Masked cytokine polypeptide

Abstract

The present disclosure provides cytokines or functional fragments thereof. In some embodiments, the cytokine or functional fragment thereof is engineered to be partially masked at one or more receptor binding sites of the cytokine or functional fragment thereof by a masking moiety. In some embodiments, the cytokine is engineered to be protease-activatable at a target site (such as in a tumor microenvironment) by including a proteolytically cleavable linker. In some embodiments, the proteolytically cleavable linker connects the cytokine to the masking moiety, connects the cytokine to a half-life extension domain, and / or connects the masking moiety to a half-life extension domain. The masking moiety blocks, occludes, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the activity of the cytokine or its binding to its cognate receptor or protein. When the cleavable linker is proteolytically cleaved at the target site, the cytokine is activated to enable it to bind to its cognate receptor or protein with increased affinity.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 737,803, filed on September 27, 2018; U.S. Provisional Application No. 62 / 888,276, filed on August 16, 2019; and U.S. Provisional Application No. 62 / 891,199, filed on August 23, 2019. The content of the applications listed above is incorporated herein by reference in its entirety for all purposes.

[0003] Incorporation of Sequence Listing by Reference

[0004] This application is being filed together with a Sequence Listing in electronic format. The Sequence Listing is provided as a file named 737762000940SeqList created on September 26, 2019, and having a size of 1,024.111 KB. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. Technical Field

[0005] The present invention relates to masked cytokines and methods for their use and preparation. Background Art

[0006] Cancer is the second leading cause of death in the United States, causing more deaths than the next five leading causes (chronic respiratory diseases, stroke, accidents, Alzheimer's disease, and diabetes). Despite significant progress, especially in targeted therapies, there is still a great deal of work to be done in this area. Immunotherapy and immuno - oncology, a branch of this field, are emerging as viable and exciting treatment options for malignancies. Specifically, it is now recognized that one hallmark of cancer is immune evasion, and a great deal of effort has been made to identify targets and develop therapies against these targets to re - activate the immune system to recognize and treat cancer.

[0007] Cytokine therapy is an effective strategy for stimulating the immune system to induce anti - tumor cytotoxicity. For example, aldesleukin, a recombinant form of interleukin - 2 (IL - 2), has been approved by the FDA for the treatment of metastatic renal cell carcinoma and melanoma. Unfortunately, the half - life of cytokines administered to patients is typically very short, thus requiring frequent dosing. For example, the product label for aldesleukin sold under the trade name Proleukin states that the drug has a half - life of 85 minutes in patients receiving a 5 - minute intravenous (IV) infusion. In addition, administering high doses of cytokines can cause adverse health outcomes, such as vascular leak, through systemic immune activation. These findings suggest a need to develop cytokine therapeutics that effectively target tumors without the side effects associated with systemic immune activation. To address this need, the present disclosure provides masked cytokines, their compositions, and methods of use.

[0008] All references cited herein, including patent applications, patent publications, and scientific literature, are incorporated herein by reference in their entirety as if each individual reference were specifically and individually incorporated by reference. SUMMARY OF THE INVENTION

[0009] The disclosed invention relates to the engineering and use of masked cytokines or functional fragments thereof. In some embodiments, the cytokine or functional fragment thereof is engineered to be partially masked at one or more receptor binding sites of the cytokine or functional fragment thereof by a masking moiety. In some embodiments, the cytokine is engineered to be protease-activated at a target site (such as in the tumor microenvironment) by including a proteolytically cleavable linker. In some embodiments, the proteolytically cleavable linker connects the cytokine to the masking moiety, connects the cytokine to a half-life extension domain, and / or connects the masking moiety to the half-life extension domain. The masking moiety blocks, occludes, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the activity of the cytokine or its binding to its cognate receptor or protein. When the cleavable linker is proteolytically cleaved at the target site, the cytokine is activated, enabling it to bind to its cognate receptor or protein with increased affinity.

[0010] One embodiment includes a masked cytokine comprising a masking moiety; and a cytokine or functional fragment thereof, wherein the masking moiety is connected to the cytokine or functional fragment thereof via a linker. In one aspect, the masked cytokine further comprises a half-life extension domain connected to the masking moiety or the cytokine or functional fragment thereof.

[0011] In one embodiment, the masked cytokine comprises, in the N- to C-terminal or C- to N-terminal direction: a) a masking moiety; b) a first linker; c) a cytokine or functional fragment thereof; and d) a half-life extension domain. In another embodiment, the masked cytokine comprises, in the N- to C-terminal or C- to N-terminal direction: a) a masking moiety; b) a first linker; c) a cytokine or functional fragment thereof; d) a second linker; and e) a half-life extension domain. In another embodiment, the masked cytokine comprises, in the N- to C-terminal or C- to N-terminal direction: a) a cytokine or functional fragment thereof; b) a first linker; c) a masking moiety; and d) a half-life extension domain.

[0012] In another embodiment, the masked cytokine comprises, in the N- to C-terminal or C- to N-terminal direction: a) a cytokine or functional fragment thereof; b) a first linker; c) a masking moiety; d) a second linker; and e) a half-life extension domain.

[0013] In one embodiment, the cytokine or its functional fragment is an IL-2 polypeptide or its functional fragment or an IL-15 polypeptide or its functional fragment.

[0014] In one embodiment, the masked cytokine introduces one or more amino acid substitutions into the amino acid sequence of an IL-2 or IL-15 polypeptide or its functional fragment. In one embodiment, the amino acid substitution reduces the affinity of the IL-2 polypeptide or its functional fragment for CD25 (IL-2Rα).

[0015] In one embodiment, the IL-2 polypeptide or its functional fragment comprises an amino acid sequence generated by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or its functional fragment that increase the affinity of the IL-2 polypeptide or its functional fragment for IL-2Rβ or IL-2Rγ.

[0016] In one embodiment, the half-life extension domain is an antibody or its fragment, and in some embodiments, one or more amino acid substitutions alter the effector function.

[0017] In one embodiment, the antibody or its fragment is the fragment crystallizable domain (Fc domain) or its fragment.

[0018] In one embodiment, the half-life extension domain is a polyamino acid sequence, such as a PAS polypeptide or an XTEN polypeptide.

[0019] In one embodiment, the masked cytokine comprises a second masking moiety, wherein the second masking moiety is linked to the cytokine or its functional fragment via a second linker.

[0020] In one embodiment, the masked cytokine comprises, in the N- to C-terminal or C- to N-terminal direction: a) a first masking moiety; b) a first linker; c) a cytokine or its functional fragment; d) a second linker; e) a second masking moiety; and f) a half-life extension domain.

[0021] In one embodiment, the masked cytokine comprises, in the N- to C-terminal or C- to N-terminal direction: a) a first masking moiety; b) a first linker; c) a cytokine or its functional fragment; d) a second linker; e) a second masking moiety; f) a third linker; and g) a half-life extension domain.

[0022] In one embodiment, the masked cytokine comprises, in the N- to C-terminal or C- to N-terminal direction: a) a second masking moiety; b) a second linker; c) a cytokine or its functional fragment; d) a first linker; e) a first masking moiety; and f) a half-life extension domain.

[0023] In one embodiment, the masked cytokine comprises, in the N- to C-terminal or C- to N-terminal direction: a) a second masking moiety; b) a second linker; c) a cytokine or a functional fragment thereof; d) a first linker; e) a first masking moiety; f) a third linker; and g) a half-life extension domain.

[0024] In one embodiment, the half-life extension domain is an albumin polypeptide or a functional fragment thereof.

[0025] In one embodiment, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of: ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, CTR1, CTSA, CTSW, CTSB, CTSC, CTSD, ESP1, CTSG, CTSH, GZMA, GZMB, GZMH, CTSK, GZMM, CTSL, CTSS, CTSV, CTSZ, HTRA4, KLK10, KLK11, KLK13, KLK14, KLK2, KLK4, DPP4, KLK6, KLK7, KLKB1, ECE1, ECE2, ECEL1, MASP2, MEP1A, MEP1B, ELANE, FAP, GZMA, MMP11, GZMK, HGFAC, HPN, HTRA1, MMP11, MMP16, MMP17, MMP19, HTRA2, MMP20, MMP21, HTRA3, HTRA4, KEL, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, KLK5, MMP3, MMP7, MMP8, MMP9, LGMN, LNPEP, MASP1, PAPPA, PAPPA2, PCSK1, NAPSA, PCSK5, PCSK6, MME, MMP1, MMP10, PLAT, PLAU, PLG, PRSS1, PRSS12, PRSS2, PRSS21, PRSS3, PRSS33, PRSS4, PRSS55, PRSS57, MMP12, PRSS8, PRSS9, PRTN3, MMP13, MMP14, ST14,TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1, and TPSG1.

[0026] In one embodiment, the half-life extending domain is conjugated to an agent such as an inhibitor of tubulin polymerization, a DNA damaging agent or a DNA synthesis inhibitor, a maytansinoid, an auristatin, a pyrrolobenzodiazepine (PBD) dimer, calicheamicin, duocarmycin, an indolinobenzodiazepine dimer, or an exatecan derivative Dxd.

[0027] In one embodiment, the half-life extending domain is conjugated to an immunostimulatory agent such as a stimulator of interferon genes (STING) agonist, such as a cyclic dinucleotide (CDN), such as cGAMP, c-di-AMP, c-di-GMP, cAIMP, c-di-IMP, or 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, or a toll-like receptor (TLR) agonist, such as TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10.

[0028] In one embodiment, the masked cytokine comprises a first half-life extending domain and a second half-life extending domain, wherein the masking moiety is linked to the first half-life extending domain, and wherein the cytokine or a functional fragment thereof is linked to the second half-life extending domain.

[0029] In one embodiment, the first half-life extending domain and the second half-life extending domain contain modifications that promote the association of the first and second half-life extending domains.

[0030] One embodiment includes a nucleic acid encoding a masked cytokine as described herein. Another embodiment includes a vector comprising the nucleic acid. In another embodiment, a host cell comprises the nucleic acid. Another embodiment includes one or more nucleic acids encoding a masked cytokine as described herein. Another embodiment includes a vector comprising the one or more nucleic acids. Another embodiment includes one or more vectors comprising the one or more nucleic acids.

[0031] In another embodiment, a method for preparing a masked cytokine is provided, comprising culturing a host cell under conditions that produce a masked cytokine. In one embodiment, the masked cytokine produced by the host cell is recovered. One embodiment includes a masked cytokine prepared by the disclosed method.

[0032] In one embodiment, a composition comprising the disclosed masked cytokine is provided. In another embodiment, the composition further comprises an anti-inflammatory agent or an anti-cancer agent, such as a PD-1 inhibitor, an EGFR inhibitor, a HER2 inhibitor, a VEGFR inhibitor, a CTLA-4 inhibitor, a BTLA inhibitor, a B7H4 inhibitor, a B7H3 inhibitor, a CSFIR inhibitor, an HVEM inhibitor, a CD27 inhibitor, a KIR inhibitor, an NKG2A inhibitor, an NKG2D agonist, a TWEAK inhibitor, an ALK inhibitor, a CD52-targeting antibody, a CCR4-targeting antibody, a PD-L1 inhibitor, a KIT inhibitor, a PDGFR inhibitor, a BAFF inhibitor, an HDAC inhibitor, a VEGF ligand inhibitor, a CD19-targeting molecule, a FOLR1-targeting molecule, a DLL3-targeting molecule, a DKK1-targeting molecule, a MUC1-targeting molecule, a MUC16-targeting molecule, a PSMA-targeting molecule, a MSLN-targeting molecule, a NY-ES0-1-targeting molecule, a B7H3-targeting molecule, a B7H4-targeting molecule, a BCMA-targeting molecule, a CD29-targeting molecule, a CD151-targeting molecule, a CD123-targeting molecule, a CD33-targeting molecule, a CD37-targeting molecule, a CDH19-targeting molecule, a CEA-targeting molecule, Claudin 18.2 targeting molecules, CLEC12A targeting molecules, EGFRVIII targeting molecules, EPCAM targeting molecules, EPHA2 targeting molecules, FCRH5 targeting molecules, FLT3 targeting molecules, GD2 targeting molecules, glypican-3 targeting molecules, gpA33 targeting molecules, GPRC5D targeting molecules, IL-23R targeting molecules, IL-1RAP targeting molecules, MCSP targeting molecules, RON targeting molecules, ROR1 targeting molecules, STEAP2 targeting molecules, TfR targeting molecules, CD166 targeting molecules, TPBG targeting molecules, TROP2 targeting molecules, proteasome inhibitors, ABL inhibitors, CD30 inhibitors, FLT3 inhibitors, MET inhibitors, RET inhibitors, IL-1β inhibitors, MEK inhibitors, ROS1 inhibitors, BRAF inhibitors, CD38 inhibitors, RANKL inhibitors, B4GALNT1 inhibitors, SLAMF7 inhibitors, IDH2 inhibitors, mTOR inhibitors, CD20 targeting antibodies, BTK inhibitors, PI3K inhibitors, FLT3 inhibitors, PARP inhibitors, CDK4 inhibitors, CDK6 inhibitors, FGFR inhibitors, RAF inhibitors, JAK1 inhibitors, JAK2 inhibitors, JAK3 inhibitors, IL-6 inhibitors, IL-17 inhibitors, Smoothened inhibitors, IL-6R inhibitors, BCL2 inhibitors, PTCH inhibitors, PIGF inhibitors, TGFB inhibitors, CD28 agonists, CD3 agonists, CD40 agonists, GITR agonists, OX40 agonists, VISTA agonists, CD137 agonists, LAG3 inhibitors, TIM3 inhibitors, TIGIT inhibitors or IL-2R inhibitors.

[0033] In one embodiment, the anti-inflammatory agent is a cyclooxygenase (COX) inhibitor, such as a COX-1 and / or COX-2 inhibitor, such as SC-560, FR122047, P6, mebenamic acid, TFAP, flurbiprofen, ketoprofen, celecoxib, rofecoxib, meloxicam, piroxicam, deracoxib, parecoxib, valdecoxib, etoricoxib, chromene derivatives, chroman derivatives, N-(2-cyclohexyloxynitrophenyl)methanesulfonamide, parecoxib, lumiracoxib, RS 57067, T-614, BMS-347070, JTE-522, S-2474, SVT-2016, CT-3, ABT-963, SC-58125, nimesulide, flusulide, NS-398, L-745337, RWJ-63556, L-784512, darbepoetin, CS-502, LAS-34475, LAS-34555, S-33516, diclofenac, mefenamic acid, SD-8381, ibuprofen, naproxen, ketorolac, indomethacin, aspirin, naproxen, tolmetin, piroxicam or meclofenamate.

[0034] In one embodiment, the anti-inflammatory agent is an NF-κB inhibitor, such as an IKK complex inhibitor, an IκB degradation inhibitor, an NF-κB nuclear translocation inhibitor, a p65 acetylation inhibitor, an NF-κB DNA binding inhibitor, an NF-κB transactivation inhibitor or a p53 induction inhibitor.

[0035] In one embodiment, the NF-κB inhibitor is TPCA-1, NF-κB activation inhibitor VI (BOT-64), BMS-345541, amlexanox, SC-514 (GK-01140), IMD-0354, IKK-16, BAY-11-7082, MG-115, MG-132, lactacystin, epoxomicin, parthenolide, carfilzomib, MLN-4924 (pevonedistat), JSH-23 rolipram, gallic acid, anacardic acid, GYY-4137, p-XSC, CV-3988, prostaglandin E2 (PGE2), LY-294002, wortmannin, mesalazine, quinacrine or flupirtine.

[0036] One embodiment includes a pharmaceutical composition comprising the disclosed masked cytokine and a pharmaceutically acceptable carrier.

[0037] Another embodiment includes a kit comprising the disclosed masked cytokine.

[0038] Another embodiment includes a method of treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of the disclosed masked cytokine composition for treating a disease or disorder such as a neoplastic disease, e.g., cancer. In one embodiment, the cancer is leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, bone cancer (e.g., osteosarcoma, chondrosarcoma, Ewing's sarcoma), bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer, testicular cancer, adrenal cancer, adenoid cystic carcinoma, anal cancer, brain cancer, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, oral cancer, thyroid cancer, retinoblastoma, parathyroid cancer, pituitary cancer, cholangiocarcinoma or uterine cancer. In some embodiments, the cancer is selected from the group consisting of: lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g., triple negative, ER positive, ER negative, chemotherapy resistant, Herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobular carcinoma, primary, metastatic), ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, squamous cell lung cancer, adenocarcinoma, large cell lung cancer, small cell lung cancer, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., of the head, neck or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma or multiple myeloma. Additional examples include thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, gastric cancer, uterine cancer or medulloblastoma, Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, Waldenström's macroglobulinemia, primary brain tumor, cancer, malignant pancreatic islet tumor, malignant carcinoid, urinary bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical cancer, endocrine or exocrine pancreatic tumors, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, Paget's disease of the nipple, phyllodes tumor, lobular carcinoma, ductal carcinoma, pancreatic stellate cell carcinoma, hepatic stellate cell carcinoma or prostate cancer.

[0039] Another embodiment includes a method of treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of the disclosed masked cytokine composition, wherein the inflammatory or autoimmune disease is selected from the group consisting of: atherosclerosis, obesity, inflammatory bowel disease (IBD), rheumatoid arthritis, allergic encephalitis, psoriasis, atopic dermatitis, osteoporosis, peritonitis, hepatitis, lupus, celiac disease, Sjogren's syndrome, polymyalgia rheumatica, multiple sclerosis (MS), ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis and temporal arteritis, graft-versus-host disease (GVHD), asthma, COPD, paraneoplastic autoimmune disease, cartilage inflammation, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, Crohn's disease, ulcerative colitis, celiac disease, sinusitis, sinusitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barre disease, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, transplant rejection, kidney injury, hepatitis C-induced vasculitis or spontaneous pregnancy loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1A and Figure 1BShows the structure of exemplary embodiments of a masked cytokine, the masked cytokine comprising a masking moiety, a cytokine or a functional fragment thereof ("cytokine"), a half-life extension domain, and a first linker, the first linker comprising a first cleavable peptide ("1CP"), a first N-terminal spacer domain ("1NSD"), and a first C-terminal spacer domain ("1CSD"). These exemplary embodiments also include a second linker, the second linker comprising a second cleavable peptide ("2CP"), a second N-terminal spacer domain ("2NSD"), and a second C-terminal spacer domain ("2CSD"). As shown by the arrows, although the exemplary embodiments show the masking moiety connected to the first linker and the cytokine or a functional fragment thereof connected to the first linker and the second linker, the masking moiety and the cytokine or a functional fragment thereof can be interchanged such that the cytokine or a functional fragment thereof is connected to the first linker and the masking moiety is connected to the first linker and the second linker. Figure 1A Shows the structure of an exemplary embodiment of a masked cytokine as a monomer. Figure 1B Shows the structure of an exemplary embodiment of a masked cytokine as a homodimer formed by disulfide bonds.

[0041] Figure 2A and Figure 2B Shows the structure of exemplary embodiments of a masked cytokine, the masked cytokine comprising a first masking moiety ("Mask 1"), a cytokine or a functional fragment thereof ("cytokine"), a second masking moiety ("Mask 2"), a half-life extension domain, a first linker, and a second linker, the first linker comprising a first cleavable peptide ("1CP"), a first N-terminal spacer domain ("1NSD"), and a first C-terminal spacer domain ("1CSD"), the second linker comprising a second cleavable peptide ("2CP"), a second N-terminal spacer domain ("2NSD"), and a second C-terminal spacer domain ("2CSD"). These exemplary embodiments also include a third linker, the third linker comprising a third cleavable peptide ("3CP"), a third N-terminal spacer domain ("3NSD"), and a third C-terminal spacer domain ("3CSD"). Figure 2A Shows the structure of an exemplary embodiment of a masked cytokine as a monomer. Figure 2B Shows the structure of an exemplary embodiment of a masked cytokine as a homodimer formed by disulfide bonds.

[0042] Figure 3A Shows the structure of exemplary embodiments of a masked cytokine comprising a masking moiety, a cytokine or a functional fragment thereof ("cytokine"), a first half-life extension domain, and a second half-life extension domain. Figure 3AThe exemplary embodiments shown also include a first linker and a second linker, the first linker including a first cleavable peptide (“1CP”), a first N-terminal spacer domain (“1NSD”), and a first C-terminal spacer domain (“1CSD”), and the second linker including a second cleavable peptide (“2CP”), a second N-terminal spacer domain (“2NSD”), and a second C-terminal spacer domain (“2CSD”). Exemplary first and second half-life extension domains include “knobs into holes” modifications that promote the association of the first half-life extension domain with the second half-life extension domain, as indicated by the “holes” in the first half-life extension domain and the “knobs” in the second half-life extension domain. It is also shown that the first half-life extension domain and the second half-life extension domain are at least partially associated due to the formation of disulfide bonds. It is to be understood that while the “hole” is depicted as part of the first half-life extension domain (connected to the masking portion) and the “knob” is depicted as part of the second half-life extension domain (connected to the cytokine), the “hole” and the “knob” may alternatively be included in the second half-life extension domain and the first half-life extension domain, respectively, such that the “hole” is part of the second half-life extension domain (connected to the cytokine) and the “knob” is part of the first half-life extension domain (connected to the masking portion). Figure 3B Shows the structure of an exemplary embodiment of a masked cytokine comprising a masking portion, a cytokine or a functional fragment thereof (“cytokine”), a first half-life extension domain, a second half-life extension domain, and a third linker, the third linker including a third N-terminal spacer domain (“3NSD”) and a third C-terminal spacer domain (“3CSD”). The third linker connects the first half-life extension domain to the second half-life extension domain. Figure 3B The exemplary embodiments shown also include a first linker and a second linker, the first linker including a first cleavable peptide (“1CP”), a first N-terminal spacer domain (“1NSD”), and a first C-terminal spacer domain (“1CSD”), and the second linker including a second cleavable peptide (“2CP”), a second N-terminal spacer domain (“2NSD”), and a second C-terminal spacer domain (“2CSD”).

[0043] Figure 4 Shown formed by disulfide bonds Figure 3A The dimer of the exemplary masked cytokine shown in

[0044] Figure 5AShows the structure of an exemplary embodiment of a masked cytokine comprising a first masking portion (“Mask 1”), a first half-life extension domain, a second masking portion (“Mask 2”), a cytokine or a functional fragment thereof (“cytokine”), and a second half-life extension domain. Figure 5A The exemplary embodiment shown in Figure 5A also includes a first linker, a second linker, and a third linker. The first linker comprises a first cleavable peptide (“1CP”), a first N-terminal spacer domain (“1NSD”), and a first C-terminal spacer domain (“1CSD”). The second linker comprises a second cleavable peptide (“2CP”), a second N-terminal spacer domain (“2NSD”), and a second C-terminal spacer domain (“2CSD”). The third linker comprises a third cleavable peptide (“3CP”), a third N-terminal spacer domain (“3NSD”), and a third C-terminal spacer domain (“3CSD”). It is to be understood that although the “hole” is depicted as part of the first half-life extension domain (connected to the masking portion) and the “knob” is depicted as part of the second half-life extension domain (connected to the cytokine), the “hole” and “knob” can alternatively be included in the second half-life extension domain and the first half-life extension domain, respectively, such that the “hole” is part of the second half-life extension domain (connected to the cytokine) and the “knob” is part of the first half-life extension domain (connected to the masking portion). Figure 5B Shows the structure of an exemplary embodiment of a masked cytokine comprising a first masking portion (“Mask 1”), a first half-life extension domain, a second masking portion (“Mask 2”), a cytokine or a functional fragment thereof (“cytokine”), a second half-life extension domain, and a fourth linker. The fourth linker connects the first half-life extension domain to the second half-life extension domain and comprises a fourth N-terminal spacer domain (“4NSD”) and a fourth C-terminal spacer domain (“4CSD”). Figure 5B The exemplary embodiment shown in Figure 5B also includes a first linker, a second linker, and a third linker. The first linker comprises a first cleavable peptide (“1CP”), a first N-terminal spacer domain (“1NSD”), and a first C-terminal spacer domain (“1CSD”). The second linker comprises a second cleavable peptide (“2CP”), a second N-terminal spacer domain (“2NSD”), and a second C-terminal spacer domain (“2CSD”). The third linker comprises a third cleavable peptide (“3CP”), a third N-terminal spacer domain (“3NSD”), and a third C-terminal spacer domain (“3CSD”). As Figure 5A and 5BAs shown by the arrows in [description], although the exemplary embodiments show that the second masking portion is connected to the third linker, and the cytokine or its functional fragment is connected to the third linker and the second linker, the first masking portion and the cytokine or its functional fragment can be interchanged such that the cytokine or its functional fragment is connected to the third linker, and the first masking portion is connected to the third linker and the second linker.

[0045] Figure 6 Showing formed by disulfide bonds Figure 5A The dimer of the exemplary masked cytokine shown in [description].

[0046] Figures 7A - 7E Showing exemplary embodiments of the masked cytokine before (left) and after (right) protease cleavage in the tumor microenvironment. Figures 7A - 7D Showing an exemplary embodiment of the masked IL-2 cytokine, and Figure 7E Showing an exemplary embodiment of the masked IL-15 cytokine. The masking portion is released by protease cleavage (e.g., IL-2Rβ, as shown in Figure 7A , 7B and 7D), or IL-2 is released ( Figure 7C ), or IL-15 is released ( Figure 7E ).

[0047] Figure 8 Showing SDS-PAGE analysis of flow-through (FT) samples (i.e., proteins not bound to the protein A column) and elution (E) samples (i.e., proteins bound to the protein A column and eluted therefrom) after the production and purification of exemplary constructs (AK304, AK305, AK307, AK308, AK309, AK310, AK311, AK312, AK313, AK314, and AK315).

[0048] Figures 9A - 9D Showing the results of SPR analysis testing the binding of exemplary masked IL-2 polypeptide constructs (AK215 and AK216) or rhIL2 control to CD25-Fc. Figure 9A Showing the interaction between AK215 and CD25-Fc, Figure 9B Showing the interaction between AK216 and CD25-Fc, and Figure 9C Showing the interaction between recombinant human IL2 (rhIL2) control and CD25-Fc. Figure 9D Providing a table summarizing the data obtained for the association constant (ka), dissociation constant (kd), equilibrium dissociation constant (KD), and Chi 2 values and U values for each interaction.

[0049] Figures 10A - 10DResults of SPR analysis showing binding of the test exemplary masked IL-2 polypeptide constructs (AK216 and AK218) or rhIL2 control to CD122-Fc. Figure 10A Showing the interaction between AK216 and CD122-Fc, Figure 10B showing the interaction between AK218 and CD122-Fc, and Figure 10C showing the interaction between recombinant human IL2 (rhIL2) control and CD122-Fc. Figure 10D Providing a table summarizing the data obtained for the association constant (ka), dissociation constant (kd), equilibrium dissociation constant (KD), and Chi 2 value and U value for each interaction.

[0050] Figure 11A Showing exemplary embodiments of the masked cytokine before (left) and after (right) protease cleavage in the tumor microenvironment. Figure 11B Showing SDS-PAGE analysis of an exemplary masked IL-2 polypeptide construct incubated in the absence (left lane) or presence (right lane) of MMP10 protease, which shows the release of IL-2 from the Fc moiety.

[0051] Figures 12A - 12D Showing STAT5 activation (%) in PBMCs treated with constructs AK032, AK035, AK041 or rhIL-2 as a control. Levels of STAT5 activation (%) are shown for NK cells, CD8+ T cells, effector T cells (Teff) and regulatory T cells (Treg), as determined after incubation with rhIL-2 ( Figure 12A ), AK032 ( Figure 12B ), AK035 ( Figure 12C ), or AK041 ( Figure 12D ).

[0052] Figures 13A - 13C Showing STAT5 activation (%) in PBMCs treated with construct AK081 or AK032. The AK081 construct with and without prior exposure to MMP10 was tested. An isotype control and an IL-2-free negative control were also tested. Levels of STAT5 activation (%) are shown for NK cells ( Figure 13A ), CD8+ T cells ( Figure 13C ), and CD4+ T cells ( Figure 13B ).

[0053] Figures 14A - 14DResults of STAT5 activation studies in PBMCs using constructs AK081 and AK111 and controls including rhIL-2 and anti-RSV antibody are shown. An untreated control was also tested. EC50 (pM) for rhIL-2, AK081, and AK111 treatment are also shown. For CD4+FoxP3+CD25+ cells ( Figure 14A ), CD8+ cells ( Figure 14B ), and CD4+FoxP3-CD25- cells ( Figure 14C ), STAT5 activation (%) is shown. Figure 14D EC50 (pM) and fold change data for AK081, AK111 constructs, and rhIL-2 control are provided.

[0054] Figures 15A - 15D Results of STAT5 activation studies in PBMCs using constructs AK167 and AK168 and controls including rhIL-2 and anti-RSV antibody are shown. An untreated control was also tested. EC50 (pM) for rhIL-2, AK167, and AK168 treatment are also shown. For CD4+FoxP3+CD25+ cells ( Figure 15A ), CD8+ cells ( Figure 15B ), and CD4+FoxP3-CD25- cells ( Figure 15C ), STAT5 activation (%) is shown. Figure 15D EC50 (pM) and fold change data for AK167 and AK168 constructs and rhIL-2 control are provided.

[0055] Figures 16A - 16D STAT5 activation (%) in PBMCs treated with constructs AK165 or AK166 or isotype control or IL-2-Fc control with (+MMP10) or without prior exposure to MMP10 protease is shown. The legend as shown in Figure 16A also applies to Figure 16B , and the legend as shown in Figure 16C also applies to Figure 16D . For CD4+FoxP3+ T regulatory cells ( Figure 16A ), CD4+FoxP3- T helper cells ( Figure 16B ), CD8+ cytotoxic T cells ( Figure 16C ), and CD56+ NK cells ( Figure 16D ), STAT5 activation (%) is shown.

[0056] Figures 17A - 17CShown is STAT5 activation (%) in PBMCs treated with construct AK109 or AK110 or isotype control or IL-2-Fc control with (+MMP10) or without prior exposure to MMP10 protease. As Figure 17B the legend shown in Figure 17A also applies to Figure 17A NK cells ( Figure 17B ), CD8 cells ( Figure 17C ), and CD4 cells (

[0057] Figures 18A - 18D ). Results of STAT5 activation studies in PBMCs using constructs AK211, AK235, AK253, AK306, AK310, AK314, and AK316 and rhIL-2 control are shown. For CD3+CD4+FoxP3+ cells ( Figure 18A ), CD3+CD4+FoxP3- cells ( Figure 18B ), and CD3+CD8+ cells ( Figure 18C ), STAT5 activation (%) is shown. Figure 18D EC50 data are provided for each test construct and the rhIL-2 control.

[0058] Figures 19A - 19D Results of STAT5 activation studies in PBMCs using protease-activated constructs AK081, AK167, AK216, AK218, AK219, AK220, and AK223 and rhIL-2 control are shown. For CD4+FoxP3+CD25+ regulatory T cells ( Figure 19A ), CD4+FoxP3-CD25- cells ( Figure 19B ), and CD8+ cells ( Figure 19C ), STAT5 activation (%) is shown. Figure 19D EC50 data are provided for each test construct and the rhIL-2 control.

[0059] Figures 20A - 20C Shown is STAT5 activation (%) in PBMCs treated with construct AK081, AK189, AK190, or AK210 or anti-RSV control. As Figure 20A the legend shown in Figure 20B also applies to 20C and Figure 20A regulatory T cells ( Figure 20B ), CD4 helper T cells ( Figure 20C ), and CD8 cells (

[0060] Figures 21A - 21CSTAT5 activation (%) in PBMCs treated with display constructs AK167, AK191, AK192, or AK193 or anti-RSV control. As Figure 21A The legend shown in Figure 21B also applies to 21C . STAT5 activation (%) is shown for regulatory T cells ( Figure 21A ), CD4 helper T cells ( Figure 21B ), and CD8 cells ( Figure 21C ).

[0061] Figure 22A and 22B show the results of reporter bioassays on exemplary masked IL-15 polypeptide constructs AK248 with (+MMP) or without prior exposure to activating protease or rhIL-15 as a control. Figure 22A show the results of reporter bioassays using the HEK-Blue IL2 reporter cell line, and Figure 22B show the results of reporter bioassays of IL-15 bioassays using the murine CTLL2 cell line.

[0062] Figures 23A - 23D show the results of pharmacokinetic studies in tumor-bearing mice using constructs AK032, AK081, AK111, AK167, or AK168 or anti-RSV control. Figure 23A Provide a simplified depiction of the structure of each construct tested. Figure 23B Fc levels (μg / mL) in plasma are shown by detecting human IgG, Figure 23C Fc-CD122 levels (μg / mL) in plasma are shown by detecting human CD122, and Figure 23D Fc-IL2 levels (μg / mL) in plasma are shown by detecting human IL-2. Anti-human IG is used as a capture antibody before the detection step.

[0063] Figures 24A - 24D show the results of pharmacokinetic studies in tumor-bearing mice using constructs AK167, AK191, AK197, AK203, AK209, or AK211 or anti-RSV control. Figure 24A Provide a simplified depiction of the structure of each construct tested. Figure 24B Fc levels (μg / mL) in plasma are shown by detecting human IgG, Figure 24C Fc-IL2 levels (μg / mL) in plasma are shown by detecting human IL-2, and Figure 24D Fc-CD122 levels (μg / mL) in plasma are shown by detecting human CD122. Anti-human IG is used as a capture antibody before the detection step.

[0064] Figures 25A - 25L Results of a study showing the in vivo responses testing the percentages of CD4, CD8, NK, and Treg in spleen, blood, and tumor using AK032, AK081, AK111, AK167, or AK168 constructs or anti-RSV IgG control. For spleen tissue, the % CD8 cells of CD3 cells ( Figure 25A ), the % CD4 of CD3 cells ( Figure 25B ), the % NK cells of CD3- cells ( Figure 25C ), and the % FoxP3 of CD4 cells ( Figure 25D ) are shown. For blood, the % CD8 cells of CD3 cells ( Figure 25E ), the % CD4 of CD3 cells ( Figure 25F ), the % NK cells of CD3- cells ( Figure 25G ), and the % FoxP3 of CD4 cells ( Figure 25H ) are shown. For tumor tissue, the % CD8 cells of CD3 cells ( Figure 25I ), the % CD4 of CD3 cells ( Figure 25J ), the % NK cells of CD3- cells ( Figure 25K ), and the % FoxP3 of CD4 cells ( Figure 25L ) are shown.

[0065] Figures 26A - 26L Results of a study showing the in vivo responses testing the percentages of CD4, CD8, NK, and Treg in spleen, blood, and tumor using AK167, AK168, AK191, AK197, AK203, AK209, or AK211 constructs or anti-RSV IgG control. For spleen tissue, the % CD8 cells of CD3 cells ( Figure 26A ), the % CD4 of CD3 cells ( Figure 26B ), the % NK cells of CD3- cells ( Figure 26C ), and the % FoxP3 of CD4 cells ( Figure 26D ) are shown. For blood, the % CD8 cells of CD3 cells ( Figure 26E ), the % CD4 of CD3 cells ( Figure 26F ), the % NK cells of CD3- cells ( Figure 26G ), and the % FoxP3 of CD4 cells ( Figure 26H ) are shown. For tumor tissue, the % CD8 cells of CD3 cells ( Figure 26I ), the % CD4 of CD3 cells ( Figure 26J ), the % NK cells of CD3- cells ( Figure 26K ), and the % FoxP3 of CD4 cells ( Figure 26L ) are shown.

[0066] Figures 27A - 27L Results of a study showing the in vivo responses testing the percentages of CD4, CD8, NK, and Treg in spleen, blood, and tumor using AK235, AK191, AK192, AK193, AK210, AK189, AK190, or AK211 constructs or anti-RSV IgG control. For spleen tissue, the % CD8 cells of CD3 cells ( Figure 27A ), the % CD4 of CD3 cells ( Figure 27B ), the % NK cells of CD3− cells ( Figure 27C ), and the % FoxP3 of CD4 cells ( Figure 27D ) are shown. For blood, the % CD8 cells of CD3 cells ( Figure 27E ), the % CD4 of CD3 cells ( Figure 27F ), the % NK cells of CD3− cells ( Figure 27G ), and the % FoxP3 of CD4 cells ( Figure 27H ) are shown. For tumor tissue, the % CD8 cells of CD3 cells ( Figure 27I ), the % CD4 of CD3 cells ( Figure 27J ), the % NK cells of CD3− cells ( Figure 27K ), and the % FoxP3 of CD4 cells ( Figure 27L ) are shown.

[0067] Figures 28A - 28I Results showing in vivo T cell activation in spleen, blood, and tumor using AK235, AK191, AK192, AK193, AK210, AK189, AK190, or AK211 constructs. In spleen, blood, and tumor, T cell activation was measured as the mean fluorescence intensity (MFI) of CD25 in CD8+ T cells ( Figure 28A ; Figure 28D ; Figure 28G ), CD4+ T cells ( Figure 28B ; Figure 28E ; Figure 28H ), or Foxp3+ cells ( Figure 28C ; Figure 28F ; Figure 28I ). Statistical analysis was performed using one-way ANOVA compared to the non-cleavable AK211 construct.

[0068] Figures 29A - 29D Results of a study testing in vivo cleavage of the exemplary masked IL-2 polypeptide constructs AK168 (cleavable peptide sequence: MPYDLYHP; SEQ ID NO: 96) and AK209 (cleavable peptide sequence: VPLSLY; SEQ ID NO: 135). Figure 29EPharmacokinetic study results showing total plasma IgG concentration (μg / mL) of the total level for AK167, AK168, and AK209 constructs and the level of the non-cleaved form of each construct.

[0069] Figures 30A - 30D Results of in vivo studies evaluating vascular leakage using the exemplary masked IL-2 polypeptide constructs AK111 or AK168 or the unmasked IL-2 polypeptide constructs AK081 or AK167 or an anti-RSV control. Figure 30A Showing the percentage of weight loss (%) and Figure 30B 、 30C and 30D show the weights (grams) of the liver, lung, and spleen, respectively.

[0070] Figure 31A and 31B Results of in vivo studies evaluating vascular leakage, as indicated by measuring the extent of dye leakage into liver and lung tissues after administration of the AK081, AK111, AK167, or AK168 constructs or an anti-RSV control. The extent of dye leakage into the liver ( Figure 31A ) and lung ( Figure 31B ) was measured based on absorbance at 650 nm.

[0071] Figure 32A and 32B Results of in vivo studies evaluating vascular leakage, as indicated by measuring the extent of perivascular monocyte invasion into liver and lung tissues after administration of the AK081, AK111, AK167, or AK168 constructs or an anti-RSV control. The average number of monocytes in the liver ( Figure 32A ) and the average number of monocytes in the lung ( Figure 32B ) are depicted respectively.

[0072] Figure 33A and 33B Results of syngeneic tumor model studies evaluating tumor volume and body weight during treatment with the AK032, AK081, AK111, AK167, or AK168 constructs or an anti-RSV control. Figure 33A Showing data on tumor volume during treatment, and Figure 33B Showing data on the percentage change in body weight (%) during treatment. Detailed Description

[0073] The present disclosure provides cytokines or functional fragments thereof. In some embodiments, the cytokine or functional fragment thereof is engineered to be partially masked at one or more receptor-binding sites of the cytokine or functional fragment thereof by a masking moiety. In some embodiments, the cytokine is engineered to be protease-activatable at a target site (such as in the tumor microenvironment) by including a proteolytically cleavable linker. In some embodiments, the proteolytically cleavable linker connects the cytokine to the masking moiety, connects the cytokine to a half-life extension domain, and / or connects the masking moiety to the half-life extension domain. The masking moiety blocks, occludes, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the activity of the cytokine or its binding to its cognate receptor or protein. When the cleavable linker is proteolytically cleaved at the target site, the cytokine is activated, enabling it to bind to its cognate receptor or protein with increased affinity.

[0074] By using masking moieties, the systemic side effects of the administered cytokine can be reduced by interfering with the cytokine's binding ability. For example, high-dose recombinant IL-2 (aldesleukin) has been approved by the FDA for the treatment of metastatic renal cell carcinoma and melanoma, but has associated severe cardiovascular, hepatic, pulmonary, gastrointestinal, neurological, and hematological side effects. Preclinical studies have shown that, for example, IL-2-induced pulmonary edema is caused by the interaction between IL-2 and the IL-2Rα (CD25) subunit of the IL-2 receptor (IL-2R) on pulmonary endothelial cells, and that IL-2-mediated pulmonary edema can be eliminated by interfering with the ability of IL-2 to bind IL-2Rα. See Krieg et al., (2010) PNAS, 107(26):11906-11911. Thus, in some embodiments where the cytokine or functional fragment thereof is an IL-2 polypeptide, a masking moiety that blocks, sequesters, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the activity of the IL-2 cytokine or its binding to IL-2Rα is used. To further reduce the systemic effects of the masked IL-2 polypeptide, the masked IL-2 polypeptide can further include a masking moiety that blocks, sequesters, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the activity of the IL-2 cytokine or its binding to the IL-2Rβ and / or IL-2Rγ subunits of the IL-2 receptor. Similar strategies are also used for other cytokines by interfering with their ability to bind certain proteins (e.g., receptor subunits) associated with adverse systemic side effects. In addition, by masking a cytokine with a linker that includes a cleavable peptide, the binding ability interfered with by using the masking moiety can be restored by cleaving the cleavable peptide in the tumor microenvironment. Thus, in some embodiments, the masked cytokines provided herein are engineered to precisely target the tumor microenvironment with pharmacological activity by taking advantage of the high local concentration of active proteases that is one of the hallmarks of cancer. This feature of the tumor microenvironment is used to convert a systemically inert molecule into a locally active cytokine. Activation of the cytokine in the tumor microenvironment significantly reduces the systemic toxicity that may be associated with administering the active form of the drug to a subject.

[0075] For all purposes, all publications (including patent documents, scientific papers, and databases) mentioned in this application are hereby incorporated by reference in their entirety to the same extent as if each individual publication were incorporated by reference separately. If the definitions set forth herein are contrary to or otherwise inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated by reference herein, the definitions set forth herein shall control.

[0076] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0077] I. Masked Cytokines

[0078] In some embodiments, the present disclosure provides a masked cytokine comprising (a) a masking moiety; and (b) a cytokine or a functional fragment thereof, wherein the masking moiety is linked to the cytokine or the functional fragment thereof via a first linker. In some embodiments, the masked cytokine further comprises a half-life extending domain linked to the masking moiety or the cytokine or the functional fragment thereof. In some embodiments, the half-life extending domain is linked to the masking moiety or the IL-2 polypeptide or the functional fragment thereof via a second linker.

[0079] In some embodiments, a masked cytokine comprises, in the N- to C-terminal direction: (a) a masking moiety; (b) a first linker; (c) a cytokine or a functional fragment thereof; and (d) a half-life extending domain. In some embodiments, a masked cytokine comprises, in the C- to N-terminal direction: (a) a masking moiety; (b) a first linker; (c) a cytokine or a functional fragment thereof; and (d) a half-life extending domain. In some embodiments, a masked cytokine comprises, in the N- to C-terminal direction: (a) a masking moiety; (b) a first linker; (c) a cytokine or a functional fragment thereof; (d) a second linker; and (e) a half-life extending domain. In some embodiments, a masked cytokine comprises, in the C- to N-terminal direction: (a) a masking moiety; (b) a first linker; (c) a cytokine or a functional fragment thereof; (d) a second linker; and (e) a half-life extending domain. In some embodiments, a masked cytokine comprises, in the N- to C-terminal direction: (a) a cytokine or a functional fragment thereof; (b) a first linker; (c) a masking moiety; and (d) a half-life extending domain. In some embodiments, a masked cytokine comprises, in the C- to N-terminal direction: (a) a cytokine or a functional fragment thereof; (b) a first linker; (c) a masking moiety; and (d) a half-life extending domain. In some embodiments, a masked cytokine comprises, in the N- to C-terminal direction: (a) a cytokine or a functional fragment thereof; (b) a first linker; (c) a masking moiety; (d) a second linker; and (e) a half-life extending domain. In some embodiments, a masked cytokine comprises, in the C- to N-terminal direction: (a) a cytokine or a functional fragment thereof; (b) a first linker; (c) a masking moiety; (d) a second linker; and (e) a half-life extending domain. In some embodiments, a masked cytokine comprises, in the N- to C-terminal direction: (a) a half-life extending domain; (b) a first linker; (c) a masking moiety; (d) a second linker; and (e) a cytokine or a functional fragment thereof. In some embodiments, a masked cytokine comprises, in the N- to C-terminal direction: (a) a half-life extending domain; (b) a cytokine or a functional fragment thereof; (c) a first linker; and (d) a masking moiety. In some embodiments, a masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 585-597, 602, 610-614, 627-636, 642, and 643. In some embodiments, a masked cytokine comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 585-597, 602, 610-614, 627-636, 642, and 643.In some embodiments, the masked cytokine is any of the exemplary constructs described in Table 4 or Table 5, or a variant produced by modifying any of the exemplary constructs described in Table 4 or Table 5, such as by incorporating one or more additional components into the structure of the constructs taught herein.

[0080] In some embodiments, the present disclosure also provides a masked cytokine comprising (a) a first masking moiety; (b) a cytokine or a functional fragment thereof, wherein the first masking moiety is linked to the cytokine or a functional fragment thereof via a first linker; and (c) a second masking moiety, wherein the second masking moiety is linked to the cytokine or a functional fragment thereof via a second linker. In some embodiments, the masked cytokine further comprises a half-life extending domain linked to the first masking moiety or the second masking moiety. In some embodiments, the half-life extending domain is linked to the first masking moiety or the second masking moiety via a third linker.

[0081] In some embodiments, the present disclosure also provides a masked cytokine comprising (a) a first masking moiety, wherein the first masking moiety is linked to a first half-life extending domain; (b) a cytokine or a functional fragment thereof, wherein the cytokine or a functional fragment thereof is linked to a second half-life extending domain; and (c) a second masking moiety, wherein the second masking moiety is linked to the first masking moiety. In some embodiments, the first masking moiety is linked to the first half-life extending domain via a first linker. In some embodiments, the second masking moiety is linked to the first masking moiety via a second linker. In some embodiments, the cytokine or a functional fragment thereof is linked to the second half-life extending domain via a third linker. In some embodiments, the first linker comprises a cleavable peptide. In some embodiments, the second linker comprises a cleavable peptide. In some embodiments, the third linker comprises a cleavable peptide.

[0082] In some embodiments, a masked cytokine comprises, in the N- to C-terminal direction: (a) a first masking moiety; (b) a first linker; (c) a cytokine or a functional fragment thereof; (d) a second linker; (e) a second masking moiety; and (f) a half-life extension domain. In some embodiments, a masked cytokine comprises, in the C- to N-terminal direction: (a) a first masking moiety; (b) a first linker; (c) a cytokine or a functional fragment thereof; (d) a second linker; (e) a second masking moiety; and (f) a half-life extension domain. In some embodiments, a masked cytokine comprises, in the N- to C-terminal direction: (a) a first masking moiety; (b) a first linker; (c) a cytokine or a functional fragment thereof; (d) a second linker; (e) a second masking moiety; (f) a third linker; and (g) a half-life extension domain. In some embodiments, a masked cytokine comprises, in the C- to N-terminal direction: (a) a first masking moiety; (b) a first linker; (c) a cytokine or a functional fragment thereof; (d) a second linker; (e) a second masking moiety; (f) a third linker; and (g) a half-life extension domain. In some embodiments, a masked cytokine comprises, in the N- to C-terminal direction: (a) a half-life extension domain; (b) a first linker; (c) a first masking moiety; (d) a second linker; (e) a cytokine or a functional fragment thereof; (f) a third linker; and (g) a second masking moiety. In some embodiments, a masked cytokine comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 567 and 598 - 601. In some embodiments, a masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 567 and 598 - 601. In some embodiments, a masked cytokine is any of the exemplary constructs described in Table 6 or Table 7, or a variant produced by modifying any of the exemplary constructs described in Table 6 or Table 7, such as by incorporating one or more additional components into the structure of a construct taught herein.

[0083] In some embodiments, the present disclosure also provides a masked cytokine comprising: (a) a first half-life extension domain and a second half-life extension domain; (b) a masking moiety; and (c) a cytokine or a functional fragment thereof, wherein the masking moiety is linked to the first half-life extension domain, the cytokine or the functional fragment thereof is linked to the second half-life extension domain, and the first and second half-life extension domains contain modifications that promote the association of the first and second half-life extension domains. In some embodiments, the masking moiety is linked to the first half-life extension domain via a first linker, and / or the cytokine or the functional fragment thereof is linked to the second half-life extension domain via a second linker. In some embodiments, the first half-life extension domain is optionally linked to the second half-life extension domain via a third linker. In some embodiments, the masked cytokine comprises: (a) a first half-life extension domain having the amino acid sequence of SEQ ID NO: 155 and a second half-life extension domain having the amino acid sequence of SEQ ID NO: 156; (b) a masking moiety having the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or a functional fragment thereof having the amino acid sequence of SEQ ID NO: 3, wherein the masking moiety is linked to the first half-life extension domain via a first linker having the amino acid sequence of SEQ ID NO: 28, the cytokine or the functional fragment thereof is linked to the second half-life extension domain via a second linker having the amino acid sequence of SEQ ID NO: 811, the first and second half-life extension domains contain modifications that promote the association of the first and second half-life extension domains, and the masked cytokine has the amino acid sequences of SEQ ID NOs: 266 and 267. In some embodiments, the masked cytokine comprises: (a) a first half-life extension domain having the amino acid sequence of SEQ ID NO: 155 and a second half-life extension domain having the amino acid sequence of SEQ ID NO: 156; (b) a masking moiety having the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or a functional fragment thereof having the amino acid sequence of SEQ ID NO: 260, wherein the masking moiety is linked to the first half-life extension domain via a first linker having the amino acid sequence of SEQ ID NO: 28, the cytokine or the functional fragment thereof is linked to the second half-life extension domain via a second linker having the amino acid sequence of SEQ ID NO: 262, the first and second half-life extension domains contain modifications that promote the association of the first and second half-life extension domains, and the masked cytokine has the amino acid sequences of SEQ ID NOs: 266 and 267. In some embodiments, the masked cytokine has the amino acid sequence of SEQ ID NO: 266.In some embodiments, the masked cytokine comprises the amino acid sequence of SEQ ID NO: 267. In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 266 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 266. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 266 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0084] In some embodiments, the masked cytokine comprises (a) a first half-life extension domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life extension domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a masking moiety comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 3, wherein the masking moiety is linked to the first half-life extension domain via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or the functional fragment thereof is linked to the second half-life extension domain via a second linker comprising the amino acid sequence of SEQ ID NO: 807, the first half-life extension domain and the second half-life extension domain contain modifications that promote the association of the first and second half-life extension domains, and the masked cytokine comprises the amino acid sequences of SEQ ID NO: 679 and 267. In some embodiments, the masked cytokine comprises (a) a first half-life extension domain comprising the amino acid sequence of SEQ ID NO: 156 and a second half-life extension domain comprising the amino acid sequence of SEQ ID NO: 155; (b) a masking moiety comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 3, wherein the masking moiety is linked to the first half-life extension domain via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or the functional fragment thereof is linked to the second half-life extension domain via a second linker comprising the amino acid sequence of SEQ ID NO: 807, the first half-life extension domain and the second half-life extension domain contain modifications that promote the association of the first and second half-life extension domains, and the masked cytokine comprises the amino acid sequences of SEQ ID NO: 679 and 267.

[0085] In some embodiments, the masked cytokine comprises (a) a first half-life extension domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life extension domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a masking moiety comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 3, wherein the masking moiety is linked to the first half-life extension domain via a first linker, the cytokine or the functional fragment thereof is linked to the second half-life extension domain via a second linker, the first half-life extension domain and the second half-life extension domain contain modifications that promote the association of the first and second half-life extension domains, and wherein the first linker and / or the second linker comprises a cleavable peptide. In some embodiments, the masked cytokine comprises (a) a first half-life extension domain comprising the amino acid sequence of SEQ ID NO: 156 and a second half-life extension domain comprising the amino acid sequence of SEQ ID NO: 155; (b) a masking moiety comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 3, wherein the masking moiety is linked to the first half-life extension domain via a first linker, the cytokine or the functional fragment thereof is linked to the second half-life extension domain via a second linker, the first half-life extension domain and the second half-life extension domain contain modifications that promote the association of the first and second half-life extension domains, and wherein the first linker and / or the second linker comprises a cleavable peptide.

[0086] In some embodiments, the masked cytokine comprises (a) a first half-life extension domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life extension domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a masking moiety comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 1, wherein the masking moiety is linked to the first half-life extension domain via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or the functional fragment thereof is linked to the second half-life extension domain via a second linker comprising the amino acid sequence of SEQ ID NO: 812, the first and second half-life extension domains contain modifications that promote the association of the first and second half-life extension domains, and the masked cytokine comprises the amino acid sequences of SEQ ID NO: 689 and 267. In some embodiments, the masked cytokine comprises (a) a first half-life extension domain comprising the amino acid sequence of SEQ ID NO: 156 and a second half-life extension domain comprising the amino acid sequence of SEQ ID NO: 155; (b) a masking moiety comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 1, wherein the masking moiety is linked to the first half-life extension domain via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or the functional fragment thereof is linked to the second half-life extension domain via a second linker comprising the amino acid sequence of SEQ ID NO: 812, the first and second half-life extension domains contain modifications that promote the association of the first and second half-life extension domains, and the masked cytokine comprises the amino acid sequences of SEQ ID NO: 689 and 267.

[0087] In some embodiments, the masked cytokine comprises (a) a first half-life extension domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life extension domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a masking moiety comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 1, wherein the masking moiety is linked to the first half-life extension domain via a first linker, the cytokine or the functional fragment thereof is linked to the second half-life extension domain via a second linker, the first half-life extension domain and the second half-life extension domain contain modifications that promote the association of the first and second half-life extension domains, and wherein the first linker and / or the second linker comprises a cleavable peptide. In some embodiments, the masked cytokine comprises (a) a first half-life extension domain comprising the amino acid sequence of SEQ ID NO: 156 and a second half-life extension domain comprising the amino acid sequence of SEQ ID NO: 155; (b) a masking moiety comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 1, wherein the masking moiety is linked to the first half-life extension domain via a first linker, the cytokine or the functional fragment thereof is linked to the second half-life extension domain via a second linker, the first half-life extension domain and the second half-life extension domain contain modifications that promote the association of the first and second half-life extension domains, and wherein the first linker and / or the second linker comprises a cleavable peptide.

[0088] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 562 and 563. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 562 and an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 563.

[0089] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 608 and 603. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 608, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 603.

[0090] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 604 and 603. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 604, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 603.

[0091] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 605 and 603. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 605, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 603.

[0092] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 606 and 603. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 606, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 603.

[0093] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 615 and 617. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 615, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 617.

[0094] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 618 and 620. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 618, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 620.

[0095] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 621 and 623. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 621 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 623.

[0096] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 624 and 626. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 624 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 626.

[0097] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 608 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 608 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0098] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 663 and 664. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 663, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 664.

[0099] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 665 and 666. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 665, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 666.

[0100] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 667 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 667, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0101] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 669 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 669, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0102] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 670 and 671. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 670, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

[0103] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 672 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 672, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0104] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 673 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 673, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0105] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 674 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 674, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0106] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 675 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 675, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0107] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 676 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 676 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0108] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 677 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 677 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0109] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 678 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 678 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0110] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 679 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 679, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0111] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 680 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 680, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0112] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 681 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 681, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0113] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 682 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 682, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0114] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 683 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 683, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0115] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 684 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 684, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0116] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 685 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 685 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0117] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 686 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 686 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0118] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 687 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 687 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0119] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 688 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 688, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0120] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 689 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 689, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0121] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 690 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 690, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0122] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 266 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 266, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0123] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 692 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 692, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0124] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 693 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 693, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0125] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 694 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 694, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0126] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 695 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 695, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0127] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 696 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 696, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0128] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 697 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 697, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0129] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 698 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 698, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0130] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 699 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 699, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0131] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 700 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 700, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0132] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 701 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 701, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0133] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 702 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 702, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0134] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 703 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 703, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0135] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 704 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 704, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0136] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 705 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 705, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0137] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 706 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 706 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0138] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 707 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 707 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0139] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 708 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 708 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0140] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 709 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 709, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0141] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 710 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 710, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0142] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 711 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 711, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0143] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 712 and 667. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 712, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 667.

[0144] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 713 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 713, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0145] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 714 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 714, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0146] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 716 and 699. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 716 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 699.

[0147] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 717 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 717 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0148] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 718 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 718 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0149] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 719 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 719, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0150] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 720 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 720, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0151] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 722 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 722, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0152] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 723 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 723, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0153] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 726 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 720, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0154] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 728 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 728, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0155] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 729 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 729, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0156] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 730 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 730, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0157] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 731 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 731, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0158] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 732 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 732, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0159] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 733 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 733, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0160] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 734 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 734, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0161] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 735 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 735, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0162] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 736 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 736, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0163] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 737 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 737, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0164] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 738 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 738, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0165] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 739 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 739, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0166] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 740 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 740, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0167] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 741 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 741, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0168] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 742 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 742, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0169] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 743 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 743, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0170] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 744 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 744, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0171] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 745 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 745, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0172] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 746 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 746, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0173] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 674 and 828. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 674, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 828.

[0174] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 674 and 829. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 674, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 829.

[0175] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 726 and 830. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 726, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 830.

[0176] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 726 and 829. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 726, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 829.

[0177] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 747 and 671. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 747, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

[0178] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 715 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 715, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

[0179] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 715 and 671. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 715, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

[0180] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 748 and 671. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 748, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

[0181] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 749 and 671. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 749, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

[0182] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 750 and 671. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 750, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

[0183] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 751 and 671. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 751, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

[0184] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 752 and 671. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 752, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

[0185] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 753 and 671. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 753 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

[0186] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 754 and 671. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 754 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

[0187] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 758 and 671. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 758 and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

[0188] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 759 and 671. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 759, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

[0189] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 760 and 671. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 760, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

[0190] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 761 and 671. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 761, and comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

[0191] In some embodiments, the masked cytokine is an exemplary masked cytokine construct as described in any of Tables 8-11, or a variant produced by modifying any of the exemplary constructs described in Tables 8-11, such as by incorporating one or more additional components into the structure of the constructs taught herein.

[0192] In some embodiments, the present disclosure also provides a masked cytokine comprising (a) a first half-life extension domain and a second half-life extension domain; (b) a first masking moiety and a second masking moiety; and (c) a cytokine or a functional fragment thereof, wherein the first masking moiety is linked to the first half-life extension domain, the second masking moiety is linked to the cytokine or a functional fragment thereof, the second masking moiety or the cytokine or a functional fragment thereof is linked to the second half-life extension domain, and the first half-life extension domain and the second half-life extension domain contain modifications that promote the association of the first and second half-life extension domains. In some embodiments, the first masking moiety is linked to the first half-life extension domain via a first linker, and / or the second masking moiety or the cytokine or a functional fragment thereof is linked to the second half-life extension domain via a second linker. In some embodiments, the second masking moiety is linked to the cytokine or a functional fragment thereof via a third linker. In some embodiments, the first half-life extension domain is optionally linked to the second half-life extension domain via a fourth linker. In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 755 and 616. In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 756 and 616. In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NO: 757 and 616. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 755 and an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 616. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 756 and an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 616.In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 757 and an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 616.

[0193] In some embodiments, the masked cytokine comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 265 - 267, 556 - 720, 722, 723, 726, 728 - 761, and 828 - 830. In some embodiments, the masked cytokine comprises two different amino acid sequences selected from the group consisting of SEQ ID NOs: 265 - 267, 556 - 720, 722, 723, 726, 728 - 761, and 828 - 830. In some embodiments, the masked cytokine comprises an amino acid sequence associated with any of the exemplary constructs described in Tables 4 - 11.

[0194] Each component of the masked cytokines provided herein is discussed in more detail below.

[0195] A. Cytokine

[0196] Cytokines or functional fragments thereof are provided herein. The cytokine or functional fragment thereof can be any cytokine, any functional fragment of any cytokine, or any natural or non - natural variant of any cytokine. Cytokines are small polypeptides that act in cell signaling, particularly in cells of the immune system. Examples of cytokines can include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors.

[0197] Cytokines can be classified in a variety of ways, such as based on their three - dimensional structure. Examples include the β - trefoil fold class, the short - chain four - helix bundle class, and the long - chain four - helix bundle class.

[0198] The β - trefoil fold class includes cytokines characterized by three βββ - loop - β units (a total of 12 β - strands) that form a barrel structure with a hairpin cap on the barrel. Examples of cytokines in the β - trefoil fold class include IL - 1α, IL - 1β, IL - 1 receptor antagonist (IL - 1RA), IL - 18, IL - 33, IL - 36α, IL - 36β, IL - 36γ, IL - 36 receptor antagonist (IL - 36RA), IL - 37, and IL - 38.

[0199] Short-chain and long-chain four-helix bundle classes are characterized by monomeric helical bundles of four amphipathic helices with a unique up-up-down-down topological orientation. Short-chain four-helix bundle classes are characterized by shorter helices, such as those with lengths of 10-20 amino acid residues, while long-chain four-helix bundle classes are characterized by longer helices, such as those with lengths of 20-30 amino acid residues. Examples of cytokines in the short-chain four-helix bundle class include IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-13, IL-15, IL-21, granulocyte-macrophage colony-stimulating factor (GM-CSF), and macrophage colony-stimulating factor (M-CSF). Examples of cytokines in the long-chain four-helix bundle class include IL-6, IL-11, IL-12, growth hormone (GH), erythropoietin (EPO), prolactin (PRL), leukemia inhibitory factor (LIF), oncostatin (OSM), and thrombopoietin (TPO).

[0200] In contrast to the short-chain and long-chain four-helix bundle classes that are monomers, some cytokines are homodimeric. Examples of dimeric cytokines include IL-10 and IFN-γ (gamma).

[0201] Some cytokines are classified as heterodimeric. Examples of heterodimeric cytokines include IL-12 and IL-23.

[0202] Some cytokines, such as IL-15, also function by binding to and being presented by membrane-bound cytokine receptors.

[0203] In some embodiments, the cytokine or a functional fragment thereof is IL-2, or a functional fragment or variant of IL-2. In some embodiments, the cytokine or a functional fragment thereof is IL-15, or a functional fragment or variant of IL-15. In some embodiments, the cytokine or a functional fragment thereof is selected from the group consisting of IL-2, IL-10, IL-12, IL-15, IL-18, interferon (IFN)-α (alpha), IFN-β (beta), and IFN-γ (gamma). In some embodiments, the cytokine or a functional fragment thereof is a functional fragment or variant of IL-2, IL-10, IL-12, IL-15, IL-18, IFN-α (alpha), IFN-β (beta), or IFN-γ (gamma).

[0204] In some embodiments, the cytokine or a functional fragment thereof is selected from the group consisting of IL-1α, IL-1β, IL-1 receptor antagonist (IL-1RA), IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-36 receptor antagonist (IL-36RA), IL-37, and IL-38.

[0205] In some embodiments, the cytokine or its functional fragment is selected from the group consisting of: IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-13, IL-15, IL-21, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), IL-6, IL-11, IL-12, growth hormone (GH), erythropoietin (EPO), prolactin (PRL), leukemia inhibitory factor (LIF), oncostatin (OSM), and thrombopoietin (TPO).

[0206] In some embodiments, the cytokine or its functional fragment is selected from the group consisting of: CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CCL1e, CCL2, CCL3, CCL3L1, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CX3CL1, XCL1, and XCL2. In some embodiments, the cytokine or its functional fragment is a functional fragment or variant of CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CCL1e, CCL2, CCL3, CCL3L1, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CX3CL1, XCL1, or XCL2.

[0207] In some embodiments, the cytokine or its functional fragment is selected from IFN-α, IFN-β, IFN-γ, IFN-ε, IFN-κ, The group consisting of IFN-ω (omega), IFN-τ (tau), IFN-ζ (zeta), IFN-δ (delta), and IFN-λ (lambda). In some embodiments, the cytokine or a functional fragment thereof is IFN-α (alpha), IFN-β (beta), IFN-γ (gamma), IFN-ε (epsilon), IFN-κ (kappa), A functional fragment or variant of IFN-ω (omega), IFN-τ (tau), IFN-ζ (zeta), IFN-δ (delta), or IFN-λ (lambda).

[0208] In some embodiments, the cytokine or a functional fragment thereof is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A, IL-28B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, and IL-37. In some embodiments, the cytokine or a functional fragment thereof is a functional fragment or variant of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17A, IL-17C, IL-17D, IL-17F, IL-17A / F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A, IL-28B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, or IL-37.

[0209] In some embodiments, the cytokine or a functional fragment thereof is selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor α (TNF-α), transforming growth factor β (TGF-β), IFN-γ (gamma), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, and IL-12. In some embodiments, the cytokine or a functional fragment thereof is a functional fragment or variant of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor α (TNF-α), transforming growth factor β (TGF-β), IFN-γ (gamma), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, or IL-12.

[0210] In some embodiments, the cytokine or a functional fragment thereof is selected from the group consisting of TNF-α (alpha), TNF-β (beta), TNF-γ (gamma), CD252, CD154, CD178, CD70, CD153, 4-1BB-L, TRAIL, RANKL, APO3L, CD256, CD257, CD258, TL1, AITRL, and EDA1. In some embodiments, the cytokine or a functional fragment thereof is a functional fragment or variant of TNF-α (alpha), TNF-β (beta), TNF-γ (gamma), CD252, CD154, CD178, CD70, CD153, 4-1BB-L, TRAIL, RANKL, APO3L, CD256, CD257, CD258, TL1, AITRL, and EDA1.

[0211] The cytokine or a functional fragment thereof comprises an amino terminus and a carboxyl terminus. In some embodiments, the half-life extension domain is linked to the amino terminus or the carboxyl terminus of the cytokine or a functional fragment thereof. In some embodiments, the masking moiety is linked to the amino terminus or the carboxyl terminus of the cytokine or a functional fragment thereof. In some embodiments, the linker is linked to the amino terminus or the carboxyl terminus of the cytokine or a functional fragment thereof. In some embodiments, the cleavable peptide of the linker is linked to the amino terminus or the carboxyl terminus of the cytokine or a functional fragment thereof. In some embodiments, the N-terminal spacer domain or the C-terminal spacer domain of the linker is linked to the amino terminus or the carboxyl terminus of the cytokine or a functional fragment thereof.

[0212] Exemplary embodiments of the cytokine or a functional fragment thereof in the form of an IL-2 polypeptide or a functional fragment thereof and an IL-15 polypeptide or a functional fragment thereof are provided in detail below.

[0213] 1. IL-2 Polypeptide

[0214] In some embodiments, the cytokine or a functional fragment thereof is an IL-2 polypeptide or a functional fragment thereof. In eukaryotic cells, IL-2 is synthesized as a 153-amino acid precursor polypeptide, which is then processed into mature IL-2 by removal of amino acid residues 1-20. This yields the mature form of IL-2 consisting of 133 amino acids (amino acid residues 21-153), which is secreted in a mature active form.

[0215] In some embodiments, the IL-2 polypeptide or a functional fragment thereof is any naturally occurring interleukin-2 (IL-2) protein or a modified variant thereof that is capable of binding to the interleukin-2 receptor (IL-2R) or a component thereof (e.g., the IL-2Rα chain) or otherwise exhibits an affinity for the interleukin-2 receptor (IL-2R) or a component thereof (e.g., the IL-2Rα chain). In some embodiments, the IL-2 polypeptide or a functional fragment thereof is the mature form of IL-2 consisting of amino acid residues 21-153 of SEQ ID NO: 159. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 160. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8 and 260. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 260. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 160, 230, 243-251, 260, 775-792, and 813-822. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 230. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 230. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0216] In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence produced by making at least one amino acid modification to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 160, 230, 243-251, 260, 775-792, and 813-822. Each of the at least one amino acid modifications can be any amino acid modification, such as a substitution, insertion, or deletion. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence produced by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 230, 243-251, 260, 775-792, and 813-822. In some embodiments, the IL-2 peptide or a functional fragment thereof comprises an amino acid sequence comprising a serine (S), glycine (G), or alanine (A) residue at amino acid residue 125. In some embodiments, the IL-2 peptide or a functional fragment thereof comprises an amino acid sequence comprising an alanine (A) residue at amino acid residue 3. For example, in some embodiments, the IL-2 peptide or a functional fragment thereof comprises an amino acid sequence produced by introducing a T3A amino acid substitution into an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8 and 160. In some embodiments, the IL-2 peptide or a functional fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 243-251 and 260. In some embodiments, the IL-2 peptide or a functional fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 775-792 and 813-822.

[0217] In some embodiments, the IL-2 peptide or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions that reduce the affinity of the IL-2 peptide or a functional fragment thereof for IL-2Rα (CD25) as compared to the amino acid sequence of wild-type IL-2. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that one or more of the amino acid residues 38, 42, 45, and 62 is alanine (A). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that the amino acid residues 38, 42, 45, and 62 are alanine (A). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that the amino acid residues 38, 42, 45, and 62 are alanine (A), and the amino acid residue 125 is serine (S), glycine (G), or alanine (A). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that the amino acid residues 38 and 42 are alanine (A), and the amino acid residue 125 is serine (S), glycine (G), or alanine (A). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from substituting alanine with amino acid residues R38, F42, Y45, and E62 in the amino acid sequence of SEQ ID NO: 160 or 251. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from substituting alanine (A) with amino acid residues R38, F42, Y45, and E62 and substituting serine (S), glycine (G), or alanine (A) with amino acid residue C125 in the amino acid sequence of SEQ ID NO: 160 or 251. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that the amino acid residue 42 is lysine (K).In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 42 is lysine (K), and amino acid residue 125 is serine (S), glycine (G), or alanine (A). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residues 42 and 45 are alanine (A), and amino acid residue 72 is glycine (G). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residues 42 and 45 are alanine (A), amino acid residue 72 is glycine (G), and amino acid residue 125 is serine (S), glycine (G), or alanine (A). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 62 is arginine (R) or serine (S). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 42 is glutamate (E). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 43 is alanine (A). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 45 is asparagine (N), arginine (R), or alanine (A).In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 45 is alanine (A) and amino acid residue 62 is serine (S). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 42 is serine (S) and amino acid residue 62 is serine (S). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 38 is glycine (G), amino acid residue 45 is alanine (A), and amino acid residue 62 is serine (S).

[0218] In some embodiments, the IL-2 peptide or a functional fragment thereof comprises an amino acid sequence produced by introducing one or more of the following amino acid substitutions into any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: R38A, F42A, F42E, F42K, K43A, Y45A, Y45N, Y45R, E62A, E62R, E62S, L72G, C125S, C125G, and C125A.

[0219] In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions that enhance the affinity of the IL-2 polypeptide or a functional fragment thereof for IL-2Rβ (CD122) as compared to the amino acid sequence of wild-type IL-2. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 80 is phenylalanine (F), amino acid residue 81 is aspartic acid (D), amino acid residue 85 is valine (V), amino acid residue 86 is valine (V), or amino acid residue 92 is phenylalanine (F), or a combination thereof. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 80 is phenylalanine (F), amino acid residue 81 is aspartic acid (D), amino acid residue 85 is valine (V), amino acid residue 86 is valine (V), or amino acid residue 92 is phenylalanine (F), or a combination thereof, and amino acid residue 125 is serine (S), glycine (G), or alanine (A). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 80 is phenylalanine (F), amino acid residue 81 is aspartic acid (D), amino acid residue 85 is valine (V), amino acid residue 86 is valine (V), and amino acid residue 92 is phenylalanine (F). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 80 is phenylalanine (F), amino acid residue 81 is aspartic acid (D), amino acid residue 85 is valine (V), amino acid residue 86 is valine (V), amino acid residue 92 is phenylalanine (F), and amino acid residue 125 is serine (S), glycine (G), or alanine (A).In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence produced by one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 18 is cysteine (C). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence produced by one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 20 is alanine (A), leucine (L), or phenylalanine (F). In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence produced by one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 16 is isoleucine. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence produced by one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 29 is leucine (L).

[0220] In some embodiments, the IL-2 peptide or a functional fragment thereof comprises an amino acid sequence produced by introducing one or more of the following amino acid substitutions into any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: L18C, D20A, D20L, D20F, H16I, N29L, L80F, R81D, L85V, I86V, I92F, C125S, C125G, and C125A.

[0221] In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions that decrease the affinity of the IL-2 peptide or a functional fragment thereof for IL-2Rα (CD25) compared to the amino acid sequence of wild-type IL-2 and one or more amino acid substitutions that increase the affinity of the IL-2 polypeptide or a functional fragment thereof for IL-2Rβ (CD122) compared to the amino acid sequence of wild-type IL-2. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822 such that one or more of amino acid residues 38, 42, 45, and 62 is alanine (A), amino acid residue 42 is lysine (K), amino acid residue 72 is glycine (G), amino acid residue 80 is phenylalanine (F), amino acid residue 81 is aspartic acid (D), amino acid residue 85 is valine (V), amino acid residue 86 is valine (V), or amino acid residue 92 is phenylalanine (F), or a combination thereof. Thus, in some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence comprising one or more of the following amino acid substitutions compared to the mature form of wild-type IL-2: D20A, D20L, D20F, H16I, L18C, N29L, R38A, F42A, F42E, F42K, K43A, Y45A, Y45N, Y45R, E62A, E62R, E62S, L72G, L80F, R81D, L85V, I86V, I92F, C125S, C125G, and C125A.

[0222] In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 38 is glycine (G), alanine (A), lysine (K), or tryptophan (W), amino acid residue 42 is alanine (A), lysine (K), or isoleucine (I), amino acid residue 45 is alanine (A) or asparagine (N), amino acid residue 62 is alanine (A) or leucine (L), or amino acid residue 68 is valine (V), or a combination thereof. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 38 is lysine (K), amino acid residue 42 is glutamine (Q), amino acid residue 45 is glutamic acid (E), or amino acid residue 68 is valine (V), or a combination thereof. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 38 is alanine (A), amino acid residue 42 is isoleucine (I), amino acid residue 45 is asparagine (N), amino acid residue 62 is leucine (L), or amino acid residue 68 is valine (V), or a combination thereof. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 38 is lysine (K), amino acid residue 42 is lysine (K), amino acid residue 45 is arginine (R), amino acid residue 62 is leucine (L), or amino acid residue 68 is valine (V), or a combination thereof. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 38 is alanine (A) or lysine (K), amino acid residue 42 is alanine (A), amino acid residue 45 is alanine (A), or amino acid residue 62 is alanine (A), or a combination thereof.In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822, such that amino acid residue 42 is isoleucine (I), amino acid residue 45 is glutamic acid (E), or amino acid residue 68 is valine (V), or a combination thereof.

[0223] In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from introducing or further introducing a C125S, C125G, or C125A substitution into the amino acid sequence of any one of the IL-2 polypeptides or functional fragments thereof described herein.

[0224] In some embodiments, for the purpose of removing O-glycosylation sites, one or more amino acid residues are removed from the amino acid sequence of the IL-2 polypeptide or a functional fragment thereof. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from deletion of the first three amino acid residues (residues 1-3) of the amino acid sequence of any IL-2 polypeptide or functional fragment thereof disclosed herein. In some embodiments, for the purpose of removing O-glycosylation sites, one or more amino acid residues are substituted into the amino acid sequence of the IL-2 polypeptide or a functional fragment thereof. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence resulting from introducing an amino acid substitution into one or more of the first three amino acid residues (residues 1-3) of the amino acid sequence of any IL-2 polypeptide or functional fragment thereof disclosed herein.

[0225] The IL-2 polypeptide or a functional fragment thereof comprises an amino terminus and a carboxyl terminus. In some embodiments, a half-life extension domain is linked to the amino terminus or carboxyl terminus of the IL-2 polypeptide or a functional fragment thereof. In some embodiments, a masking moiety is linked to the amino terminus or carboxyl terminus of the IL-2 polypeptide or a functional fragment thereof. In some embodiments, a linker is linked to the amino terminus or carboxyl terminus of the IL-2 polypeptide or a functional fragment thereof. In some embodiments, a cleavable peptide of the linker is linked to the amino terminus or carboxyl terminus of the IL-2 polypeptide or a functional fragment thereof. In some embodiments, an N-terminal spacer domain or a C-terminal spacer domain of the linker is linked to the amino terminus or carboxyl terminus of the IL-2 polypeptide or a functional fragment thereof.

[0226] 2. IL-15 Polypeptide

[0227] In some embodiments, the cytokine or a functional fragment thereof is an IL-15 polypeptide or a functional fragment thereof. In eukaryotic cells, IL-15 is synthesized as a 162-amino acid precursor polypeptide, which is then processed to mature IL-15 by removal of amino acid residues 1-48. This yields the mature form of IL-15, consisting of 114 amino acids (amino acid residues 49-162), which is secreted in a mature, active form (see SEQ ID NO: 167).

[0228] In some embodiments, the IL-15 polypeptide or a functional fragment thereof is any naturally occurring interleukin-15 (IL-15) protein or a modified variant thereof that is capable of binding to the interleukin-15 receptor (IL-15R) or a component thereof (e.g., the IL-15Rα, IL-2Rβ, and / or IL-2Rγ chain) or otherwise exhibiting an affinity for the interleukin-15 receptor (IL-15R) or a component thereof (e.g., the IL-15Rα, IL-2Rβ, and / or IL-2Rγ chain). In some embodiments, the IL-15 polypeptide or a functional fragment thereof is the mature form of IL-15 consisting of amino acid residues 49-162 of SEQ ID NO: 166. In some embodiments, the IL-15 polypeptide or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 167. In some embodiments, the IL-15 polypeptide or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 166 or 167.

[0229] In some embodiments, the IL-15 polypeptide or a functional fragment thereof comprises an amino acid sequence generated by at least one amino acid modification to the amino acid sequence of SEQ ID NO: 167. Each of the at least one amino acid modifications can be any amino acid modification, such as a substitution, insertion, or deletion. In some embodiments, the IL-15 polypeptide or a functional fragment thereof comprises an amino acid sequence generated by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid substitutions in the amino acid sequence of SEQ ID NO: 167.

[0230] The IL-15 polypeptide or a functional fragment thereof comprises an amino terminus and a carboxyl terminus. In some embodiments, the half-life extension domain is linked to the amino terminus or the carboxyl terminus of the IL-15 polypeptide or a functional fragment thereof. In some embodiments, the masking moiety is linked to the amino terminus or the carboxyl terminus of the IL-15 polypeptide or a functional fragment thereof. In some embodiments, the linker is linked to the amino terminus or the carboxyl terminus of the IL-15 polypeptide or a functional fragment thereof. In some embodiments, the cleavable peptide of the linker is linked to the amino terminus or the carboxyl terminus of the IL-15 polypeptide or a functional fragment thereof. In some embodiments, the N-terminal spacer domain or the C-terminal spacer domain of the linker is linked to the amino terminus or the carboxyl terminus of the IL-15 polypeptide or a functional fragment thereof.

[0231] B. Masking moiety

[0232] As used herein, a masking moiety refers to a moiety that is capable of binding to a cytokine or a functional fragment thereof or otherwise exhibiting an affinity for a cytokine or a functional fragment thereof such that, in some embodiments, the binding reduces the affinity of the cytokine or a functional fragment thereof for its cognate receptor or protein. For example, a masking moiety for an IL-2 polypeptide or a functional fragment thereof is capable of binding to the IL-2 polypeptide or a functional fragment thereof or otherwise exhibiting an affinity for the IL-2 polypeptide or a functional fragment thereof such that, in some embodiments, the binding reduces the affinity of the IL-2 polypeptide or a functional fragment thereof for its cognate receptor or protein (e.g., IL-2R or a component thereof, such as the IL-2Rα and / or IL-2Rβ chains). When bound to a cytokine or a functional fragment thereof, the masking moiety blocks, sequesters, inhibits (e.g., reduces) or otherwise prevents (e.g., masks) the activity of the cytokine or a functional fragment thereof or its binding to its cognate receptor or protein. In some embodiments that include a first masking moiety and a second masking moiety, the presence of one of the masking moieties (e.g., the first masking moiety) blocks, sequesters, inhibits (e.g., reduces) or otherwise prevents (e.g., masks) the activity of the cytokine or a functional fragment thereof or its binding to its cognate receptor or protein, while in some embodiments, the other masking moiety (e.g., the second masking moiety) may remain associated with the cytokine or a functional fragment thereof after cleavage of the first masking moiety and still allow or even potentially facilitate the binding of the cytokine or a functional fragment thereof to its cognate receptor or protein at the site where the first masking moiety was bound. See, for example Figure 7B . Methods for determining the degree of binding of a protein (e.g., a cytokine) to a cognate protein (e.g., a cytokine receptor) are well known in the art.

[0233] The masked cytokines provided herein comprise a masking moiety. The masking moiety comprises an amino terminus and a carboxyl terminus. In some embodiments, the masked cytokine comprises a single masking moiety. In some embodiments, the masked cytokine comprises more than one masking moiety, each of which can be any of the masking moieties described herein. In some embodiments, the masked cytokine comprises a first masking moiety and a second masking moiety. It is to be understood that, for example, reference to "the masking moiety" or "a masking moiety" can refer to the masking moiety in a masked cytokine that comprises a single masking moiety, or it can refer to the first masking moiety in a masked cytokine that comprises a first masking moiety and a second masking moiety, or it can refer to the second masking moiety in a masked cytokine that comprises a first masking moiety and a second masking moiety, or it can refer to the first masking moiety and the second masking moiety in a masked cytokine that comprises a first masking moiety and a second masking moiety.

[0234] In some embodiments, the masking moiety is linked to the cytokine or a functional fragment thereof. In some embodiments, the masking moiety is linked to the cytokine or a functional fragment thereof via a first linker. In some embodiments, the masked cytokine comprises a half-life extending domain linked to the masking moiety. In some embodiments, the masked cytokine comprises a half-life extending domain linked to the masking moiety via a second linker. In some embodiments, the masking moiety is linked to the cytokine or a functional fragment thereof and is linked to the half-life extending domain. In some embodiments, the cytokine or a functional fragment thereof is linked to the amino terminus of the masking peptide, and the half-life extending domain is linked to the carboxyl terminus of the masking peptide. In some embodiments, the cytokine or a functional fragment thereof is linked to the carboxyl terminus of the masking peptide, and the half-life extending domain is linked to the amino terminus of the masking peptide. In some embodiments, the half-life extending domain is linked to the amino terminus or the carboxyl terminus of the masking moiety. In some embodiments, the second linker is linked to the amino terminus or the carboxyl terminus of the masking moiety. In some embodiments, the masking moiety is linked to the first linker and is linked to the second linker. In some embodiments, the first linker is linked to the amino terminus of the masking peptide, and the second linker is linked to the carboxyl terminus of the masking peptide. In some embodiments, the first linker is linked to the carboxyl terminus of the masking peptide, and the second linker is linked to the amino terminus of the masking peptide.

[0235] In some embodiments that include a first masking moiety and a second masking moiety, the first masking moiety is linked to a cytokine or a functional fragment thereof, and the second masking moiety is linked to a cytokine or a functional fragment thereof. In some embodiments that include a first masking moiety and a second masking moiety, the first masking moiety is linked to a cytokine or a functional fragment thereof via a first linker, and the second masking moiety is linked to a cytokine or a functional fragment thereof via a second linker. In some embodiments, the masked cytokine includes a half-life extension domain linked to the first masking moiety or the second masking moiety. In some embodiments, the masked cytokine includes a half-life extension domain linked to the first masking moiety or the second masking moiety via a third linker. In some embodiments, the first linker is linked to the amino terminus or the carboxyl terminus of the first masking moiety. In some embodiments, the second linker is linked to the amino terminus or the carboxyl terminus of the second masking moiety. In some embodiments, the half-life extension domain is linked to the amino terminus or the carboxyl terminus of the first masking moiety. In some embodiments, the half-life extension domain is linked to the amino terminus or the carboxyl terminus of the second masking moiety. In some embodiments, the third linker is linked to the amino terminus or the carboxyl terminus of the first masking moiety. In some embodiments, the third linker is linked to the amino terminus or the carboxyl terminus of the second masking moiety.

[0236] In some embodiments that include a first masking moiety and a second masking moiety, the first masking moiety is linked to a first half-life extension domain, the second masking moiety is linked to the first masking moiety, and the cytokine or a functional fragment thereof is linked to a second half-life extension domain. In some embodiments that include a first masking moiety and a second masking moiety, the first masking moiety is linked to the first half-life extension domain via a first linker, the second masking moiety is linked to the first masking moiety via a second linker, and the cytokine or a functional fragment thereof is linked to the second half-life extension domain via a third linker. In some embodiments, the first linker is linked to the amino terminus or the carboxyl terminus of the first half-life extension domain. In some embodiments, the second linker is linked to the amino terminus or the carboxyl terminus of the second masking moiety. In some embodiments, the second half-life extension domain is linked to the amino terminus or the carboxyl terminus of the cytokine or a functional fragment thereof. In some embodiments, the first half-life extension domain is linked to the amino terminus or the carboxyl terminus of the first masking moiety. In some embodiments, the third linker is linked to the amino terminus or the carboxyl terminus of the second half-life extension domain. In some embodiments, the third linker is linked to the amino terminus or the carboxyl terminus of the cytokine or a functional fragment thereof.

[0237] In some embodiments that include a first masking moiety and a second masking moiety, the second masking moiety is linked to a first half-life extending domain, the first masking moiety is linked to the second masking moiety, and a cytokine or a functional fragment thereof is linked to a second half-life extending domain. In some embodiments that include a first masking moiety and a second masking moiety, the second masking moiety is linked to the first half-life extending domain via a first linker, the first masking moiety is linked to the second masking moiety via a second linker, and a cytokine or a functional fragment thereof is linked to the second half-life extending domain via a third linker. In some embodiments, the first linker is linked to the amino terminus or carboxyl terminus of the first half-life extending domain. In some embodiments, the second linker is linked to the amino terminus or carboxyl terminus of the first masking moiety. In some embodiments, the second half-life extending domain is linked to the amino terminus or carboxyl terminus of the cytokine or a functional fragment thereof. In some embodiments, the first half-life extending domain is linked to the amino terminus or carboxyl terminus of the second masking moiety. In some embodiments, the third linker is linked to the amino terminus or carboxyl terminus of the second half-life extending domain. In some embodiments, the third linker is linked to the amino terminus or carboxyl terminus of the cytokine or a functional fragment thereof.

[0238] In some embodiments that include a first half-life extending domain and a second half-life extending domain, a masking moiety is linked to the first half-life extending domain. In some embodiments, the first half-life extending domain is linked to the amino terminus or carboxyl terminus of the masking moiety. In some embodiments, the masking moiety is linked to the first half-life extending domain via a first linker. In some embodiments, the first linker is linked to the amino terminus or carboxyl terminus of the masking moiety.

[0239] In some embodiments that include a first half-life extending domain, a second half-life extending domain, a first masking moiety, and a second masking moiety, the first masking moiety is linked to the first half-life extending domain, and the second masking moiety is linked to a cytokine or a functional fragment thereof. In some embodiments, the second masking moiety is further linked to the second half-life extending domain. In some embodiments, the first masking moiety is linked to the first half-life extending domain via a first linker, and / or the second masking moiety or the cytokine or a functional fragment thereof is linked to the second half-life extending domain via a second linker. In some embodiments, the second masking moiety is linked to the cytokine or a functional fragment thereof via a third linker.

[0240] In some embodiments, the first half-life extending domain is linked to the amino terminus or carboxy terminus of the first masking moiety. In some embodiments, the cytokine or a functional fragment thereof is linked to the amino terminus or carboxy terminus of the second masking moiety. In some embodiments, the first linker is linked to the amino terminus or carboxy terminus of the first masking moiety. In some embodiments, the second linker is linked to the amino terminus or carboxy terminus of the second masking moiety. In some embodiments, the third linker is linked to the amino terminus or carboxy terminus of the second masking moiety. In some embodiments, the second linker is linked to the amino terminus of the second masking moiety and the third linker is linked to the carboxy terminus of the second masking moiety. In some embodiments, the second linker is linked to the carboxy terminus of the second masking moiety and the third linker is linked to the amino terminus of the second masking moiety.

[0241] In some embodiments, the masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, 161-165, 187-218, 221-229, 231, and 261. In some embodiments, the masking moiety comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, 161-165, 187-218, 221-229, 231, and 261. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 261. In some embodiments, the masking moiety comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 261.

[0242] In some embodiments, the masked cytokine comprises a first masking moiety and a second masking moiety. In some embodiments, the first masking moiety comprises the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 187-218, 221-229, and 261. In some embodiments, the first masking moiety comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking moiety comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 187-218, 221-229, and 261. In some embodiments, the first masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 187-218, 221-229, and 261, and the second masking moiety comprises the amino acid sequence of SEQ ID NO: 9 or 231. In some embodiments, the first masking moiety comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 187-218, 221-229, and 261, and the second masking moiety comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231. In some embodiments, the first masking moiety comprises the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking moiety comprises the amino acid sequence of SEQ ID NO: 261. In some embodiments, the second masking moiety comprises the amino acid sequence of SEQ ID NO: 9 or 231, and the first masking moiety comprises the amino acid sequence of SEQ ID NO: 261.In some embodiments, the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 261, and the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231. In some embodiments, the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 261, and the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231.

[0243] In some embodiments, the first masking portion comprises the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 161 - 165, 221 - 226, and 261. In some embodiments, the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 161 - 165, 221 - 226, and 261. In some embodiments, the first masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 161 - 165, 221 - 226, and 261, and the second masking portion comprises the amino acid sequence of SEQ ID NO: 9 or 231. In some embodiments, the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 161 - 165, 221 - 226, and 261, and the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231. In some embodiments, the first masking portion comprises the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 161 - 165, 221 - 226, 261, 826, and 827.In some embodiments, the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 221 - 226, 261, 826, and 827. In some embodiments, the second masking portion comprises the amino acid sequence of SEQ ID NO: 9 or 231, and the first masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 221 - 226, 261, 826, and 827. In some embodiments, the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231, and the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 221 - 226, 261, 826, and 827.

[0244] In some embodiments, the first masking portion comprises the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 187 - 218. In some embodiments, the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 187 - 218. In some embodiments, the first masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 187 - 218, and the second masking portion comprises the amino acid sequence of SEQ ID NO: 9 or 231. In some embodiments, the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 187 - 218, and the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231.

[0245] In some embodiments, the first masking portion comprises the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 227-229. In some embodiments, the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 227-229. In some embodiments, the first masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 227-229, and the second masking portion comprises the amino acid sequence of SEQ ID NO: 9 or 231. In some embodiments, the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 227-229, and the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231.

[0246] In some embodiments, the first masking portion comprises the amino acid sequence of SEQ ID NO: 9, and the second masking portion comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the first masking portion comprises the amino acid sequence of SEQ ID NO: 10, and the second masking portion comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9, and the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10, and the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the masking portion comprises an amino acid sequence produced by one or more of the following amino acid substitutions to SEQ ID NO: 10 or 261: C122S, C168S, R42A, K71A, T73A, T74A, V75A, H133A, Y134A, R137D, Q162W, E170A and Q188A. In some embodiments, the masking portion comprising an amino acid sequence produced by one or more amino acid substitutions comprises the amino acid sequence of SEQ ID NO: 826.

[0247] In some embodiments, the masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 161 - 165, 219 - 229, 232 - 234, 261, and 823 - 825. In some embodiments, the masking moiety comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 161 - 165, 219 - 229, 232 - 234, 261, and 823 - 825. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 261. In some embodiments, the masking moiety comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 261. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 826 or 827, or comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 826 or 827.

[0248] In some embodiments, the masked cytokine comprises a first masking moiety and a second masking moiety. In some embodiments, the first masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825, and the second masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 219 - 229, and 261. In some embodiments, the first masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 219 - 229, and 261, and the second masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825. In some embodiments, the first masking moiety comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 219 - 229, and 261, and the second masking moiety comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825. In some embodiments, the second masking moiety comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 219 - 229, and 261, and the first masking moiety comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825.

[0249] In some embodiments, the first masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825, and the second masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 219 - 229, 261, 826, and 827. In some embodiments, the second masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825, and the first masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 219 - 229, 261, 826, and 827. In some embodiments, the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 219 - 229, 261, 826, and 827, and the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825. In some embodiments, the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 219 - 229, 261, 826, and 827, and the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825. In some embodiments, the first masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825, and the second masking portion comprises the amino acid sequence of SEQ ID NO: 826 or 827. In some embodiments, the second masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825, and the first masking portion comprises the amino acid sequence of SEQ ID NO: 826 or 827.

[0250] In some embodiments, the first masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825, and the second masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 221 - 226, and 261. In some embodiments, the first masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 221 - 226, and 261, and the second masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825. In some embodiments, the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 221 - 226, and 261, and the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825. In some embodiments, the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161 - 165, 221 - 226, and 261, and the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825.

[0251] In some embodiments, the first masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825, and the second masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 227 - 229. In some embodiments, the first masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 227 - 229, and the second masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825. In some embodiments, the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 227 - 229, and the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825. In some embodiments, the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 227 - 229, and the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825.

[0252] In some embodiments, the first masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825, and the second masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 219 and 220. In some embodiments, the first masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 219 and 220, and the second masking portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825. In some embodiments, the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 219 and 220, and the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825. In some embodiments, the second masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 219 and 220, and the first masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825.

[0253] In some embodiments, the masking moiety comprises an amino acid sequence produced by introducing one or more of the following amino acid substitutions into the amino acid sequence of any of SEQ ID NOs: 232 - 234 and 823 - 825: R24A, R26A, K34A, S40A, L42A, and P67A. In some embodiments, the masking moiety comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825, and is further modified by introducing one or more of the following amino acid substitutions into the amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 232 - 234 and 823 - 825: R24A, R26A, K34A, S40A, L42A, and P67A.

[0254] In some embodiments, the masking moiety comprises IL-2Rα (also known as CD25) or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-2. In some embodiments, the masking moiety comprises IL-2Rα (also known as CD25) or a fragment, portion or variant thereof, and comprises the amino acid sequence of SEQ ID NO: 9 or 231. In some embodiments, the masking moiety comprises IL-2Rβ (also known as CD122) or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-2 and / or IL-15. In some embodiments, the masking moiety comprises IL-2Rβ (also known as CD122) or a fragment, portion or variant thereof, and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 161-165, 221-226, 261, 826 and 827. In some embodiments, the masking moiety comprises IL-2Rγ (also known as CD132) or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-2 and / or IL-15. In some embodiments, the masking moiety comprises IL-15Rα (also known as CD215) or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-15. In some embodiments, the masking moiety comprises IL-15Rα (also known as CD215) or a fragment, portion or variant thereof, and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 232-234 and 823-825. In some embodiments, the masking moiety comprises the antigen-binding domain of an antibody or a fragment thereof. In some embodiments, the masking moiety comprises the antigen-binding domain of an anti-IL-2 antibody or a fragment thereof. In some embodiments, the masking moiety comprises the antigen-binding domain of an anti-IL-15 antibody or a fragment thereof.

[0255] In some embodiments that include a first masking portion and a second masking portion, the first masking portion comprises an IL-2Rα or a fragment, portion, or variant thereof that retains or otherwise exhibits an affinity for IL-2, and the second masking portion comprises an IL-2Rβ or a fragment, portion, or variant thereof that retains or otherwise exhibits an affinity for IL-2. In some embodiments that include a first masking portion and a second masking portion, the first masking portion comprises an IL-2Rβ or a fragment, portion, or variant thereof that retains or otherwise exhibits an affinity for IL-2, and the second masking portion comprises an IL-2Rα or a fragment, portion, or variant thereof that retains or otherwise exhibits an affinity for IL-2. In some embodiments that include a first masking portion and a second masking portion, the first masking portion comprises an IL-2Rα or a fragment, portion, or variant thereof that retains or otherwise exhibits an affinity for IL-2, and the second masking portion comprises an IL-2Rγ or a fragment, portion, or variant thereof that retains or otherwise exhibits an affinity for IL-2. In some embodiments that include a first masking portion and a second masking portion, the first masking portion comprises an IL-2Rγ or a fragment, portion, or variant thereof that retains or otherwise exhibits an affinity for IL-2, and the second masking portion comprises an IL-2Rα or a fragment, portion, or variant thereof that retains or otherwise exhibits an affinity for IL-2. In some embodiments that include a first masking portion and a second masking portion, the first masking portion comprises an IL-2Rα or a fragment, portion, or variant thereof that retains or otherwise exhibits an affinity for IL-2, and the second masking portion comprises an antigen-binding domain of an anti-IL-2 antibody or a fragment thereof. In some embodiments that include a first masking portion and a second masking portion, the first masking portion comprises an antigen-binding domain of an anti-IL-2 antibody or a fragment thereof, and the second masking portion comprises an IL-2Rα or a fragment, portion, or variant thereof that retains or otherwise exhibits an affinity for IL-2.

[0256] In some embodiments that include a first masking moiety and a second masking moiety, the first masking moiety comprises an IL-15Rα or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15, and the second masking moiety comprises an IL-2Rβ or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15. In some embodiments that include a first masking moiety and a second masking moiety, the first masking moiety comprises an IL-2Rβ or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15, and the second masking moiety comprises an IL-15Rα or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15. In some embodiments that include a first masking moiety and a second masking moiety, the first masking moiety comprises an IL-15Rα or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15, and the second masking moiety comprises an IL-2Rγ or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15. In some embodiments that include a first masking moiety and a second masking moiety, the first masking moiety comprises an IL-2Rγ or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15, and the second masking moiety comprises an IL-15Rα or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15. In some embodiments that include a first masking moiety and a second masking moiety, the first masking moiety comprises an IL-15Rα or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15, and the second masking moiety comprises an antigen-binding domain of an anti-IL-15 antibody or a fragment thereof. In some embodiments that include a first masking moiety and a second masking moiety, the first masking moiety comprises an antigen-binding domain of an anti-IL-15 antibody or a fragment thereof, and the second masking moiety comprises an IL-15Rα or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15.

[0257] C. Linker

[0258] As used herein, a linker refers to a peptide of more than two amino acids that is used to link together two components of any masked cytokine as described herein. In some embodiments, the linker comprises a cleavable peptide. In some embodiments, the linker comprises an amino-terminal spacer domain (N-terminal spacer domain) and / or a carboxy-terminal spacer domain (C-terminal spacer domain). In some embodiments, the linker comprises an N-terminal spacer domain, a cleavable peptide, and / or a C-terminal spacer domain. In some embodiments, the linker consists of an N-terminal spacer domain. In some embodiments, the linker consists of a C-terminal spacer domain. In some embodiments, the linker consists of an N-terminal spacer domain and a C-terminal spacer domain. In some embodiments, the linker consists of a cleavable peptide. In some embodiments, the linker consists of an N-terminal spacer domain, a cleavable peptide, and a C-terminal spacer domain. In some embodiments, the linker consists of an N-terminal spacer domain and a cleavable peptide. In some embodiments, the linker consists of a cleavable peptide and a C-terminal spacer domain. In some embodiments, the linker consists of a cleavable peptide.

[0259] In some embodiments, a masked cytokine comprises a linker. In some embodiments, a masked cytokine comprises a single linker. In some embodiments, a masked cytokine comprises more than one linker, each of which can be any linker as described herein. In some embodiments, a masked cytokine comprises a first linker and a second linker. In some embodiments, a masked cytokine comprises a first linker, a second linker, and a third linker. In some embodiments, a masked cytokine comprises a first linker, a second linker, a third linker, and a fourth linker. For example, reference to "the linker" or "a linker" can refer to the linker in a masked cytokine that comprises a single linker, or it can refer to the first linker in a masked cytokine that comprises a first linker and a second linker, or it can refer to the second linker in a masked cytokine that comprises a first linker and a second linker, or it can refer to the first linker and the second linker in a masked cytokine that comprises a first linker and a second linker, or it can refer to the first linker, the second linker, and / or the third linker in a masked cytokine that comprises a first linker, a second linker, and a third linker, or it can refer to the first linker, the second linker, the third linker, and / or the fourth linker in a masked cytokine that comprises a first linker, a second linker, a third linker, and a fourth linker.

[0260] It is to be understood that each of the following terms is considered a "linker" as described herein: linker, first linker, second linker, third linker, and fourth linker, and any other numbered linker (e.g., fifth linker, sixth linker, etc.). Thus, in some embodiments, a masked cytokine can comprise a linker, a first linker, a second linker, a third linker, and / or a fourth linker, each of which is considered a "linker" as described herein, and any linker described herein can be a linker, a first linker, a second linker, a third linker, and / or a fourth linker as described herein.

[0261] It is also to be understood that two components "linked" together include embodiments in which the two components are directly linked together and embodiments in which the two components are linked together via a linker as described herein. For example, "a half-life extension domain linked to a masking moiety" is interpreted to mean that the amino acid sequence of the half-life extension domain is directly linked to the amino acid sequence of the masking moiety, or the amino acid sequence of the half-life extension domain is linked to the amino acid sequence of the masking moiety via a linker, any linker as described herein.

[0262] A linker comprises an amino terminus and a carboxyl terminus. In some embodiments, the masking moiety is linked to the amino terminus or the carboxyl terminus of the linker. In some embodiments, a cytokine or a functional fragment thereof is linked to the amino terminus or the carboxyl terminus of the linker. In some embodiments, a cytokine or a functional fragment thereof is linked to the amino terminus of the linker, and the masking moiety is linked to the carboxyl terminus of the linker. In some embodiments, a cytokine or a functional fragment thereof is linked to the carboxyl terminus of the linker, and the masking moiety is linked to the amino terminus of the linker. In some embodiments, a half-life extension domain is linked to the amino terminus or the carboxyl terminus of the linker. In some embodiments, a half-life extension domain is linked to the amino terminus of the linker, and the masking moiety is linked to the carboxyl terminus of the linker. In some embodiments, a half-life extension domain is linked to the carboxyl terminus of the linker, and the masking moiety is linked to the amino terminus of the linker. In some embodiments, a half-life extension domain is linked to the amino terminus of the linker, and a cytokine or a functional fragment thereof is linked to the carboxyl terminus of the linker. In some embodiments, a half-life extension domain is linked to the carboxyl terminus of the linker, and a cytokine or a functional fragment thereof is linked to the amino terminus of the linker.

[0263] In some embodiments that include a first masking moiety and a second masking moiety each linked to a cytokine or a functional fragment thereof, the first masking moiety or the second masking moiety is linked to the amino terminus of a linker, and the cytokine or a functional fragment thereof is linked to the carboxyl terminus of the linker. In some embodiments that include a first masking moiety and a second masking moiety each linked to a cytokine or a functional fragment thereof, the first masking moiety or the second masking moiety is linked to the carboxyl terminus of a linker, and the cytokine or a functional fragment thereof is linked to the amino terminus of the linker.

[0264] In some embodiments that include a first half-life extending domain and a second half-life extending domain, the masking moiety is linked to the amino terminus of a linker, and the first half-life extending domain or the second half-life extending domain is linked to the carboxyl terminus of the linker. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the masking moiety is linked to the carboxyl terminus of a linker, and the first half-life extending domain or the second half-life extending domain is linked to the amino terminus of the linker. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the cytokine or a functional fragment thereof is linked to the amino terminus of a linker, and the first half-life extending domain or the second half-life extending domain is linked to the carboxyl terminus of the linker. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the cytokine or a functional fragment thereof is linked to the carboxyl terminus of a linker, and the first half-life extending domain or the second half-life extending domain is linked to the amino terminus of the linker.

[0265] In some embodiments that include a first masking moiety linked to a first half-life extending domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein the second masking moiety or the cytokine or its functional fragment is linked to a second half-life extending domain, the first masking moiety is linked to the amino terminus of the linker, and the first half-life extending domain is linked to the carboxyl terminus of the linker. In some embodiments that include a first masking moiety linked to a first half-life extending domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein the second masking moiety or the cytokine or its functional fragment is linked to a second half-life extending domain, the first masking moiety is linked to the carboxyl terminus of the linker, and the first half-life extending domain is linked to the amino terminus of the linker. In some embodiments that include a first masking moiety linked to a first half-life extending domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein the second masking moiety or the cytokine or its functional fragment is linked to a second half-life extending domain, the second masking moiety is linked to the amino terminus of the linker, and the cytokine or its functional fragment is linked to the carboxyl terminus of the linker. In some embodiments that include a first masking moiety linked to a first half-life extending domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein the second masking moiety or the cytokine or its functional fragment is linked to a second half-life extending domain, the second masking moiety is linked to the carboxyl terminus of the linker, and the cytokine or its functional fragment is linked to the amino terminus of the linker. In some embodiments that include a first masking moiety linked to a first half-life extending domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein the second masking moiety or the cytokine or its functional fragment is linked to a second half-life extending domain, the second masking moiety is linked to the amino terminus of the linker, and the second half-life extending domain is linked to the carboxyl terminus of the linker. In some embodiments that include a first masking moiety linked to a first half-life extending domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein the second masking moiety or the cytokine or its functional fragment is linked to a second half-life extending domain, the second masking moiety is linked to the carboxyl terminus of the linker, and the second half-life extending domain is linked to the amino terminus of the linker. In some embodiments that include a first masking moiety linked to a first half-life extending domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein the second masking moiety or the cytokine or its functional fragment is linked to a second half-life extending domain, the cytokine or its functional fragment is linked to the amino terminus of the linker, and the second half-life extending domain is linked to the carboxyl terminus of the linker.In some embodiments that include a first masking moiety linked to a first half-life extending domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein the second masking moiety or the cytokine or a functional fragment thereof is linked to a second half-life extending domain, the cytokine or a functional fragment thereof is linked to the carboxyl terminus of a linker, and the second half-life extending domain is linked to the amino terminus of the linker.

[0266] In some embodiments that include a first masking moiety linked to a first half-life extending domain via a first linker, a second masking moiety linked to the first masking moiety via a second linker, and a cytokine or a functional fragment thereof linked to a second half-life extending domain via a third linker, the first masking moiety is linked to the carboxyl terminus of the first linker, and the first half-life extending domain is linked to the amino terminus of the first linker, the second masking moiety is linked to the carboxyl terminus of the second linker, the first masking moiety is linked to the amino terminus of the second linker, the cytokine or a functional fragment thereof is linked to the carboxyl terminus of the third linker, and the second half-life extending domain is linked to the amino terminus of the third linker.

[0267] In some embodiments that include a first masking moiety linked to a first half-life extending domain via a first linker, a second masking moiety linked to the first masking moiety via a second linker, and a cytokine or a functional fragment thereof linked to a second half-life extending domain via a third linker, the first masking moiety is linked to the amino terminus of the first linker, and the first half-life extending domain is linked to the carboxyl terminus of the first linker, the second masking moiety is linked to the amino terminus of the second linker, the first masking moiety is linked to the carboxyl terminus of the second linker, the cytokine or a functional fragment thereof is linked to the amino terminus of the third linker, and the second half-life extending domain is linked to the carboxyl terminus of the third linker.

[0268] In some embodiments that include a second masking moiety linked to a first half-life extending domain via a first linker, a first masking moiety linked to the second masking moiety via a second linker, and a cytokine or a functional fragment thereof linked to a second half-life extending domain via a third linker, the second masking moiety is linked to the carboxyl terminus of the first linker, and the first half-life extending domain is linked to the amino terminus of the first linker, the first masking moiety is linked to the carboxyl terminus of the second linker, the second masking moiety is linked to the amino terminus of the second linker, the cytokine or a functional fragment thereof is linked to the carboxyl terminus of the third linker, and the second half-life extending domain is linked to the amino terminus of the third linker.

[0269] In some embodiments comprising a second masking moiety linked to a first half-life extending domain via a first linker, a first masking moiety linked to the second masking moiety via a second linker, and a cytokine or a functional fragment thereof linked to a second half-life extending domain via a third linker, the second masking moiety is linked to the amino terminus of the first linker, and the first half-life extending domain is linked to the carboxyl terminus of the first linker, the first masking moiety is linked to the amino terminus of the second linker, the second masking moiety is linked to the carboxyl terminus of the second linker, the cytokine or a functional fragment thereof is linked to the amino terminus of the third linker, and the second half-life extending domain is linked to the carboxyl terminus of the third linker.

[0270] In some embodiments comprising a first half-life extending domain and a second half-life extending domain, the first half-life extending domain is linked to the second half-life extending domain. In some embodiments comprising a first half-life extending domain and a second half-life extending domain, the first half-life extending domain is linked to the second half-life extending domain via a linker.

[0271] In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-153, 235-242, 262-264, 268-320, 323-338, 340-354, 356-555, 668, 691, 724, 725, 727, 762-771, 794, and 797-812. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-153, 235-242, 264, 268-317, 323-338, 340-347, 356-415, 420-491, 494-501, 504-535, 538-555, 727, 794, and 799. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-19, 262, 263, 318-320, 348-354, 416-419, 492, 493, 502, 503, 536, 537, 668, 691, 724, 725, 762-771, 797, 798, and 800-812. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 262. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, and 269. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-33. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-44. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-95. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 235. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 268. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 269. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 263, 318-322, 339, 348-355, 416-419, 492, 493, 502, 503, 536, 537, 668, 691, 724, 725, 727, 762-771, 794, 795, and 797-812.

[0272] In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 794, 795, and 797 - 812. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 270 - 302, 306 - 317, 342 - 347, 356 - 415, 420 - 491, 494 - 501, 504 - 535, and 538 - 555.

[0273] In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 - 153, 235 - 242, 262 - 264, 268 - 320, 323 - 338, 340 - 354, 356 - 555, 668, 691, 724, 725, 727, 762 - 771, 794, and 797 - 812. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 - 19, 262, 263, 318 - 320, 348 - 354, 416 - 419, 492, 493, 502, 503, 536, 537, 668, 691, 724, 725, 762 - 771, 797, 798, and 800 - 812. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 - 33. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 - 44. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 45 - 95. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 235.In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 262. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 263, 318 - 320, 348 - 354, 416 - 419, 492, 493, 502, 503, 536, 537, 668, 691, 724, 725, 762 - 771, 797, 798, and 800 - 812. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 318 - 320. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 348 - 354. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 416 - 419. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 492, 493, 502, 503, 536, 537. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 668, 691, 724, 725, 762 - 771, 797, 798, and 800 - 812.In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 762 - 771, 797, 798, and 800 - 812. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 762 - 771. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 797, 798, and 800 - 812. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 800 - 812.

[0274] Some embodiments of the cleavable peptides and spacer domains of the linker are described in more detail below.

[0275] 1. Cleavable Peptides

[0276] In some embodiments, the linker comprises a cleavable peptide. In some embodiments having more than one linker comprising a cleavable peptide, the cleavable peptides may be referred to as a first cleavable peptide, a second cleavable peptide, or a third cleavable peptide, each of which is considered a "cleavable peptide" and may be any of the cleavable peptides described herein. For example, reference to "the cleavable peptide" or "a cleavable peptide" may refer to the cleavable peptide in a masked cytokine comprising a single cleavable peptide, or it may refer to the first cleavable peptide in a masked cytokine comprising a first and a second cleavable peptide, or it may refer to the second cleavable peptide in a masked cytokine comprising a first and a second cleavable peptide, or it may refer to the first and second cleavable peptides in a masked cytokine comprising a first and a second cleavable peptide, or it may refer to the first, second, and / or third cleavable peptides in a masked cytokine comprising a first, a second, and a third cleavable peptide. It is contemplated that in some embodiments, there may be more than three cleavable peptides according to the teachings herein.

[0277] A cleavable peptide is a polypeptide that includes cleavage sites such as protease cleavage sites. In some embodiments, the cleavable peptide comprises more than one cleavage site. As used herein, a "cleavage site" refers to a recognizable site for cleaving a portion of the cleavable peptide seen in any linker that includes the cleavable peptide described herein. Thus, cleavage sites are seen in the sequence of the cleavable peptide as described herein. In some embodiments, the cleavage site is an amino acid sequence that is recognized and cleaved by a cleavage agent. Exemplary cleavage agents include proteins, enzymes, DNases, RNases, metals, acids, and bases.

[0278] In some embodiments, the cleavable peptide comprises a protease cleavage site. In some embodiments, the protease cleavage site is a tumor-associated protease cleavage site. As provided herein, a "tumor-associated protease cleavage site" is an amino acid sequence recognized by a protease, the expression of which is specific or upregulated for tumor cells or their tumor cell environment. In some embodiments, the protease cleavage site is a cleavage site recognized by one or more enzymes selected from the group consisting of: ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, CTR1, CTSA, CTSW, CTSB, CTSC, CTSD, ESP1, CTSG, CTSH, GZMA, GZMB, GZMH, CTSK, GZMM, CTSL, CTSS, CTSV, CTSZ, HTRA4, KLK10, KLK11, KLK13, KLK14, KLK2, KLK4, DPP4, KLK6, KLK7, KLKB1, ECE1, ECE2, ECEL1, MASP2, MEP1A, MEP1B, ELANE, FAP, GZMA, MMP11, GZMK, HGFAC, HPN, HTRA1, MMP11, MMP16, MMP17, MMP19, HTRA2, MMP20, MMP21, HTRA3, HTRA4, KEL, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, KLK5, MMP3, MMP7, MMP8, MMP9, LGMN, LNPEP, MASP1, PAPPA, PAPPA2, PCSK1, NAPSA, PCSK5, PCSK6, MME,MMP1, MMP10, PLAT, PLAU, PLG, PRSS1, PRSS12, PRSS2, PRSS21, PRSS3, PRSS33, PRSS4, PRSS55, PRSS57, MMP12, PRSS8, PRSS9, PRTN3, MMP13, MMP14, ST14, TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1, and TPSG1. In some embodiments, the protease cleavage site is a cleavage site recognized by one or more enzymes selected from the group consisting of: ADAM17, HTRA1, PRSS1, FAP, GZMK, NAPSA, MMP1, MMP2, MMP9, MMP10, MMP7, MMP12, MMP28, ADAMTS9, HGFAC, and HTRA3.

[0279] In certain embodiments, the protease cleavage site is a matrix metalloproteinase (MMP) cleavage site, an a disintegrin and metalloproteinase (ADAM) metalloprotease cleavage site, a prostate specific antigen (PSA) protease cleavage site, a urokinase type plasminogen activator (uPA) protease cleavage site, a membrane-type serine protease 1 (MT-SP1) protease cleavage site, a protein sheddase protease cleavage site (ST14), or a podoplanin protease cleavage site. In certain embodiments, the matrix metalloproteinase (MMP) cleavage site is an MMP9 cleavage site, an MMP13 cleavage site, or an MMP2 cleavage site. In certain embodiments, the a disintegrin and metalloproteinase (ADAM) metalloprotease cleavage site is an ADAM9 metalloprotease cleavage site, an ADAM10 metalloprotease cleavage site, or an ADAM17 metalloprotease cleavage site. The protease cleavage site can be specified by a specific amino acid sequence.

[0280] In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of: ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, CTR1, CTSA, CTSW, CTSB, CTSC, CTSD, ESP1, CTSG, CTSH, GZMA, GZMB, GZMH, CTSK, GZMM, CTSL, CTSS, CTSV, CTSZ, HTRA4, KLK10, KLK11, KLK13, KLK14, KLK2, KLK4, DPP4, KLK6, KLK7, KLKB1, ECE1, ECE2, ECEL1, MASP2, MEP1A, MEP1B, ELANE, FAP, GZMA, MMP11, GZMK, HGFAC, HPN, HTRA1, MMP11, MMP16, MMP17, MMP19, HTRA2, MMP20, MMP21, HTRA3, HTRA4, KEL, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, KLK5, MMP3, MMP7, MMP8, MMP9, LGMN, LNPEP, MASP1, PAPPA, PAPPA2, PCSK1, NAPSA, PCSK5, PCSK6, MME, MMP1, MMP10, PLAT, PLAU, PLG, PRSS1, PRSS12, PRSS2, PRSS21, PRSS3, PRSS33, PRSS4, PRSS55, PRSS57, MMP12, PRSS8, PRSS9, PRTN3, MMP13, MMP14, ST14,TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1, and TPSG1. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of: ADAM17, HTRA1, PRSS1, FAP, GZMK, NAPSA, MMP1, MMP2, MMP9, MMP10, MMP7, MMP12, MMP28, ADAMTS9, HGFAC, and HTRA3. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of uPA and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of a proteolytic enzyme and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of legumain and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of a proteolytic enzyme and uPA. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of uPA and legumain. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of a proteolytic enzyme and legumain. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of uPA, a proteolytic enzyme, and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of uPA, legumain, and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of a proteolytic enzyme, legumain, and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of a proteolytic enzyme, legumain, and uPA.,

[0281] In some embodiments, the cleavable peptide is a substrate of a protease that is co-localized in a region or tissue expressing a cytokine receptor. The cytokine receptor can be any cytokine receptor. In some embodiments, the cytokine receptor is selected from the group consisting of: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCR1, CX3CR1, IL-1RAP, IL-1RAPL1, IL-1RAPL2, IL-1RL1, IL-1RL2, IL-1R1, IL-1R2, IL-2R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, IL-4R, IL-5Rα, IL-6R, IL-6ST, IL-7R, IL-9R, IL-10Rα, IL-10Rβ, IL-11Rα, IL-12Rβ1, IL-12Rβ2, IL-13Rα1, IL-13Rα2, IL-15Rα, IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE, IL-18RAP, IL-18R1, IL-20Rα, IL-20Rβ, IL-21R, IL-22Rα1, IL-22Rα2, IL-23R, IL-27Rα, IL-28Rα, IL-31RA, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, GMRα(CD116), CD131, GHR, PRLR, EPOR, LIFR(CD118), OSMRβ, TPO-R(CD110), CSF-1R, EDAR, TNFRSF1A, TNFRSF1B, LTBR, TNFRSF4, CD40, FAS, TNFRSF6B, CD27, TNFRSF8, TNFRSF9, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11A, TNFRSF11B, TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF14, NGFR, TNFRSF17, TNFRSF18, TNFRSF19, RELT, TNFRSF21, TNFRSF25 and EDA2R.

[0282] In some embodiments, the cleavable peptide is a pentamer (i.e., a peptide of 5 amino acids in length), a hexamer (i.e., a peptide of 6 amino acids in length), a heptamer (i.e., a peptide of 7 amino acids in length), an octamer (i.e., a peptide of 8 amino acids in length), a nonamer (i.e., a peptide of 9 amino acids in length), a decamer (i.e., a peptide of 10 amino acids in length), an undecamer (i.e., a peptide of 11 amino acids in length), a dodecamer (i.e., a peptide of 12 amino acids in length), a tridecamer (i.e., a peptide of 13 amino acids in length), a tetradecamer (i.e., a peptide of 14 amino acids in length), a pentadecamer (i.e., a peptide of 15 amino acids in length), a hexadecamer (i.e., a peptide of 16 amino acids in length), a heptadecamer (i.e., a peptide of 17 amino acids in length), or an octadecamer (i.e., a peptide of 18 amino acids in length). Exemplary cleavable peptide sequences are shown in Table 1.

[0283] Table 1. Exemplary cleavable peptide sequences

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291] In some embodiments, the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 96 - 153, 236 - 242, 264, 270 - 302, 306 - 317, 342 - 347, 356 - 415, 420 - 491, 494 - 501, 504 - 535, and 538 - 555. In some embodiments, the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 270 - 302, 306 - 317, 342 - 347, 356 - 415, 420 - 491, 494 - 501, 504 - 535, and 538 - 555. In some embodiments, the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 281, 293, 297, 399 - 415, 420 - 491, 494 - 501, 504 - 535, and 538 - 555. In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 264. In some embodiments, the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 96 - 153, 264, 270 - 302, 306 - 317, 342 - 347, 356 - 415, 420 - 491, 494 - 501, 504 - 535, and 538 - 555 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 236 - 242. In some embodiments, the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 96 - 131 and 264 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 236 - 242. In some embodiments, the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 96 - 131 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 236 - 242. In embodiments where the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 96 - 153, 264, 270 - 302, 306 - 317, 342 - 347, 356 - 415, 420 - 491, 494 - 501, 504 - 535, and 538 - 555 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 236 - 242, the two amino acid sequences can be linked in any order.For example, in embodiments where the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 96 - 153, 236 - 242, 264, 270 - 302, 306 - 317, 342 - 347, 356 - 415, 420 - 491, 494 - 501, 504 - 535, and 538 - 555 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 236 - 242, the amino acid sequence selected from the group consisting of SEQ ID NOs: 96 - 153, 236 - 242, 264, 270 - 302, 306 - 317, 342 - 347, 356 - 415, 420 - 491, 494 - 501, 504 - 535, and 538 - 555 comprises an N-terminus and a C-terminus, and the amino acid sequence selected from the group consisting of SEQ ID NOs: 236 - 242 is linked to the N-terminus or C-terminus of the amino acid sequence selected from the group consisting of SEQ ID NOs: 96 - 153, 264, 270 - 302, 306 - 317, 342 - 347, 356 - 415, 420 - 491, 494 - 501, 504 - 535, and 538 - 555. In some embodiments where the cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 96 - 131 and 264 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 236 - 242, the amino acid sequence selected from the group consisting of SEQ ID NOs: 96 - 131 and 264 comprises an N-terminus and a C-terminus, and the amino acid sequence selected from the group consisting of SEQ ID NOs: 236 - 242 is linked to the N-terminus or C-terminus of the amino acid sequence selected from the group consisting of SEQ ID NOs: 96 - 131 and 264.

[0292] In some embodiments, one or more additional amino acids are incorporated into the amino acid sequence of the cleavable peptide by addition. In some embodiments, the one or more amino acids incorporated into the amino acid sequence of the cleavable peptide by addition are selected from the group consisting of: hydrophilic amino acids (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine), hydrophobic amino acids (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, glycine, or proline), polar amino acids (e.g., serine, threonine, cysteine, asparagine, glutamine, or tyrosine), nonpolar amino acids (e.g., glycine, alanine, valine, proline, leucine, isoleucine, methionine, tryptophan, or phenylalanine), amino acids having aliphatic side chains (e.g., glycine, alanine, valine, leucine, or isoleucine), amino acids having hydroxyl-containing side chains (e.g., serine or threonine), amino acids having sulfur-containing side chains (e.g., cysteine or methionine), charged amino acids (e.g., arginine, lysine, aspartic acid, or glutamic acid), uncharged amino acids (e.g., serine, threonine, asparagine, or glutamine), aromatic amino acids (e.g., tyrosine, tryptophan, or phenylalanine), cyclic amino acids (e.g., proline), acidic amino acids (e.g., aspartic acid, asparagine, glutamic acid, or glutamine), basic amino acids (e.g., histidine, lysine, or arginine), and bulky amino acids (e.g., phenylalanine, tyrosine, or tryptophan). In some embodiments, glycine (G) is incorporated by addition to the N-terminus and / or C-terminus of the amino acid sequence of the cleavable peptide. In some embodiments, glycine (G) and proline (P) are incorporated by addition to the N-terminus and / or C-terminus of the amino acid sequence of the cleavable peptide, such as by incorporating the amino acid sequence GP or PG into the N-terminus and / or C-terminus of the amino acid sequence of the cleavable peptide.

[0293] The cleavable peptide comprises an amino terminus and a carboxyl terminus. In some embodiments, the cleavable peptide is linked to an N-terminal spacer domain and / or a C-terminal spacer domain. In some embodiments, the N-terminal spacer domain is linked to the amino terminus of the cleavable peptide. In some embodiments, the C-terminal spacer domain is linked to the carboxyl terminus of the cleavable peptide. In some embodiments, the N-terminal spacer domain is linked to the amino terminus of the cleavable peptide and the C-terminal spacer domain is linked to the carboxyl terminus of the cleavable peptide. In some embodiments, the N-terminal spacer domain is linked to the amino terminus of the cleavable peptide and the C-terminal spacer domain is linked to the carboxyl terminus of the cleavable peptide.

[0294] In some embodiments, the amino terminus of the cleavable peptide is linked to a component other than the N-terminal spacer domain. In some embodiments, the carboxy terminus of the cleavable peptide is linked to a component other than the C-terminal spacer domain. In some embodiments, the masking moiety is linked to the amino terminus or the carboxy terminus of the cleavable peptide. In some embodiments, a cytokine or a functional fragment thereof is linked to the amino terminus or the carboxy terminus of the cleavable peptide. In some embodiments, the half-life extension domain is linked to the amino terminus or the carboxy terminus of the cleavable peptide. In some embodiments, the first half-life extension domain is linked to the amino terminus or the carboxy terminus of the cleavable peptide. In some embodiments, the second half-life extension domain is linked to the amino terminus or the carboxy terminus of the cleavable peptide.

[0295] 2. Spacer Domain

[0296] In some embodiments, the linker comprises a spacer domain. As used herein, in some embodiments, "spacer domain" may refer to the N-terminal spacer domain and / or the C-terminal spacer domain. In some embodiments, the linker comprises the N-terminal spacer domain and / or the C-terminal spacer domain. In some embodiments, the linker comprises the N-terminal spacer domain. In some embodiments, the linker comprises the C-terminal spacer domain. In some embodiments, the linker comprises the N-terminal spacer domain and the C-terminal spacer domain. In some embodiments, the linker comprises the N-terminal spacer domain and the cleavable peptide. In some embodiments, the linker comprises the cleavable peptide and the C-terminal spacer domain. In some embodiments, the linker comprises the N-terminal spacer domain, the cleavable peptide, and the C-terminal spacer domain.

[0297] In some embodiments, such as some embodiments having more than one N-terminal spacer domain, the N-terminal spacer domain may be referred to as a first N-terminal spacer domain, a second N-terminal spacer domain, or a third N-terminal spacer domain, each of which is considered an "N-terminal spacer domain" and may be any N-terminal spacer domain described herein. Similarly, in some embodiments, such as some embodiments having more than one C-terminal spacer domain, the C-terminal spacer domain may be referred to as a first C-terminal spacer domain, a second C-terminal spacer domain, or a third C-terminal spacer domain, each of which is considered a "C-terminal spacer domain" and may be any C-terminal spacer domain described herein. For example, reference to an "N-terminal spacer domain" or "the N-terminal spacer domain" may refer to the N-terminal spacer domain in a masked cytokine comprising a single N-terminal spacer domain, or it may refer to the first N-terminal spacer domain in a masked cytokine comprising a first N-terminal spacer domain and a second N-terminal spacer domain, or it may refer to the second N-terminal spacer domain in a masked cytokine comprising a first N-terminal spacer domain and a second N-terminal spacer domain, or it may refer to the first N-terminal spacer domain and the second N-terminal spacer domain in a masked cytokine comprising a first N-terminal spacer domain and a second N-terminal spacer domain, or it may refer to the first N-terminal spacer domain, the second N-terminal spacer domain, and / or the third N-terminal spacer domain in a masked cytokine comprising a first N-terminal spacer domain, a second N-terminal spacer domain, and a third N-terminal spacer domain.

[0298] Each of the N-terminal spacer domain and the C-terminal spacer domain comprises an amino terminus and a carboxyl terminus. In some embodiments, the masking moiety is linked to the amino terminus or the carboxyl terminus of the N-terminal spacer domain. In some embodiments, the cytokine or a functional fragment thereof is linked to the amino terminus or the carboxyl terminus of the N-terminal spacer domain. In some embodiments, the half-life extending domain is linked to the amino terminus or the carboxyl terminus of the N-terminal spacer domain. In some embodiments, the first half-life extending domain is linked to the amino terminus or the carboxyl terminus of the N-terminal spacer domain. In some embodiments, the second half-life extending domain is linked to the amino terminus or the carboxyl terminus of the N-terminal spacer domain.

[0299] In some embodiments, the masking moiety is linked to the amino terminus or carboxy terminus of the C-terminal spacer domain. In some embodiments, the cytokine or a functional fragment thereof is linked to the amino terminus or carboxy terminus of the C-terminal spacer domain. In some embodiments, the half-life extending domain is linked to the amino terminus or carboxy terminus of the C-terminal spacer domain. In some embodiments, the first half-life extending domain is linked to the amino terminus or carboxy terminus of the C-terminal spacer domain. In some embodiments, the second half-life extending domain is linked to the amino terminus or carboxy terminus of the C-terminal spacer domain.

[0300] In some embodiments, the masking moiety is linked to the amino terminus of the N-terminal spacer domain and the half-life extending domain is linked to the carboxy terminus of the N-terminal spacer domain. In some embodiments, the cytokine or a functional fragment thereof is linked to the amino terminus of the N-terminal spacer domain and the half-life extending domain is linked to the carboxy terminus of the N-terminal spacer domain. In some embodiments, the masking moiety is linked to the carboxy terminus of the N-terminal spacer domain and the half-life extending domain is linked to the amino terminus of the N-terminal spacer domain. In some embodiments, the cytokine or a functional fragment thereof is linked to the carboxy terminus of the N-terminal spacer domain and the half-life extending domain is linked to the amino terminus of the N-terminal spacer domain.

[0301] In some embodiments, the masking moiety is linked to the amino terminus of the C-terminal spacer domain and the half-life extending domain is linked to the carboxy terminus of the C-terminal spacer domain. In some embodiments, the cytokine or a functional fragment thereof is linked to the amino terminus of the C-terminal spacer domain and the half-life extending domain is linked to the carboxy terminus of the C-terminal spacer domain. In some embodiments, the masking moiety is linked to the carboxy terminus of the C-terminal spacer domain and the half-life extending domain is linked to the amino terminus of the C-terminal spacer domain. In some embodiments, the cytokine or a functional fragment thereof is linked to the carboxy terminus of the C-terminal spacer domain and the half-life extending domain is linked to the amino terminus of the C-terminal spacer domain.

[0302] In some embodiments that include a first masking moiety and a second masking moiety, the first masking moiety or the second masking moiety is linked to the amino terminus of the N-terminal spacer domain and the cytokine or a functional fragment thereof is linked to the carboxy terminus of the N-terminal spacer domain. In some embodiments that include a first masking moiety and a second masking moiety, the first masking moiety or the second masking moiety is linked to the carboxy terminus of the N-terminal spacer domain and the cytokine or a functional fragment thereof is linked to the amino terminus of the N-terminal spacer domain.

[0303] In some embodiments that include a first masking portion and a second masking portion, the first masking portion or the second masking portion is linked to the amino terminus of the C-terminal spacer domain, and the cytokine or its functional fragment is linked to the carboxyl terminus of the C-terminal spacer domain. In some embodiments that include a first masking portion and a second masking portion, the first masking portion or the second masking portion is linked to the carboxyl terminus of the C-terminal spacer domain, and the cytokine or its functional fragment is linked to the amino terminus of the C-terminal spacer domain.

[0304] In some embodiments that include a first masking portion and a second masking portion, the first masking portion is linked to the amino terminus of the C-terminal spacer domain, and the second masking portion is linked to the carboxyl terminus of the C-terminal spacer domain. In some embodiments that include a first masking portion and a second masking portion, the first masking portion is linked to the carboxyl terminus of the C-terminal spacer domain, and the second masking portion is linked to the amino terminus of the C-terminal spacer domain.

[0305] In some embodiments that include a first masking portion and a second masking portion, the half-life extending domain is linked to the amino terminus of the N-terminal spacer domain, and the first masking portion or the second masking portion is linked to the carboxyl terminus of the N-terminal spacer domain. In some embodiments that include a first masking portion and a second masking portion, the half-life extending domain is linked to the carboxyl terminus of the N-terminal spacer domain, and the first masking portion or the second masking portion is linked to the amino terminus of the N-terminal spacer domain.

[0306] In some embodiments that include a first masking portion and a second masking portion, the first masking portion is linked to the amino terminus of the N-terminal spacer domain, and the second masking portion is linked to the carboxyl terminus of the N-terminal spacer domain. In some embodiments that include a first masking portion and a second masking portion, the first masking portion is linked to the carboxyl terminus of the N-terminal spacer domain, and the second masking portion is linked to the amino terminus of the N-terminal spacer domain.

[0307] In some embodiments that include a first masking portion and a second masking portion, the half-life extending domain is linked to the amino terminus of the C-terminal spacer domain, and the first masking portion or the second masking portion is linked to the carboxyl terminus of the C-terminal spacer domain. In some embodiments that include a first masking portion and a second masking portion, the half-life extending domain is linked to the carboxyl terminus of the C-terminal spacer domain, and the first masking portion or the second masking portion is linked to the amino terminus of the C-terminal spacer domain.

[0308] In some embodiments that include a first half-life extending domain and a second half-life extending domain, the masking moiety is linked to the amino terminus of the N-terminal spacer domain, and the first half-life extending domain or the second half-life extending domain is linked to the carboxyl terminus of the N-terminal spacer domain. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the masking moiety is linked to the carboxyl terminus of the N-terminal spacer domain, and the first half-life extending domain or the second half-life extending domain is linked to the amino terminus of the N-terminal spacer domain. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the cytokine or a functional fragment thereof is linked to the amino terminus of the N-terminal spacer domain, and the first half-life extending domain or the second half-life extending domain is linked to the carboxyl terminus of the N-terminal spacer domain. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the cytokine or a functional fragment thereof is linked to the carboxyl terminus of the N-terminal spacer domain, and the first half-life extending domain or the second half-life extending domain is linked to the amino terminus of the N-terminal spacer domain.

[0309] In some embodiments that include a first half-life extending domain and a second half-life extending domain, the masking moiety is linked to the amino terminus of the C-terminal spacer domain, and the first half-life extending domain or the second half-life extending domain is linked to the carboxyl terminus of the C-terminal spacer domain. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the masking moiety is linked to the carboxyl terminus of the C-terminal spacer domain, and the first half-life extending domain or the second half-life extending domain is linked to the amino terminus of the C-terminal spacer domain. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the cytokine or a functional fragment thereof is linked to the amino terminus of the C-terminal spacer domain, and the first half-life extending domain or the second half-life extending domain is linked to the carboxyl terminus of the C-terminal spacer domain. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the cytokine or a functional fragment thereof is linked to the carboxyl terminus of the C-terminal spacer domain, and the first half-life extending domain or the second half-life extending domain is linked to the amino terminus of the C-terminal spacer domain.

[0310] In some embodiments that include a first half-life extending domain, a second half-life extending domain, a first masking moiety, and a second masking moiety, the first masking moiety is linked to the amino terminus of the N-terminal spacer domain, and the first half-life extending domain is linked to the carboxy terminus of the N-terminal spacer domain. In some embodiments that include a first half-life extending domain, a second half-life extending domain, a first masking moiety, and a second masking moiety, the first masking moiety is linked to the carboxy terminus of the N-terminal spacer domain, and the first half-life extending domain is linked to the amino terminus of the N-terminal spacer domain. In some embodiments that include a first half-life extending domain, a second half-life extending domain, a first masking moiety, and a second masking moiety, the first masking moiety is linked to the amino terminus of the C-terminal spacer domain, and the first half-life extending domain is linked to the carboxy terminus of the C-terminal spacer domain. In some embodiments that include a first half-life extending domain, a second half-life extending domain, a first masking moiety, and a second masking moiety, the first masking moiety is linked to the carboxy terminus of the C-terminal spacer domain, and the first half-life extending domain is linked to the amino terminus of the C-terminal spacer domain.

[0311] In some embodiments that include a first half-life extending domain, a second half-life extending domain, a first masking moiety, and a second masking moiety, the second masking moiety is linked to the amino terminus of the N-terminal spacer domain, and the cytokine or its functional fragment is linked to the carboxy terminus of the N-terminal spacer domain. In some embodiments that include a first half-life extending domain, a second half-life extending domain, a first masking moiety, and a second masking moiety, the second masking moiety is linked to the carboxy terminus of the N-terminal spacer domain, and the cytokine or its functional fragment is linked to the amino terminus of the N-terminal spacer domain. In some embodiments that include a first half-life extending domain, a second half-life extending domain, a first masking moiety, and a second masking moiety, the second masking moiety is linked to the amino terminus of the C-terminal spacer domain, and the cytokine or its functional fragment is linked to the carboxy terminus of the C-terminal spacer domain. In some embodiments that include a first half-life extending domain, a second half-life extending domain, a first masking moiety, and a second masking moiety, the second masking moiety is linked to the carboxy terminus of the C-terminal spacer domain, and the cytokine or its functional fragment is linked to the amino terminus of the C-terminal spacer domain.

[0312] In some embodiments that include a first half-life extending domain, a second half-life extending domain, a first masking moiety, and a second masking moiety, the second half-life extending domain is linked to the amino terminus of the N-terminal spacer domain, and the cytokine or its functional fragment or the second masking moiety is linked to the carboxyl terminus of the N-terminal spacer domain. In some embodiments that include a first half-life extending domain, a second half-life extending domain, a first masking moiety, and a second masking moiety, the second half-life extending domain is linked to the carboxyl terminus of the N-terminal spacer domain, and the cytokine or its functional fragment or the second masking moiety is linked to the amino terminus of the N-terminal spacer domain. In some embodiments that include a first half-life extending domain, a second half-life extending domain, a first masking moiety, and a second masking moiety, the second half-life extending domain is linked to the amino terminus of the C-terminal spacer domain, and the cytokine or its functional fragment or the second masking moiety is linked to the carboxyl terminus of the C-terminal spacer domain. In some embodiments that include a first half-life extending domain, a second half-life extending domain, a first masking moiety, and a second masking moiety, the second half-life extending domain is linked to the carboxyl terminus of the C-terminal spacer domain, and the cytokine or its functional fragment or the second masking moiety is linked to the amino terminus of the C-terminal spacer domain.

[0313] In some embodiments that include a first half-life extending domain and a second half-life extending domain, the first half-life extending domain is linked to the second half-life extending domain via a linker. In some embodiments, the linker that links the first half-life extending domain and the second half-life extending domain includes an N-terminal spacer domain and / or a C-terminal spacer domain. In some embodiments, the linker that links the first half-life extending domain and the second half-life extending domain includes a cleavable peptide and an N-terminal spacer domain and / or a C-terminal spacer domain. In some embodiments that include a first half-life extending domain and a second half-life extending domain linked together via a linker, the linker includes an amino terminus and a carboxyl terminus, the first half-life extending domain is linked to the amino terminus of the linker, and the second half-life extending domain is linked to the carboxyl terminus of the linker. In some embodiments that include a first half-life extending domain and a second half-life extending domain linked together via a linker, the linker includes an amino terminus and a carboxyl terminus, the first half-life extending domain is linked to the carboxyl terminus of the linker, and the second half-life extending domain is linked to the amino terminus of the linker.

[0314] In some embodiments, the N-terminal spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, 269, 303-305, 323-338, 340, 341, 727, 794, and 799. In some embodiments, the C-terminal spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, 269, 303-305, 323-338, 340, 341, 727, 794, and 799. In some embodiments, the N-terminal spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, 269, 303-305, 323-338, 340, 341, 727, 794, and 799, and the C-terminal spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, 269, 303-305, 323-338, 340, 341, 727, 794, and 799.

[0315] In some embodiments, the N-terminal spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, and 269. In some embodiments, the C-terminal spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, and 269. In some embodiments, the N-terminal spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, and 269, and the C-terminal spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, and 269. In some embodiments, the linker comprises an N-terminal spacer domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, and 269 and a C-terminal spacer domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, and 269. In some embodiments, the linker comprises an N-terminal spacer domain comprising the amino acid sequence of SEQ ID NO: 268 and a C-terminal spacer domain comprising the amino acid sequence of SEQ ID NO: 269. In some embodiments, the linker comprises an N-terminal spacer domain comprising the amino acid sequence of SEQ ID NO: 268, a cleavable peptide comprising the amino acid sequence of SEQ ID NO: 264, and a C-terminal spacer domain comprising the amino acid sequence of SEQ ID NO: 269. In some embodiments, the linker comprises an N-terminal spacer domain comprising the amino acid sequence of SEQ ID NO: 269 and a C-terminal spacer domain comprising the amino acid sequence of SEQ ID NO: 268. In some embodiments, the linker comprises an N-terminal spacer domain comprising the amino acid sequence of SEQ ID NO: 269, a cleavable peptide comprising the amino acid sequence of SEQ ID NO: 264, and a C-terminal spacer domain comprising the amino acid sequence of SEQ ID NO: 268.

[0316] In some embodiments, the N-terminal spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, and 269. In some embodiments, the N-terminal spacer domain comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20-95, 235, 268, 269, 303-305, 323-338, 340, 341, 727, 794, and 799. In some embodiments, the N-terminal spacer domain comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20-95, 235, 268, and 269. In some embodiments, the C-terminal spacer domain comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20-95, 235, 268, 269, 303-305, 323-338, 340, 341, 727, 794, and 799. In some embodiments, the C-terminal spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, and 269. In some embodiments, the C-terminal spacer domain comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20-95, 235, 268, and 269. In some embodiments, the N-terminal spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, and 269, and the C-terminal spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 268, and 269.In some embodiments, the N-terminal spacer domain comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20-95, 235, 268, and 269, and the C-terminal spacer domain comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20-95, 235, 268, and 269. In some embodiments, the N-terminal spacer domain comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 268, and the C-terminal spacer domain comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 269. In some embodiments, the N-terminal spacer domain comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 268, the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 264, and the C-terminal spacer domain comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 269. Exemplary spacer domains (e.g., N-terminal spacer domain and / or C-terminal spacer domain) are shown in Table 2.

[0317] Table 2. Exemplary Spacer Domains

[0318]

[0319]

[0320]

[0321] In some embodiments, the N-terminal spacer domain comprises an amino acid sequence produced by modifying the amino acid sequence of any N-terminal spacer domain described herein. In some embodiments, the N-terminal spacer domain comprises an amino acid sequence produced by modifying the amino acid sequence of any one of SEQ ID NOs: 20-95, 235, 268, 269, 303-305, 323-338, 340, 341, 727, 794, and 799. In some embodiments, the C-terminal spacer domain comprises an amino acid sequence produced by modifying the amino acid sequence of any C-terminal spacer domain described herein. In some embodiments, the C-terminal spacer domain comprises an amino acid sequence produced by modifying the amino acid sequence of any one of SEQ ID NOs: 20-95, 235, 268, 269, 303-305, 323-338, 340, 341, 727, 794, and 799. In some embodiments, the N-terminal spacer domain comprises an amino acid sequence produced by modifying the amino acid sequence of any one of SEQ ID NOs: 20-95, 235, 268, 269, 303-305, 323-338, 340, 341, 727, 794, and 799, and the C-terminal spacer domain comprises an amino acid sequence produced by modifying the amino acid sequence of any one of SEQ ID NOs: 20-95, 235, 268, 269, 303-305, 323-338, 340, 341, 727, 794, and 799. In some embodiments, the N-terminal spacer domain and / or the C-terminal spacer domain comprises the amino acid sequence of SEQ ID NO: 28.

[0322] In some embodiments, the N-terminal spacer domain and / or the C-terminal spacer domain consists of one or more amino acids. In some embodiments, the one or more amino acids of the N-terminal spacer domain and / or the C-terminal spacer domain are selected from the group consisting of: hydrophilic amino acids (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine or glutamine), hydrophobic amino acids (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, glycine or proline), polar amino acids (e.g., serine, threonine, cysteine, asparagine, glutamine or tyrosine), non-polar amino acids (e.g., glycine, alanine, valine, proline, leucine, isoleucine, methionine, tryptophan or phenylalanine), amino acids with aliphatic side chains (e.g., glycine, alanine, valine, leucine or isoleucine), amino acids with hydroxy-containing side chains (e.g., serine or threonine), amino acids with sulfur-containing side chains (e.g., cysteine or methionine), charged amino acids (e.g., arginine, lysine, aspartic acid or glutamic acid), uncharged amino acids (e.g., serine, threonine, asparagine or glutamine), aromatic amino acids (e.g., tyrosine, tryptophan or phenylalanine), cyclic amino acids (e.g., proline), acidic amino acids (e.g., aspartic acid, asparagine, glutamic acid or glutamine), basic amino acids (e.g., histidine, lysine or arginine), and bulky amino acids (e.g., phenylalanine, tyrosine or tryptophan). In some embodiments, the N-terminal spacer domain and / or the C-terminal spacer domain consists of glycine (G). In some embodiments, the N-terminal spacer domain and / or the C-terminal spacer domain consists of glycine (G) and proline (P) and has an amino acid sequence of GP or PG.

[0323] It is to be understood that a modification to a "spacer domain" as described in some embodiments can refer to a modification to any one or more of the spacer domains described herein (e.g., any N-terminal spacer domain and / or any C-terminal spacer domain described herein). For example, a modification to a spacer domain can refer to (a) a modification to any N-terminal spacer domain described herein, (b) a modification to any C-terminal spacer domain described herein, or (c) a modification to any N-terminal spacer domain and any C-terminal spacer domain described herein. Thus, any linker described herein that includes an N-terminal spacer domain includes embodiments in which the N-terminal spacer domain is modified according to the modifications described herein, any linker described herein that includes a C-terminal spacer domain includes embodiments in which the C-terminal spacer domain is modified according to the modifications described herein, and any linker described herein that includes an N-terminal spacer domain and a C-terminal spacer domain includes embodiments in which the N-terminal spacer domain and / or the C-terminal spacer domain is modified according to the modifications described herein.

[0324] A modification to the sequence of an N-terminal spacer domain and / or a C-terminal spacer domain can be any modification to the amino acid sequence of the spacer domain, including incorporating any additional amino acids into the sequence, substituting different amino acids with any amino acid, and / or removing any amino acids from the sequence.

[0325] In some embodiments, one or more additional amino acids are incorporated by addition into the amino acid sequence of the N-terminal spacer domain and / or the C-terminal spacer domain. In some embodiments, the one or more amino acids incorporated by addition into the amino acid sequence of the N-terminal spacer domain and / or the C-terminal spacer domain are selected from the group consisting of: hydrophilic amino acids (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine), hydrophobic amino acids (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, glycine, or proline), polar amino acids (e.g., serine, threonine, cysteine, asparagine, glutamine, or tyrosine), nonpolar amino acids (e.g., glycine, alanine, valine, proline, leucine, isoleucine, methionine, tryptophan, or phenylalanine), amino acids having aliphatic side chains (e.g., glycine, alanine, valine, leucine, or isoleucine), amino acids having hydroxy-containing side chains (e.g., serine or threonine), amino acids having sulfur-containing side chains (e.g., cysteine or methionine), charged amino acids (e.g., arginine, lysine, aspartic acid, or glutamic acid), uncharged amino acids (e.g., serine, threonine, asparagine, or glutamine), aromatic amino acids (e.g., tyrosine, tryptophan, or phenylalanine), cyclic amino acids (e.g., proline), acidic amino acids (e.g., aspartic acid, asparagine, glutamic acid, or glutamine), basic amino acids (e.g., histidine, lysine, or arginine), and bulky amino acids (e.g., phenylalanine, tyrosine, or tryptophan).

[0326] In some embodiments, one or more amino acids are substituted into the amino acid sequence of the N-terminal spacer domain and / or the C-terminal spacer domain. In some embodiments, the one or more amino acids substituted into the amino acid sequence of the spacer domain are selected from the group consisting of: hydrophilic amino acids (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine), hydrophobic amino acids (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, glycine, or proline), polar amino acids (e.g., serine, threonine, cysteine, asparagine, glutamine, or tyrosine), nonpolar amino acids (e.g., glycine, alanine, valine, proline, leucine, isoleucine, methionine, tryptophan, or phenylalanine), amino acids with aliphatic side chains (e.g., glycine, alanine, valine, leucine, or isoleucine), amino acids with hydroxy-containing side chains (e.g., serine or threonine), amino acids with sulfur-containing side chains (e.g., cysteine or methionine), charged amino acids (e.g., arginine, lysine, aspartic acid, or glutamic acid), uncharged amino acids (e.g., serine, threonine, asparagine, or glutamine), aromatic amino acids (e.g., tyrosine, tryptophan, or phenylalanine), cyclic amino acids (e.g., proline), acidic amino acids (e.g., aspartic acid, asparagine, glutamic acid, or glutamine), basic amino acids (e.g., histidine, lysine, or arginine), and bulky amino acids (e.g., phenylalanine, tyrosine, or tryptophan).

[0327] In some embodiments, the modification of the amino acid sequence of the N-terminal spacer domain and / or the C-terminal spacer domain comprises substituting a hydrophobic amino acid with at least one hydrophilic amino acid, substituting a hydrophilic amino acid with at least one hydrophobic amino acid, substituting a nonpolar amino acid with at least one polar amino acid, substituting a polar amino acid with at least one nonpolar amino acid, substituting an uncharged amino acid with at least one charged amino acid, substituting a charged amino acid with at least one uncharged amino acid, substituting an acidic amino acid with at least one basic amino acid, substituting a basic amino acid with at least one acidic amino acid, substituting a non-bulky amino acid with at least one bulky amino acid, substituting a bulky amino acid with at least one non-bulky amino acid, substituting an aliphatic amino acid with at least one amino acid having a hydroxy-containing or sulfur-containing side chain, substituting an aromatic amino acid with at least one amino acid having a hydroxy-containing or sulfur-containing side chain, or substituting an amino acid having a hydroxy-containing or sulfur-containing side chain with at least one aromatic amino acid.

[0328] In some embodiments, one or more amino acids removed from the amino acid sequence of the N-terminal spacer domain and / or the C-terminal spacer domain are selected from the group consisting of: hydrophilic amino acids (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine or glutamine), hydrophobic amino acids (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, glycine or proline), polar amino acids (e.g., serine, threonine, cysteine, asparagine, glutamine or tyrosine), non-polar amino acids (e.g., glycine, alanine, valine, proline, leucine, isoleucine, methionine, tryptophan or phenylalanine), amino acids with aliphatic side chains (e.g., glycine, alanine, valine, leucine or isoleucine), amino acids with hydroxyl-containing side chains (e.g., serine or threonine), amino acids with sulfur-containing side chains (e.g., cysteine or methionine), charged amino acids (e.g., arginine, lysine, aspartic acid or glutamic acid), uncharged amino acids (e.g., serine, threonine, asparagine or glutamine), aromatic amino acids (e.g., tyrosine, tryptophan or phenylalanine), cyclic amino acids (e.g., proline), acidic amino acids (e.g., aspartic acid, asparagine, glutamic acid or glutamine), basic amino acids (e.g., histidine, lysine or arginine), and bulky amino acids (e.g., phenylalanine, tyrosine or tryptophan).

[0329] Any modification to the spacer domains described herein (e.g., any N-terminal spacer domain and / or any C-terminal spacer domain) can be a modification to the amino acid sequence of any N-terminal spacer domain and / or C-terminal spacer domain described herein, including any of SEQ ID NOs: 20-95, 235, 268, 269, 303-305, 323-338, 340, 341, 727, 794, and 799. Any modification to the spacer domains described herein can be made for any purpose. For example, the N-terminal spacer domain and / or the C-terminal spacer domain of a linker including a cleavable peptide can be modified to change the conformation of the linker such that the cleavage efficiency of the cleavable peptide is altered. The N-terminal spacer domain and / or the C-terminal spacer domain of a linker including a cleavable peptide can also be modified to change the structure of the linker including the cleavable peptide such that the cleavage efficiency of the cleavable peptide is altered under certain pH conditions.

[0330] D. Half-life extension domain

[0331] A long in vivo half-life is important for therapeutic proteins. Unfortunately, cytokines administered to a subject typically have a short half-life because they are generally rapidly cleared from the subject's body by mechanisms including renal clearance and endocytic degradation. Thus, in some embodiments of the masked cytokines provided herein, to extend the half-life of the cytokine in vivo, a half-life extension domain is linked to the masked cytokine.

[0332] In some embodiments, the masked cytokines provided herein comprise a half-life extension domain selected from the group consisting of an antibody and its fragments, albumin, an albumin-binding protein, an IgG-binding protein, and a polyamino acid sequence. Other mechanisms for extending the half-life of the masked cytokines available in the art are also contemplated. The half-life extension domain comprises an amino terminus and a carboxyl terminus.

[0333] In some embodiments, the masked cytokine comprises a half-life extension domain. In some embodiments, the masked cytokine comprises a single half-life extension domain. In some embodiments, the masked cytokine comprises more than one half-life extension domain, each of which can be any of the half-life extension domains described herein. In some embodiments, the masked cytokine comprises a first half-life extension domain and a second half-life extension domain. It is to be understood that reference to "a half-life extension domain" or "the half-life extension domain" can refer to the half-life extension domain in a masked cytokine comprising a single half-life extension domain, or it can refer to the first half-life extension domain in a masked cytokine comprising a first half-life extension domain and a second half-life extension domain, or it can refer to the second half-life extension domain in a masked cytokine comprising a first half-life extension domain and a second half-life extension domain, or it can refer to the first half-life extension domain and the second half-life extension domain in a masked cytokine comprising a first half-life extension domain and a second half-life extension domain.

[0334] In some embodiments that include a first half-life extending domain and a second half-life extending domain, the first half-life extending domain is linked to the second half-life extending domain. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the first half-life extending domain is linked to the second half-life extending domain via a linker. In some embodiments, the linked first and second half-life extending domains can each be any half-life extending domain described herein. For example, in some embodiments, the first half-life extending domain and / or the second half-life extending domain of the linked first and second half-life extending domains is an Fc domain or a fragment thereof. In some embodiments, the first half-life extending domain and / or the second half-life extending domain of the linked first and second half-life extending domains is an antibody or a fragment, variant, or derivative thereof.

[0335] 1. Antibodies and their fragments

[0336] By linking a masked cytokine to an antibody or a fragment thereof capable of undergoing FcRn-mediated recycling, clearance of the masked cytokine from the subject can be reduced or otherwise delayed, thereby extending the half-life of the administered masked cytokine.

[0337] In some embodiments of the masked cytokine, the half-life extending domain comprises an antibody or a fragment thereof. In some embodiments, the masked cytokine comprises more than one antibody or a fragment thereof, each of which can be any antibody or a fragment thereof described herein. In some embodiments, the masked cytokine comprises a first half-life extending domain and a second half-life extending domain, each of which comprises an antibody or a fragment thereof. It is to be understood that reference to "an antibody or a fragment thereof" or "the antibody or a fragment thereof" can refer to the antibody or a fragment thereof of the half-life extending domain in a masked cytokine that comprises a single half-life extending domain, or it can refer to the antibody or a fragment thereof of the first half-life extending domain in a masked cytokine that comprises a first half-life extending domain and a second half-life extending domain, or it can refer to the antibody or a fragment thereof of the second half-life extending domain in a masked cytokine that comprises a first half-life extending domain and a second half-life extending domain, or it can refer to the antibody or a fragment thereof of the first half-life extending domain and the antibody or a fragment thereof of the second half-life extending domain in a masked cytokine that comprises a first half-life extending domain and a second half-life extending domain.

[0338] The antibody or fragment thereof can be any antibody or fragment thereof. In some embodiments, the antibody or fragment thereof is any antibody or fragment thereof capable of undergoing FcRn-mediated recycling, such as any heavy chain polypeptide or portion thereof (e.g., Fc domain or fragment thereof) capable of undergoing FcRn-mediated recycling. However, in some embodiments of the masked cytokine comprising a first half-life extending domain and a second half-life extending domain, the first half-life extending domain or the second half-life extending domain can comprise an antibody or fragment thereof that does not bind to the FcRn receptor, such as a light chain polypeptide. For example, in some embodiments of the masked cytokine, the first half-life extending domain comprises an antibody or fragment thereof that comprises a light chain polypeptide or portion thereof that does not directly interact with the FcRn receptor, but the masked cytokine has an extended half-life due to the presence of a second half-life extending domain (such as by comprising a heavy chain polypeptide) capable of interacting with the FcRn receptor. It is well recognized in the art that FcRn-mediated recycling requires binding of the FcRn receptor to the Fc region of the antibody or fragment thereof. For example, studies have shown that residues I253, S254, H435, and Y436 (numbered according to the Kabat EU index numbering system) are important for the interaction between the human Fc region and the human FcRn complex. See, e.g., Firan, M. et al., Int. Immunol. 13 (2001) 993-1002; Shields, R.L. et al., J. Biol. Chem. 276 (2001) 6591-6604). Also, various mutants of residues 248-259, 301-317, 376-382, and 424-437 (numbered according to the Kabat EU index numbering system) have been examined and reported. Yeung, Y.A. et al. (J. Immunol. 182 (2009) 7667-7671).

[0339] In some embodiments, the antibody or fragment thereof comprises a heavy chain polypeptide or a light chain polypeptide. In some embodiments, the antibody or fragment thereof comprises a portion of a heavy chain polypeptide or a light chain polypeptide. In some embodiments, the antibody or fragment thereof comprises an Fc domain or fragment thereof. In some embodiments, the antibody or fragment thereof comprises the CH2 and CH3 domains or fragment thereof. In some embodiments, the antibody or fragment thereof comprises the constant domain of a heavy chain polypeptide. In some embodiments, the antibody or fragment thereof comprises the constant domain of a light chain polypeptide. In some embodiments, the antibody or fragment thereof comprises a heavy chain polypeptide or fragment thereof (e.g., Fc domain or fragment thereof). In some embodiments, the antibody or fragment thereof comprises a light chain polypeptide.

[0340] In some embodiments, the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 158. In some embodiments, the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 157. In some embodiments, the heavy chain polypeptide comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 158. In some embodiments, the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 168. In some embodiments, the heavy chain polypeptide comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 168. In some embodiments, the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 169. In some embodiments, the heavy chain polypeptide comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 169.

[0341] In some embodiments, the light chain polypeptide comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 157. In some embodiments, the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 170. In some embodiments, the light chain polypeptide comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 170.

[0342] In some embodiments, the antibody or fragment thereof comprises an Fc domain or fragment thereof. In some embodiments, the antibody or fragment thereof is an Fc domain or fragment thereof.

[0343] In some embodiments, the Fc domain or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 616, 619, 622, 625, 721, 772 - 774, 793, and 796. In some embodiments, the Fc domain or fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 616, 619, 622, 625, 721, 772 - 774, 793, and 796. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 154. In some embodiments, the Fc domain or fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 154. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the Fc domain or fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 155. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156. In some embodiments, the Fc domain or fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 265. In some embodiments, the Fc domain or fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 265. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155.In some embodiments, the Fc domain or fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 155. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 772, or comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 772. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 773, or comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 773. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 774, or comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 774.

[0344] In some embodiments that include a first half-life extending domain and a second half-life extending domain, the first half-life extending domain comprises an Fc domain or a fragment thereof, the Fc domain or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 154-156, 265, 616, 619, 622, 625, 721, 772-774, 793, and 796, and the second half-life extending domain comprises an Fc domain or a fragment thereof, the Fc domain or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 154-156, 265, 616, 619, 622, 625, 721, 772-774, 793, and 796. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the first half-life extending domain comprises an Fc domain or a fragment thereof, the Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 154-156, 265, 616, 619, 622, 625, 721, 772-774, 793, and 796, and the second half-life extending domain comprises an Fc domain or a fragment thereof, the Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 154-156, 265, 616, 619, 622, 625, 721, 772-774, 793, and 796. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the first half-life extending domain comprises the amino acid sequence of any of the first half-life extending domains listed in Table 4-11, and the second half-life extending domain comprises the amino acid sequence of any of the first half-life extending domains listed in Table 4-11.

[0345] In some embodiments that include a first half-life extending domain and a second half-life extending domain, the first half-life extending domain includes an Fc domain or a fragment thereof that includes the amino acid sequence of SEQ ID NO: 155, and the second half-life extending domain includes an Fc domain or a fragment thereof that includes the amino acid sequence of SEQ ID NO: 156. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the first half-life extending domain includes an Fc domain or a fragment thereof that includes the amino acid sequence of SEQ ID NO: 156, and the second half-life extending domain includes an Fc domain or a fragment thereof that includes the amino acid sequence of SEQ ID NO: 155. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the first half-life extending domain includes an Fc domain or a fragment thereof that includes an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 155, and the second half-life extending domain includes an Fc domain or a fragment thereof that includes an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 156. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the first half-life extending domain includes an Fc domain or a fragment thereof that includes an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 156, and the second half-life extending domain includes an Fc domain or a fragment thereof that includes an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 155.

[0346] In some embodiments that include a first half-life extending domain and a second half-life extending domain, the first half-life extending domain comprises an Fc domain or a fragment thereof that comprises the amino acid sequence set forth in SEQ ID NO: 155, and the second half-life extending domain comprises an Fc domain or a fragment thereof that comprises the amino acid sequence set forth in SEQ ID NO: 616. In some embodiments that include a first half-life extending domain and a second half-life extending domain, the first half-life extending domain comprises an Fc domain or a fragment thereof that comprises the amino acid sequence set forth in SEQ ID NO: 616, and the second half-life extending domain comprises an Fc domain or a fragment thereof that comprises the amino acid sequence set forth in SEQ ID NO: 155. In some embodiments that include a first half-life extending domain and a second half-...

Claims

1. A masked cytokine, comprising: a) A first half-life extension domain and a second half-life extension domain, wherein the first half-life extension domain is a first Fc domain and the second half-life extension domain is a second Fc domain; b) A masking moiety, wherein the amino acid sequence of the masking moiety is SEQ ID NO:826; and c) A cytokine, wherein the cytokine is an IL-2 polypeptide, and the amino acid sequence of the IL-2 polypeptide is SEQ ID NO:3, wherein the masking moiety is linked to the first half-life extension domain via a first linker, wherein the first linker is a cleavable peptide, and the amino acid sequence of the cleavable peptide is SEQ ID NO:96, wherein the cytokine is linked to the second half-life extension domain via a second linker, and the second linker is non-cleavable, wherein the amino acid sequence of the first Fc domain is SEQ ID NO:155 and the amino acid sequence of the second Fc domain is SEQ ID NO:156, and wherein the first half-life extension domain associates with the second half-life extension domain via a knob-into-hole modification.

2. A masked cytokine, comprising: a) A masking moiety and a first polypeptide comprising a first half-life extension domain; and b) A cytokine and a second polypeptide comprising a second half-life extension domain, wherein the first half-life extension domain is a first Fc domain and the second half-life extension domain is a second Fc domain; wherein the amino acid sequence of the masking moiety is SEQ ID NO:826; wherein the cytokine is an IL-2 polypeptide, and the amino acid sequence of the IL-2 polypeptide is SEQ ID NO:3; wherein the masking moiety is linked to the C-terminus of the first half-life extension domain via a first linker, and the first linker is non-cleavable; wherein the cytokine is linked to the C-terminus of the second half-life extension domain via a second linker, and the second linker is a cleavable peptide, and the amino acid sequence of the cleavable peptide is SEQ ID NO:135; and wherein the amino acid sequence of the first Fc domain is SEQ ID NO:155 and the amino acid sequence of the second Fc domain is SEQ ID NO:156, and wherein the first half-life extension domain associates with the second half-life extension domain via a knob-into-hole modification.

3. The masked cytokine according to claim 1, wherein the amino acid sequence of the second linker is SEQ ID NO:

339.

4. The masked cytokine according to claim 2, wherein the amino acid sequence of the first linker is SEQ ID NO:

28.

5. A pharmaceutical composition comprising the masked cytokine according to any one of claims 1, 2, 3 and 4 and a pharmaceutically acceptable carrier.

6. A kit comprising the masked cytokine according to any one of claims 1, 2, 3 and 4.

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