease-activating procytokine according to any one of claims 8 to 9, wherein the albumin is human albumin.

11. The protease-activated procytokine according to claim 2, wherein the pharmacokinetic modulator comprises PEG.

12. The protease-activated procytokine according to claim 2, wherein the pharmacokinetic modulator comprises XTEN.

13. The protease-activated procytokine according to claim 2, wherein the pharmacokinetic modulator comprises CTP.

14. A protease-activated procytokine according to any one of claims 2 to 13, wherein the protease-cleavable polypeptide sequence is located between the cytokine polypeptide sequence and the pharmacokinetic modulator.

15. A protease-activated procytokine according to any one of claims 2 to 13, wherein the pharmacokinetic modulator is located between the cytokine polypeptide sequence and the protease-cleavable polypeptide sequence.

16. A protease-activating procytokine according to any one of claims 1 to 15, comprising multiple protease-cleavable polypeptide sequences.

17. The protease-activated procytokine according to claim 16, wherein the cytokine polypeptide sequence is sandwiched between protease-cleavable polypeptide sequences.

18. The protease-activated procytokine according to claim 17, having the structure PM-CL-CY-CL-IN (N-terminus → C-terminus or C-terminus → N-terminus) (where PM is a pharmacokinetic modulator, each CL is independently a protease-cleavable polypeptide sequence, CY is a cytokine polypeptide sequence, and IN is an inhibitory polypeptide sequence).

19. A protease-activated procytokine according to any one of claims 1 to 18, comprising a targeting sequence, wherein the targeting sequence is between a cytokine polypeptide sequence and a protease-cleavable polypeptide sequence or a protease-cleavable polypeptide sequence.

20. A protease-activating procytokine according to any one of claims 1 to 19, wherein the cytokine polypeptide sequence includes modifications to inhibit disulfide bond formation, and optionally the rest includes a wild-type sequence.

21. A protease-activated procytokine according to any one of claims 1 to 20, wherein the cytokine polypeptide sequence has at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with the sequence of a wild-type cytokine polypeptide sequence or the cytokine polypeptide sequences in Table 1.

22. The protease-activating procytokine according to claim 21, wherein the cytokine polypeptide sequence is a wild-type cytokine polypeptide sequence.

23. A protease-activating procytokine according to any one of claims 1 to 22, wherein the cytokine polypeptide sequence is a monomeric cytokine or a dimeric cytokine polypeptide sequence containing monomers linked covalently (preferably via a polypeptide linker) or non-covalently.

24. A protease-activating procytokine according to any one of claims 1 to 23, wherein the inhibitory polypeptide sequence includes a cytokine-binding domain.

25. The protease-activating procytokine according to claim 24, wherein the cytokine-binding domain is the cytokine-binding domain of a cytokine receptor or the cytokine-binding domain of fibronectin.

26. The protease-activating procytokine according to claim 24, wherein the cytokine-binding domain is an immunoglobulin cytokine-binding domain.

27. The protease-activating procytokine of claim 26, wherein the immunoglobulin cytokine-binding domain comprises a light chain variable domain and a heavy chain variable domain that bind to a cytokine.

28. A protease-activating procytokine according to any one of claims 26 to 27, wherein the immunoglobulin cytokine binding domain is scFv, Fab, or VHH.

29. Protease-cleavable polypeptide sequences include metalloproteases, serine proteases, cysteine ​​proteases, aspartate proteases, threonine proteases, glutamate proteases, gelatinases, asparagine peptide lyases, cathepsins, kallikrein, plasmin, collagenases, hKl, hK10, hK15, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidine, bromelain, calpain, caspase, and Mir. 1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, regmine, plasmmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, ADAM10, ADAM17, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), ing A protease-activated procytokine according to any one of claims 1 to 28, which is recognized by tarleukin-1b-converting enzyme, thrombin, FAP (FAP-a), dipeptidyl peptidase or dipeptidyl peptidase IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, proteinase 3, mast cell chymase, mast cell tryptase or dipeptidyl peptidase.

30. A protease-activated procytokine according to any one of claims 1 to 29, wherein the protease-cleavable polypeptide sequence comprises any sequence of SEQ ID NOs. 700 to 741 or a variant having one or two mismatches compared to any sequence of SEQ ID NOs. 700 to 741.

31. A protease-activated procytokine according to any one of claims 1 to 30, wherein a protease-cleavable polypeptide sequence is recognized by a matrix metalloproteinase.

32. A protease-activated procytokine according to any one of claims 1 to 31, wherein a protease-cleavable polypeptide sequence is recognized by MMP-1.

33. A protease-activating procytokine according to any one of claims 1 to 32, wherein a protease-cleavable polypeptide sequence is recognized by MMP-2.

34. A protease-activating procytokine according to any one of claims 1 to 33, wherein a protease-cleavable polypeptide sequence is recognized by MMP-3.

35. A protease-activated procytokine according to any one of claims 1 to 34, wherein a protease-cleavable polypeptide sequence is recognized by MMP-7.

36. A protease-activated procytokine according to any one of claims 1 to 35, wherein a protease-cleavable polypeptide sequence is recognized by MMP-8.

37. A protease-activated procytokine according to any one of claims 1 to 36, wherein a protease-cleavable polypeptide sequence is recognized by MMP-9.

38. A protease-activated procytokine according to any one of claims 1 to 37, wherein a protease-cleavable polypeptide sequence is recognized by MMP-12.

39. A protease-activated procytokine according to any one of claims 1 to 38, wherein a protease-cleavable polypeptide sequence is recognized by MMP-13.

40. A protease-activated procytokine according to any one of claims 1 to 39, wherein a protease-cleavable polypeptide sequence is recognized by MMP-14.

41. A protease-activated procytokine according to any one of claims 1 to 40, wherein the protease-cleavable polypeptide sequence is recognized by one or more MMPs.

42. A protease-activated procytokine according to any one of claims 1 to 41, wherein the protease-cleavable polypeptide sequence is recognized by two, three, four, five, six, or seven copies of MMP-2, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13, and MMP-14.

43. A protease-activated procytokine according to any one of claims 1 to 42, wherein the protease-cleavable polypeptide sequence comprises a variant sequence having one or two mismatches compared to any sequence of SEQ ID NOs. 80 to 94 or any of SEQ ID NOs. 80 to 90.

44. The protease-activated procytokine of claim 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 80 or a variant sequence having one or two mismatches compared thereto.

45. A protease-activated procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 81 or a variant sequence having one or two mismatches compared thereto.

46. A protease-activated procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 82 or a variant sequence having one or two mismatches compared thereto.

47. A protease-activated procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 83 or a variant sequence having one or two mismatches compared thereto.

48. A protease-activated procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 84 or a variant sequence having one or two mismatches compared thereto.

49. A protease-activated procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 85 or a variant sequence having one or two mismatches compared thereto.

50. A protease-activated procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 86 or a variant sequence having one or two mismatches compared thereto.

51. A protease-activated procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 87 or a variant sequence having one or two mismatches compared thereto.

52. A protease-activated procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 88 or a variant sequence having one or two mismatches compared thereto.

53. A protease-activated procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 89 or a variant sequence having one or two mismatches compared thereto.

54. A protease-activated procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 90 or a variant sequence having one or two mismatches compared thereto.

55. A protease-activated procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NOs. 80 to 89 or 90.

56. A protease-activated procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO:

91.

57. A protease-activating procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO:

92.

58. A protease-activated procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO:

93.

59. A protease-activating procytokine according to any one of claims 1 to 43, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO:

94.

60. A protease-activating procytokine according to any one of claims 1 to 59, wherein the targeting sequence includes any sequence of sequence numbers 180 to 662 or a variant having one or two mismatches compared to any sequence of sequence numbers 180 to 662.

61. The protease-activating procytokine of claim 60, wherein the targeting sequence includes any sequence of sequence numbers 180 to 662.

62. A protease-activating procytokine according to any one of claims 1 to 61, wherein the targeting sequence binds to denatured collagen.

63. A protease-activating procytokine according to any one of claims 1 to 61, wherein the targeting sequence binds to collagen.

64. A protease-activating procytokine according to any one of claims 62 to 63, wherein the collagen is collagen I.

65. A protease-activating procytokine according to any one of claims 62 to 63, wherein the collagen is collagen II.

66. A protease-activated procytokine according to any one of claims 62 to 63, wherein the collagen is collagen III.

67. A protease-activated procytokine according to any one of claims 62 to 63, wherein the collagen is collagen IV.

68. A protease-activating procytokine according to any one of claims 1 to 61, wherein the targeting sequence binds to an integrin.

69. The protease-activating procytokine of claim 68, wherein the integrin is one or more of α1β1 integrin, α2β1 integrin, α3β1 integrin, α4β1 integrin, α5β1 integrin, α6β1 integrin, α7β1 integrin, α9β1 integrin, α4β7 integrin, αvβ3 integrin, αvβ5 integrin, αIIbβ3 integrin, αIIIbβ3 integrin, αMβ2 integrin, or αIIbβ3 integrin.

70. A protease-activating procytokine according to any one of claims 1 to 61, wherein the targeting sequence binds to von Willebrand factor.

71. A protease-activating procytokine according to any one of claims 1 to 61, wherein the targeting sequence binds to IgB.

72. A protease-activating procytokine according to any one of claims 1 to 61, wherein the targeting sequence binds to heparin.

73. The protease-activating procytokine of claim 72, wherein the targeting sequence is bound to heparin and syndecane, heparan sulfate proteoglycan, or integrin, and optionally the integrin is one or more of α1β1 integrin, α2β1 integrin, α3β1 integrin, α4β1 integrin, α5β1 integrin, α6β1 integrin, α7β1 integrin, α9β1 integrin, α4β7 integrin, αvβ3 integrin, αvβ5 integrin, αIIbβ3 integrin, αIIIbβ3 integrin, αMβ2 integrin, or αIIbβ3 integrin.

74. A protease-activating procytokine according to any one of claims 72 to 73, wherein syndecane is one or more of syndecane-1, syndecane-4, and syndecane-2(w).

75. A protease-activating procytokine according to any one of claims 1 to 61, wherein the targeting sequence binds to a heparan sulfate proteoglycan.

76. A protease-activating procytokine according to any one of claims 1 to 61, wherein the targeting sequence binds to a sulfated glycoprotein.

77. A protease-activating procytokine according to any one of claims 1 to 61, wherein the targeting sequence binds to hyaluronic acid.

78. A protease-activating procytokine according to any one of claims 1 to 61, wherein the targeting sequence binds to fibronectin.

79. A protease-activating procytokine according to any one of claims 1 to 61, wherein the targeting sequence binds to a cadherin.

80. A protease-activated procytokine according to any one of claims 1 to 79, wherein the targeting sequence is designed to bind to its target in a pH-sensitive manner.

81. The protease-activated procytokine of claim 80, wherein the targeting sequence has a higher affinity for its target at a pH lower than normal physiological pH, and optionally the pH lower than normal physiological pH is less than 7 or less than 6.

82. The protease-activated procytokine according to claim 81, wherein the targeting sequence has a higher affinity for its target at pH levels in the range of 5 to 7, for example, 5 to 5.5, 5.5 to 6, 6 to 6.5, or 6.5 to 7, than the normal physiological pH.

83. A protease-activating procytokine according to any one of claims 1 to 82, comprising one or more histidines in a targeting sequence, for example, one, two, three, four, five, six, seven, eight, nine, or ten histidines.

84. A protease-activating procytokine according to any one of claims 1 to 83, wherein the targeting sequence includes a variant having one or two mismatches compared to any of the sequences of sequence numbers 641 to 662 or any of the sequences of sequence numbers 641 to 662.

85. The protease-activating procytokine according to claim 84, wherein the targeting sequence includes any sequence of sequence numbers 641 to 662.

86. A protease-activating procytokine according to any one of claims 80 to 86, wherein the targeting sequence is designed to bind to an extracellular matrix component, IgB (CD79b), integrin, cadherin, heparan sulfate proteoglycan, syndecan, or fibronectin with pH sensitivity.

87. The protease-activating procytokine according to claim 86, wherein the extracellular matrix component is hyaluronic acid, heparin, heparan sulfate, or sulfated glycoprotein.

88. The protease-activated procytokine of claim 86, wherein the targeting sequence is designed to bind to fibronectin in a pH-sensitive manner.

89. A protease-activating procytokine according to any one of claims 1 to 88, wherein the cytokine polypeptide sequence is an interleukin polypeptide sequence.

90. A protease-activating procytokine according to any one of claims 1 to 89, wherein the cytokine polypeptide sequence can bind to a receptor containing CD132.

91. A protease-activating procytokine according to any one of claims 1 to 90, wherein the cytokine polypeptide sequence can bind to a receptor containing CD122.

92. A protease-activating procytokine according to any one of claims 1 to 91, wherein the cytokine polypeptide sequence can bind to a receptor containing CD25.

93. A protease-activating procytokine according to any one of claims 1 to 92, wherein the cytokine polypeptide sequence is an IL-2 polypeptide sequence.

94. The protease-activating procytokine of claim 93, wherein the IL-2 polypeptide sequence is at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identical to any sequence of SEQ ID NOs: 1 to 4.

95. The protease-activating procytokine according to claim 94, wherein the IL-2 polypeptide sequence comprises any sequence of SEQ ID NOs: 1 to 4.

96. A protease-activating procytokine according to any one of claims 93 to 95, wherein the IL-2 polypeptide sequence is a human IL-2 polypeptide sequence.

97. The protease-activating procytokine of claim 96, wherein the IL-2 polypeptide sequence comprises the sequence of SEQ ID NO:

1.

98. A protease-activating procytokine according to any one of claims 93 to 95, wherein the IL-2 polypeptide sequence comprises the sequence of SEQ ID NO:

2.

99. A protease-activating procytokine according to any one of claims 93 to 98, wherein the inhibitory polypeptide sequence includes the IL-2 binding domain of the IL-2 receptor (IL-2R).

100. The protease-activating procytokine of claim 99, wherein the inhibitory polypeptide sequence comprises an amino acid sequence having at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with any of the sequences of SEQ ID NOs. 10 to 19.

101. The protease-activating procytokine of claim 100, wherein IL-2R is human IL-2R.

102. A protease-activating procytokine according to any one of claims 93 to 98, wherein the inhibitory polypeptide sequence comprises an IL-2-binding immunoglobulin domain.

103. A protease-activating procytokine according to any one of claims 93 to 98, wherein the IL-2 binding immunoglobulin domain is a human IL-2 binding immunoglobulin domain.

104. The protease-activating procytokine of claim 103, wherein the IL-2 binding immunoglobulin domain comprises a VL region containing hypervariable regions (HVRs) HVR-1, HVR-2, and HVR-3 having sequences of SEQ ID NOs. 33, 34, and 35, respectively, and a VH region containing HVR-1, HVR-2, and HVR-3 having sequences of SEQ ID NOs. 36, 37, and 38, respectively.

105. A protease-activating procytokine according to any one of claims 102 to 104, wherein the IL-2-binding immunoglobulin domain comprises a VL region containing an amino acid sequence having at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent or 99 percent identity with the sequence of SEQ ID NO: 32, and a VH region containing an amino acid sequence having at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent or 99 percent identity with the sequence of SEQ ID NO:

33.

106. The protease-activated procytokine of claim 105, wherein the IL-2 binding immunoglobulin domain comprises a VL region containing the sequence of SEQ ID NO: 32 and a VH region containing the sequence of SEQ ID NO:

33.

107. A protease-activated procytokine according to any one of claims 102 to 104, wherein the IL-2-binding immunoglobulin domain is scFv.

108. The protease-activating procytokine of claim 107, wherein the IL-2-binding immunoglobulin domain comprises an amino acid sequence having at least 80 percent, 85 percent, 90 percent, 95 percent, 97 percent, 98 percent, or 99 percent identity with the sequence of SEQ ID NO: 30 or 31.

109. The protease-activating procytokine of claim 108, wherein the IL-2-binding immunoglobulin domain comprises the sequence of SEQ ID NO: 30 or 31.

110. The protease-activating procytokine according to claim 1, comprising any sequence of sequence numbers 803 to 852.

111. A pharmaceutical composition comprising any protease-activated procytokine according to claims 1 to 110.

112. A protease-activated procytokine or pharmaceutical composition according to any one of claims 1 to 111, for use in therapeutic purposes.

113. A protease-activated procytokine or pharmaceutical composition according to any one of claims 1 to 112, for use in the treatment of cancer.

114. A method for treating cancer, comprising administering a protease-activated procytokine or pharmaceutical composition according to any of claims 1 to 113 to a subject in need thereof.

115. Use of any protease-activated procytokine or pharmaceutical composition according to claims 1 to 110 in the manufacture of a pharmaceutical for the treatment of cancer.

116. A method, use, or protease-activated procytokine for use according to any one of claims 113 to 115, wherein the cancer is a solid tumor.

117. The method, use or use of protease-activated procytokines according to claim 115, wherein the solid tumor is metastatic and / or unresectable.

118. A method, use, or protease-activated procytokine for use according to any one of claims 113 to 117, wherein the cancer is a PD-L1 expressing cancer.

119. A method, use, or protease-activated procytokine for use according to any of claims 113 to 118, wherein the cancer is melanoma, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, gastric or gastrointestinal cancer, lymphoma, colon or colorectal cancer, endometrial cancer, thyroid cancer, or bladder cancer.

120. A method, use, or protease-activated procytokine for use according to any one of claims 113 to 119, wherein the cancer is a high-frequency microsatellite-instability cancer.

121. A method, use, or protease-activated procytokine for use according to any one of claims 113 to 120, wherein cancer is a mismatch repair deficiency.

122. A nucleic acid encoding a protease-activating procytokine according to any one of claims 1 to 110.

123. An expression vector comprising the nucleic acid of claim 121.

124. A host cell comprising the nucleic acid of claim 121 or the vector of claim 122.

125. A method for producing protease-activated procytokines, comprising culturing the host cells of claim 124 under conditions that produce protease-activated procytokines.

126. The method of claim 125, further comprising isolation of protease-activated procytokines.

127. A method for boosting T regulatory cells and / or reducing inflammatory or autoimmune activity, comprising administering any protease-activated procytokine according to claims 1 to 110 to a target area of ​​interest, for example, a target area of ​​inflammation.

128. A method for treating an inflammatory or autoimmune disease or disorder in a subject, comprising administering a protease-activated procytokine according to any one of claims 1 to 110 to a target area of ​​the subject, for example, an area of ​​inflammation or an area of ​​autoimmune activity in the subject.

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