Masking cytokine polypeptides

Abstract

Provided herein are cytokines, or functional fragments thereof, which, in some embodiments, are engineered to be masked by a masking moiety at one or more receptor binding sites of the cytokines, or functional fragments thereof. In some embodiments, the cytokines are engineered to be activatable by a protease at a target site, such as in a tumor microenvironment, by including a proteolytically cleavable linker. In some embodiments, the proteolytically cleavable linker links the cytokine to the masking moiety, links the cytokine to a half-life extending domain, and / or links the masking moiety to a half-life extending domain. The masking moiety blocks, blocks, inhibits (e.g., reduces) or otherwise prevents (e.g., masks) the activity of the cytokine or binding to its homologous receptor or protein.

Description

[0001] This application is a divisional application of the invention patent application with application date of September 27, 2019, application number 201980077964.7, and invention name “Masked Cytokine Polypeptide”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 737,803, filed September 27, 2018, U.S. Provisional Application No. 62 / 888,276, filed August 16, 2019, and U.S. Provisional Application No. 62 / 891,199, filed August 23, 2019. The contents of the above-listed applications are incorporated herein by reference in their entirety for all purposes.

[0004] Incorporated by reference into the sequence listing

[0005] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file named 737762000940SeqList, created on September 26, 2019, and is 1,024.111 KB in size. The information in the electronic sequence listing is incorporated by reference in its entirety. Technical Field

[0006] The present invention relates to masked cytokines and methods of use and preparation thereof. Background Art

[0007] Cancer is the second leading cause of death in the United States, responsible for more deaths than the next five leading causes (chronic respiratory diseases, stroke, accidents, Alzheimer's disease, and diabetes). Although significant progress has been made, particularly in targeted therapies, there is still much work to be done in this field. Immunotherapy and immuno-oncology, a subfield of this field, are becoming viable and exciting therapeutic options for treating malignancies. In particular, it is now recognized that a hallmark of cancer is immune evasion, and considerable effort has been put into identifying targets and developing therapies directed against these targets to reactivate the immune system to recognize and treat cancer.

[0008] Cytokine therapy is an effective strategy to stimulate 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 the cytokines administered to patients is usually very short, and therefore requires frequent administration. For example, the product label of aldesleukin, sold under the brand name Proleukin, indicates that the drug shows a half-life of 85 minutes in patients receiving a 5-minute intravenous (IV) infusion. In addition, the administration of high doses of cytokines may cause adverse health outcomes, such as vascular leakage, through systemic immune activation. These findings indicate the need to develop cytokine therapeutics that effectively target tumors without the side effects associated with systemic immune activation. In order to address this demand, masked cytokines, compositions thereof, and methods of use thereof are provided herein.

[0009] All references cited herein, including patent applications, patent publications, and scientific literature, are hereby incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0010] The disclosed invention relates to the engineering and use of masked cytokines or their functional fragments, in some embodiments, the cytokine or its functional fragment is engineered to be masked by a masked portion at one or more receptor binding sites of the cytokine or its functional fragment. In some embodiments, the cytokine is engineered to be activated by proteases at the target site (such as in a tumor microenvironment) by including a linker that can be proteolytically cleaved. In some embodiments, the linker that can be proteolytically cleaved connects the cytokine to a masked portion, connects the cytokine to a half-life extension domain, and / or connects the masked portion to a half-life extension domain. The masked portion blocks, closes, inhibits (e.g., reduces) or otherwise prevents (e.g., masks) the activity of the cytokine or its cognate receptor or protein. When the cleavable linker is proteolytically cleaved at the target site, the cytokine is activated so that it can bind to its cognate receptor or protein with increased affinity.

[0011] One embodiment includes a masked cytokine comprising a masking moiety and a cytokine or a functional fragment thereof, wherein the masking moiety is connected to the cytokine or its functional fragment 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 its functional fragment.

[0012] 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 a 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 a 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 a functional fragment thereof; b) a first linker; c) a masking moiety; and d) a half-life extension domain.

[0013] In another embodiment, the masked cytokine comprises, in the N- to C-terminal or 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.

[0014] In one embodiment, the cytokine or functional fragment thereof is an IL-2 polypeptide or a functional fragment thereof or an IL-15 polypeptide or a functional fragment thereof.

[0015] 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 a functional fragment thereof. In one embodiment, the amino acid substitutions reduce the affinity of the IL-2 polypeptide or a functional fragment thereof for CD25 (IL-2Rα).

[0016] In one embodiment, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence produced by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or a functional fragment thereof that increase the affinity of the IL-2 polypeptide or a functional fragment thereof for IL-2Rβ or IL-2Rγ.

[0017] In one embodiment, the half-life extending domain is an antibody or fragment thereof, and in some embodiments, one or more amino acid substitutions alter effector function.

[0018] In one embodiment, the antibody or fragment thereof is a fragment crystallizable domain (Fc domain) or a fragment thereof.

[0019] In one embodiment, the half-life extending domain is a polyamino acid sequence, such as a PAS polypeptide or an XTEN polypeptide.

[0020] In one embodiment, the masked cytokine comprises a second masking moiety, wherein the second masking moiety is linked to the cytokine or a functional fragment thereof via a second linker.

[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 a functional fragment thereof; d) a second linker; e) a second masking moiety; and f) a half-life extending domain.

[0022] 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 a functional fragment thereof; d) a second linker; e) a second masking moiety; f) a third linker; and g) a half-life extending 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; and f) a half-life extending domain.

[0024] 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 extending domain.

[0025] In one embodiment, the half-life extending domain is an albumin polypeptide or a functional fragment thereof.

[0026] 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, C ELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, CTRl, 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, TMP RSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1 and TPSG1. ,

[0027] In one embodiment, the half-life extension 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, a calicheamicin, a duocarmycin, an indolinone benzodiazepine dimer, or an exotecan derivative Dxd.

[0028] 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.

[0029] In one embodiment, the masked cytokine comprises a first half-life prolonging domain and a second half-life prolonging domain, wherein the masking moiety is linked to the first half-life prolonging domain, and wherein the cytokine or a functional fragment thereof is linked to the second half-life prolonging domain.

[0030] In one embodiment, the first half-life extending domain and the second half-life extending domain contain modifications that promote association of the first and second half-life extending domains.

[0031] One embodiment comprises a nucleic acid encoding a masked cytokine as described herein. Another embodiment comprises a vector comprising the nucleic acid. In another embodiment, a host cell comprises the nucleic acid. Another embodiment comprises one or more nucleic acids encoding a masked cytokine as described herein. Another embodiment comprises a vector comprising the one or more nucleic acids. Another embodiment comprises one or more vectors comprising the one or more nucleic acids.

[0032] In another embodiment, a method for producing a masked cytokine is provided, comprising culturing host cells under conditions that produce the masked cytokine. In one embodiment, the masked cytokine produced by the host cells is recovered. One embodiment includes a masked cytokine produced by the disclosed method.

[0033] In one embodiment, a composition comprising the disclosed masked cytokine is provided. In another embodiment, the composition further comprises an anti-inflammatory or 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, a 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, or a combination thereof. Inhibitors, HDAC inhibitors, VEGF ligand inhibitors, CD19 targeting molecules, FOLR1 targeting molecules, DLL3 targeting molecules, DKK1 targeting molecules, MUC1 targeting molecules, MUC16 targeting molecules, PSMA targeting molecules, MSLN targeting molecules, NY-ES0-1 targeting molecules, B7H3 targeting molecules, B7H4 targeting molecules, BCMA targeting molecules, CD29 targeting molecules, CD151 targeting molecules, CD123 targeting molecules, CD33 targeting molecules, CD37 targeting molecules, CDH19 targeting molecules, CEA targeting molecules, 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 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.

[0034] 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, mofezolic 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, flusulamide, NS-398, L-745337, RWJ-63556, L-784512, Dabufilon, CS-502, LAS-34475, LAS-34555, S-33516, diclofenac, mefenamic acid, SD-8381, ibuprofen, naproxen, ketorolac, indomethacin, aspirin, naproxen, tolmetin, piroxicam, or meclofenamate.

[0035] 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.

[0036] 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, cyclooxygenase, parthenolide, carfilzomib, MLN-4924 (pevonedistat), JSH-23 rolipram, gallic acid, anacardic acid, GYY-4137, p-XSC, CV-3988, prostaglandin E2 (PGE2), LY-294002, wortmannin, mesalamine, quinacrine, or flavopiridol.

[0037] One embodiment includes a pharmaceutical composition comprising the disclosed masked cytokines and a pharmaceutically acceptable carrier.

[0038] Another embodiment includes a kit comprising the disclosed masked cytokines.

[0039] 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 a masked cytokine composition disclosed for treating a disease or condition, 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 cancer, endometrial cancer, esophageal cancer, gastric cancer, oral cancer, thyroid cancer, retinoblastoma, parathyroid cancer, pituitary cancer, bile duct cancer, 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, stomach cancer, uterine cancer or medulloblastoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, essential macroglobulinemia, primary brain tumor, cancer, malignant pancreatic insulinoma, malignant carcinoid, urinary bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenocortical cancer, endocrine or exocrine pancreatic tumors, medullary thyroid cancer, medullary thyroid cancer, thyroid carcinoma), melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, Paget disease of the nipple, phyllodes tumor, lobular carcinoma, ductal carcinoma, pancreatic stellate cell carcinoma, hepatic stellate cell carcinoma, or prostate cancer.

[0040] 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 a 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 skin disease, osteoporosis, peritonitis, hepatitis, lupus, celiac disease, Sjögren's syndrome, polymyalgia rheumatica, multiple sclerosis (MS), ankylosing spondylosis, Inflammation, type 1 diabetes, alopecia areata, vasculitis and temporal arteritis, graft-versus-host disease (GVHD), asthma, COPD, paraneoplastic autoimmune diseases, cartilage inflammation, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular 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, oligoarticular 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 silver 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-Barré disease, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, transplant rejection, renal injury, hepatitis C-induced vasculitis, or spontaneous pregnancy loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1A and Figure 1BThe structures of exemplary embodiments of masked cytokines are shown, comprising a masking portion, a cytokine or a functional fragment thereof ("cytokine"), a half-life extension domain, and a 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 further comprise a second linker comprising a second cleavable peptide ("2CP"), a second N-terminal spacer domain ("2NSD"), and a second C-terminal spacer domain ("2CSD"). As indicated by the arrows, although the exemplary embodiments show that the masking portion is connected to the first linker, and the cytokine or its functional fragment is connected to the first linker and the second linker, the masking portion and the cytokine or its functional fragment can be interchangeable such that the cytokine or its functional fragment is connected to the first linker, and the masking portion is connected to the first linker and the second linker. Figure 1A The structure of an exemplary embodiment of a masked cytokine as a monomer is shown. Figure 1B Shown is the structure of an exemplary embodiment of a masked cytokine as a homodimer formed by disulfide bonds.

[0042] Figure 2A and Figure 2B The structures of exemplary embodiments of masked cytokines are shown, comprising a first masking portion ("Mask 1"), a cytokine or a functional fragment thereof ("cytokine"), a second masking portion ("Mask 2"), a half-life extension domain, a first linker comprising a first cleavable peptide ("1CP"), a first N-terminal spacer domain ("1NSD"), and a first C-terminal spacer domain ("1CSD"), and a 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 further comprise a 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 The structure of an exemplary embodiment of a masked cytokine as a monomer is shown. Figure 2B Shown is the structure of an exemplary embodiment of a masked cytokine as a homodimer formed by disulfide bonds.

[0043] Figure 3A Shown are structures of exemplary embodiments of masked cytokines comprising a masking moiety, a cytokine or functional fragment thereof ("cytokine"), a first half-life extending domain, and a second half-life extending domain. Figure 3AThe exemplary embodiment shown in also includes a first linker comprising a first cleavable peptide ("1CP"), a first N-terminal spacer domain ("1NSD"), and a first C-terminal spacer domain ("1CSD"), and a second linker comprising a second cleavable peptide ("2CP"), a second N-terminal spacer domain ("2NSD"), and a second C-terminal spacer domain ("2CSD"). The exemplary first and second half-life extending domains include "knobs into holes" modifications that promote association of the first half-life extending domain with the second half-life extending domain, as shown by the "hole" in the first half-life extending domain and the "knob" in the second half-life extending domain. It is also shown that the first half-life extending domain and the second half-life extending domain are at least partially associated due to the formation of a disulfide bond. It is to be understood that although the "hole" is depicted as part of the first half-life extension domain (attached to the masking moiety) and the "knob" is depicted as part of the second half-life extension domain (attached to the cytokine), the "hole" and "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 (attached to the cytokine) and the "knob" is part of the first half-life extension domain (attached to the masking moiety). Figure 3B Shown is the structure of an exemplary embodiment of a masked cytokine comprising a shielding moiety, a cytokine or a functional fragment thereof ("cytokine"), a first half-life extending domain, a second half-life extending domain, and a third linker comprising a third N-terminal spacer domain ("3NSD") and a third C-terminal spacer domain ("3CSD"). The third linker connects the first half-life extending domain to the second half-life extending domain. Figure 3B The exemplary embodiment shown in also includes a first linker and a second linker, wherein the first linker includes a first cleavable peptide ("1CP"), a first N-terminal spacer domain ("1NSD") and a first C-terminal spacer domain ("1CSD"), and the second linker includes a second cleavable peptide ("2CP"), a second N-terminal spacer domain ("2NSD") and a second C-terminal spacer domain ("2CSD").

[0044] Figure 4 Shows the disulfide bonds Figure 3A Dimers of exemplary masked cytokines are shown in .

[0045] Figure 5AShown is the structure of an exemplary embodiment of a masked cytokine comprising a first masking moiety ("Mask 1"), a first half-life extending domain, a second masking moiety ("Mask 2"), a cytokine or a functional fragment thereof ("cytokine"), and a second half-life extending domain. Figure 5A The exemplary embodiment shown in also includes a first linker, a second linker and a third linker, wherein the first linker includes a first cleavable peptide ("1CP"), a first N-terminal spacer domain ("1NSD") and a first C-terminal spacer domain ("1CSD"), the second linker includes a second cleavable peptide ("2CP"), a second N-terminal spacer domain ("2NSD") and a second C-terminal spacer domain ("2CSD"), and the third linker includes 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 (attached to the masking moiety) and the "knob" is depicted as part of the second half-life extension domain (attached to the cytokine), the "hole" and "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 (attached to the cytokine) and the "knob" is part of the first half-life extension domain (attached to the masking moiety). Figure 5B The structure of an exemplary embodiment of a masked cytokine comprising a first masking moiety ("Mask 1"), a first half-life extending domain, a second masking moiety ("Mask 2"), a cytokine or a functional fragment thereof ("cytokine"), a second half-life extending domain, and a fourth linker comprising a fourth N-terminal spacer domain ("4NSD") and a fourth C-terminal spacer domain ("4CSD"). The fourth linker connects the first half-life extending domain to the second half-life extending domain. Figure 5B The exemplary embodiment shown in also includes a first linker, a second linker, and a third linker, wherein the first linker includes a first cleavable peptide ("1CP"), a first N-terminal spacer domain ("1NSD"), and a first C-terminal spacer domain ("1CSD"), the second linker includes a second cleavable peptide ("2CP"), a second N-terminal spacer domain ("2NSD"), and a second C-terminal spacer domain ("2CSD"), and the third linker includes 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 , although the exemplary embodiment shows 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, so 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.

[0046] Figure 6 Shows the disulfide bonds Figure 5A Dimers of exemplary masked cytokines are shown in .

[0047] Figures 7A-7E Exemplary embodiments of masked cytokines are shown before (left) and after (right) cleavage by proteases as in the tumor microenvironment. Figures 7A-7D Exemplary embodiments of masked IL-2 cytokines are shown, and Figure 7E An exemplary embodiment of a masked IL-15 cytokine is shown. The masking portion (e.g., IL-2Rβ, e.g., Figure 7A 、 7B and 7D ), or release of IL-2 ( Figure 7C ), or release of IL-15( Figure 7E ).

[0048] Figure 8 Shown are SDS-PAGE analyses of flow-through (FT) samples (i.e., protein not bound to a Protein A column) and elution (E) samples (i.e., protein bound to and eluted from a Protein A column) following the production and purification of exemplary constructs (AK304, AK305, AK307, AK308, AK309, AK310, AK311, AK312, AK313, AK314, and AK315).

[0049] Figures 9A-9D Shown are the results of SPR analyses testing exemplary masked IL-2 polypeptide constructs (AK215 and AK216) or an rhIL2 control for binding to CD25-Fc. Figure 9A showed the interaction between AK215 and CD25-Fc, Figure 9B showed the interaction between AK216 and CD25-Fc, and Figure 9C Shown is the interaction between recombinant human IL2 (rhIL2) control and CD25-Fc. Figure 9D Provides a summary of the association constant (ka), dissociation constant (kd), equilibrium dissociation constant (KD), and Chi for each interaction 2 Table of data obtained for values and U values.

[0050] Figures 10A-10DShown are the results of SPR analyses testing exemplary masked IL-2 polypeptide constructs (AK216 and AK218) or an rhIL2 control for binding to CD122-Fc. Figure 10A showed the interaction between AK216 and CD122-Fc, Figure 10B showed the interaction between AK218 and CD122-Fc, and Figure 10C Shown is the interaction between recombinant human IL2 (rhIL2) control and CD122-Fc. Figure 10D Provides a summary of the association constant (ka), dissociation constant (kd), equilibrium dissociation constant (KD), and Chi for each interaction 2 Table of data obtained for values and U values.

[0051] Figure 11A Exemplary embodiments of masked cytokines are shown before (left) and after (right) cleavage by proteases as in the tumor microenvironment. Figure 11B Shown is SDS-PAGE analysis of exemplary masked IL-2 polypeptide constructs incubated in the absence (left lane) or presence (right lane) of MMP10 protease, demonstrating the release of IL-2 from the Fc portion.

[0052] Figures 12A-12D STAT5 activation (%) is shown in PBMCs treated with constructs AK032, AK035, AK041 or rhIL-2 as a control. The level of STAT5 activation (%) is shown for NK cells, CD8+ T cells, effector T cells (Teff) and regulatory T cells (Treg), as in the case of rhIL-2 ( Figure 12A )、AK032( Figure 12B )、AK035( Figure 12C ) or AK041( Figure 12D ) were incubated together.

[0053] Figures 13A-13C STAT5 activation (%) in PBMCs treated with constructs AK081 or AK032 is shown. AK081 constructs were tested with and without prior exposure to MMP10. Isotype controls and a negative control without IL-2 were also tested. For NK cells ( Figure 13A ), CD8+ T cells ( Figure 13C ) and CD4+ T cells ( Figure 13B ) shows the level of STAT5 activation (%).

[0054] Figures 14A-14DResults of STAT5 activation studies in PBMCs using constructs AK081 and AK111 and controls including rhIL-2 and anti-RSV antibodies are shown. Untreated controls were also tested. EC50s (pM) for rhIL-2, AK081, and AK111 treatments are also shown. For CD4+FoxP3+CD25+ cells ( Figure 14A ), CD8+ cells ( Figure 14B ) and CD4+FoxP3-CD25- cells ( Figure 14C ) shows STAT5 activation (%). Figure 14D EC50 (pM) and fold change data are provided for AK081, AK111 constructs, and rhIL-2 control.

[0055] Figures 15A-15D Results of STAT5 activation studies in PBMCs using constructs AK167 and AK168 and controls including rhIL-2 and anti-RSV antibodies are shown. Untreated controls were also tested. EC50s (pM) for rhIL-2, AK167, and AK168 treatments are also shown. For CD4+FoxP3+CD25+ cells ( Figure 15A ), CD8+ cells ( Figure 15B ) and CD4+FoxP3-CD25- cells ( Figure 15C ) shows STAT5 activation (%). Figure 15D EC50 (pM) and fold change data are provided for AK167 and AK168 constructs and rhIL-2 control.

[0056] Figures 16A-16D STAT5 activation (%) in PBMCs treated with constructs AK165 or AK166 with (+MMP10) or without prior exposure to MMP10 protease, or isotype control or IL-2-Fc control is shown. Figure 16A The legend shown in also applies to Figure 16B , and as Figure 16C The legend shown in 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 ) shows STAT5 activation (%).

[0057] Figures 17A-17CSTAT5 activation (%) in PBMCs treated with constructs AK109 or AK110 with (+MMP10) or without prior exposure to MMP10 protease, or isotype control or IL-2-Fc control is shown. Figure 17B The legend shown in also applies to Figure 17A For NK cells ( Figure 17A ), CD8 cells ( Figure 17B ) and CD4 cells ( Figure 17C ) shows STAT5 activation (%).

[0058] 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. Figure 18A ), CD3+CD4+FoxP3- cells ( Figure 18B ) and CD3+CD8+ cells ( Figure 18C ) shows STAT5 activation (%). Figure 18D EC50 data are provided for each construct tested as well as an rhIL-2 control.

[0059] 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. Figure 19A ), CD4+FoxP3-CD25- cells ( Figure 19B ) and CD8+ cells ( Figure 19C ) shows STAT5 activation (%). Figure 19D EC50 data are provided for each construct tested as well as an rhIL-2 control.

[0060] Figures 20A-20C STAT5 activation (%) in PBMCs treated with constructs AK081, AK189, AK190 or AK210 or anti-RSV control is shown. Figure 20A The legend shown in also applies to Figure 20B and 20C For regulatory T cells ( Figure 20A ), CD4 helper T cells ( Figure 20B ) and CD8 cells ( Figure 20C ) shows STAT5 activation (%).

[0061] Figures 21A-21CSTAT5 activation (%) in PBMCs treated with constructs AK167, AK191, AK192 or AK193 or anti-RSV control is shown. Figure 21A The legend shown in also applies to Figure 21B and 21C For regulatory T cells ( Figure 21A ), CD4 helper T cells ( Figure 21B ) and CD8 cells ( Figure 21C ) shows STAT5 activation (%).

[0062] Figure 22A and 22B Shown are the results of a reporter bioassay performed on an exemplary masked IL-15 polypeptide construct AK248 or rhIL-15 as a control with (+MMP) or without prior exposure to an activating protease. Figure 22A Results from a reporter bioassay using the HEK-Blue IL2 reporter cell line are shown, and Figure 22B Shown are the results of a reporter bioassay for IL-15 bioassay using the mouse CTLL2 cell line.

[0063] Figures 23A-23D Shown are the results of pharmacokinetic studies in tumor-bearing mice using constructs AK032, AK081, AK111, AK167, or AK168 or an anti-RSV control. Figure 23A A simplified depiction of the structure of each construct tested is provided. Figure 23B The Fc level in plasma (μg / mL) was shown by detecting human IgG. Figure 23C The level of Fc-CD122 in plasma (μg / mL) was shown by detecting human CD122, and Figure 23D The level of Fc-IL2 in plasma (μg / mL) was shown by detecting human IL-2. Before the detection step, anti-human IG was used as the capture antibody.

[0064] Figures 24A-24D Shown are the results of pharmacokinetic studies in tumor-bearing mice using constructs AK167, AK191 AK197, AK203, AK209, or AK211 or an anti-RSV control. Figure 24A A simplified depiction of the structure of each construct tested is provided. Figure 24B The Fc level in plasma (μg / mL) was shown by detecting human IgG. Figure 24C The level of Fc-IL2 in plasma (μg / mL) was measured by detecting human IL-2, and Figure 24D The level of Fc-CD122 in plasma (μg / mL) was shown by detecting human CD122. Before the detection step, anti-human IG was used as the capture antibody.

[0065] Figures 25A-25L Results are shown of a study testing the in vivo responses of CD4, CD8, NK, and Treg percentages in spleen, blood, and tumor using AK032, AK081, AK111, AK167, or AK168 constructs or anti-RSV IgG control. For spleen tissue, the % CD3 cells (CD8 cells) are shown. Figure 25A ), %CD4( Figure 25B ), CD3-cells %NK cells ( Figure 25C ), %FoxP3 of CD4 cells ( Figure 25D For blood, the %CD8 cells of CD3 cells are shown ( Figure 25E ), %CD4( Figure 25F ), CD3-cells %NK cells ( Figure 25G ), %FoxP3 of CD4 cells ( Figure 25H For tumor tissues, the percentage of CD3 cells and CD8 cells ( Figure 25I ), %CD4( Figure 25J ), CD3-cells %NK cells ( Figure 25K ), %FoxP3 of CD4 cells ( Figure 25L ).

[0066] Figures 26A-26L Results are shown of a study testing the in vivo responses of CD4, CD8, NK, and Treg percentages in spleen, blood, and tumors using AK167, AK168, AK191, AK197, AK203, AK209, or AK211 constructs or anti-RSV IgG controls. For spleen tissue, the % CD3 cells (CD8 cells) are shown. Figure 26A ), %CD4( Figure 26B ), CD3-cells %NK cells ( Figure 26C ), %FoxP3 of CD4 cells ( Figure 26D For blood, the %CD8 cells of CD3 cells are shown ( Figure 26E ), %CD4( Figure 26F ), CD3-cells %NK cells ( Figure 26G ), %FoxP3 of CD4 cells ( Figure 26H For tumor tissues, the percentage of CD3 cells and CD8 cells ( Figure 26I ), %CD4( Figure 26J ), CD3-cells %NK cells ( Figure 26K ), %FoxP3 of CD4 cells ( Figure 26L ).

[0067] Figures 27A-27L Results are shown of a study testing the in vivo responses of CD4, CD8, NK, and Treg percentages in spleen, blood, and tumors using AK235, AK191, AK192, AK193, AK210, AK189, AK190, or AK211 constructs or anti-RSV IgG controls. For spleen tissue, the % CD3 cells (CD8 cells) are shown. Figure 27A ), %CD4( Figure 27B ), CD3-cells %NK cells ( Figure 27C ), %FoxP3 of CD4 cells ( Figure 27D For blood, the %CD8 cells of CD3 cells are shown ( Figure 27E ), %CD4( Figure 27F ), CD3-cells %NK cells ( Figure 27G ), %FoxP3 of CD4 cells ( Figure 27H For tumor tissues, the percentage of CD3 cells and CD8 cells ( Figure 27I ), %CD4( Figure 27J ), CD3-cells %NK cells ( Figure 27K ), %FoxP3 of CD4 cells ( Figure 27L ).

[0068] Figures 28A-28I Results are shown for in vivo T cell activation in spleen, blood, and tumors using AK235, AK191, AK192, AK193, AK210, AK189, AK190, or AK211 constructs. T cell activation was measured as 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 ) Mean fluorescence intensity (MFI) of CD25 in ). Statistical analysis was performed using one-way ANOVA compared to the uncleavable AK211 construct.

[0069] Figures 29A-29D Shown are the results of studies testing the in vivo cleavage of 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 29EResults of pharmacokinetic studies showing total plasma IgG concentrations (μg / mL) for total levels of AK167, AK168, and AK209 constructs as well as levels of the uncleaved form of each construct.

[0070] Figures 30A-30D Shown are the results of in vivo studies assessing vascular leakage using exemplary masked IL-2 polypeptide constructs AK111 or AK168 or unmasked IL-2 polypeptide constructs AK081 or AK167 or an anti-RSV control. Figure 30A Shows the percentage (%) of weight loss, and Figure 30B 、 30C and 30D show the weight (g) of liver, lung and spleen, respectively.

[0071] Figure 31A and 31B Results of in vivo studies evaluating vascular leakage are shown, as indicated by measuring the extent of dye leakage into liver and lung tissue following administration of AK081, AK111, AK167, or AK168 constructs or anti-RSV control. Dye leakage into liver ( Figure 31A ) and lungs ( Figure 31B ) in the extent of leakage.

[0072] Figure 32A and 32B Results of in vivo studies evaluating vascular leakage are shown, as indicated by measuring the extent of perivascular invasion of monocytes into liver and lung tissues following administration of AK081, AK111, AK167, or AK168 constructs or anti-RSV control. The mean number of monocytes in the liver ( Figure 32A ) and the average number of monocytes in the lungs ( Figure 32B ).

[0073] Figure 33A and 33B Shown are the results of a syngeneic tumor model study assessing tumor volume and body weight during treatment with AK032, AK081, AK111, AK167, or AK168 constructs or anti-RSV control. Figure 33A Data showing tumor volume during treatment, and Figure 33B Data are shown as percent (%) change in body weight during treatment. DETAILED DESCRIPTION

[0074] Provided herein are cytokines or their functional fragments, in some embodiments, the cytokines or their functional fragments are engineered to be shielded by a masked portion at one or more receptor binding sites of the cytokine or its functional fragment. In some embodiments, the cytokine is engineered to be activated by proteases at the target site (such as in a tumor microenvironment) by including a joint that can be proteolytically cleaved. In some embodiments, the joint that can be proteolytically cleaved connects the cytokine to a masked portion, connects the cytokine to a half-life extension domain, and / or connects the masked portion to a half-life extension domain. The masked portion blocks, closes, inhibits (e.g., reduces) or otherwise prevents (e.g., masking) the activity of the cytokine or its cognate receptor or protein. When the cleavable joint is proteolytically cleaved at the target site, the cytokine is activated so that it can bind to its cognate receptor or protein with increased affinity.

[0075] By using a masking moiety, 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 is associated with serious 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 lung endothelial cells, and that IL-2-mediated pulmonary edema can be eliminated by interfering with IL-2's ability to bind to IL-2Rα. See Krieg et al., (2010) PNAS, 107(26):11906-11911. Therefore, in some embodiments where the cytokine or a functional fragment thereof is an IL-2 polypeptide, a masking moiety is used that blocks, inhibits, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the activity of the IL-2 cytokine or its binding to IL-2Rα. To further reduce the systemic effects of the masked IL-2 polypeptide, the masked IL-2 polypeptide may further include a masking moiety that blocks, inhibits, 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-2R. Similar strategies are also used for other cytokines by interfering with their ability to bind to certain proteins (e.g., receptor subunits) associated with causing adverse systemic side effects. In addition, by masking the cytokine using a linker comprising a cleavable peptide, the binding ability interfered with by the use of the masking moiety can be restored by cleavage of the cleavable peptide in the tumor microenvironment. Therefore, in some embodiments, the masked cytokines provided herein are engineered to precisely target pharmacological activity to the tumor microenvironment by utilizing the high local concentration of active proteases, which are one of the hallmarks of cancer. This feature of the tumor microenvironment is used to convert systemically inert molecules into locally active cytokines. 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.

[0076] All publications (including patent documents, scientific papers, and databases) mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in patents, applications, published applications, and other publications incorporated by reference herein, the definition set forth herein takes precedence over the definition incorporated by reference herein.

[0077] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0078] I. Masked cytokines

[0079] Provided herein, in some embodiments, are masked cytokines comprising (a) a masking moiety; and (b) a cytokine or a functional fragment thereof, wherein the masking moiety is linked to the cytokine or its functional fragment via a first linker. In some embodiments, the masked cytokine further comprises a half-life extension domain linked to the masking moiety or the cytokine or its functional fragment. In some embodiments, the half-life extension domain is linked to the masking moiety or the IL-2 polypeptide or its functional fragment via a second linker.

[0080] In some embodiments, the masked cytokine comprises, in the N- to C-terminal direction: (a) a masking portion; (b) a first linker; (c) a cytokine or a functional fragment thereof; and (d) a half-life extension domain. In some embodiments, the masked cytokine comprises, in the C- to N-terminal direction: (a) a masking portion; (b) a first linker; (c) a cytokine or a functional fragment thereof; and (d) a half-life extension domain. In some embodiments, the masked cytokine comprises, in the N- to C-terminal direction: (a) a masking portion; (b) a first linker; (c) a cytokine or a functional fragment thereof; (d) a second linker, and (e) a half-life extension domain. In some embodiments, the masked cytokine comprises, in the C- to N-terminal direction: (a) a masking portion; (b) a first linker; (c) a cytokine or a functional fragment thereof; (d) a second linker, and (e) a half-life extension domain. In some embodiments, the masked cytokine comprises, in the N- to C-terminal direction: (a) a cytokine or a functional fragment thereof; (b) a first linker; (c) a shielding portion; and (d) a half-life extension domain. In some embodiments, the masked cytokine comprises, in the C- to N-terminal direction: (a) a cytokine or a functional fragment thereof; (b) a first linker; (c) a shielding portion; and (d) a half-life extension domain. In some embodiments, the masked cytokine comprises, in the N- to C-terminal direction: (a) a cytokine or a functional fragment thereof; (b) a first linker; (c) a shielding portion; and (d) a second linker; and (e) a half-life extension domain. In some embodiments, the masked cytokine comprises, in the C- to N-terminal direction: (a) a cytokine or a functional fragment thereof; (b) a first linker; (c) a shielding portion; (d) a second linker; and (e) a half-life extension domain. In some embodiments, the masked cytokine comprises, in the N- to C-terminal direction: (a) a half-life extension 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, the masked cytokine comprises, in the N- to C-terminal direction: (a) a half-life extension domain; (b) a cytokine or a functional fragment thereof; (c) a first linker; and (d) a masking moiety. 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 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 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 is 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 construct according to the teachings herein.

[0081] Also provided herein in some embodiments are masked cytokines comprising (a) a first masking moiety; (b) a cytokine or a functional fragment thereof, wherein the first masking moiety is connected to the cytokine or its functional fragment via a first joint; and (c) a second masking moiety, wherein the second masking moiety is connected to the cytokine or its functional fragment via a second joint. In some embodiments, the masked cytokine further comprises a half-life extension domain connected to the first masking moiety or the second masking moiety. In some embodiments, the half-life extension domain is connected to the first masking moiety or the second masking moiety via a third joint.

[0082] Also provided herein in some embodiments are masked cytokines comprising (a) a first masking portion, wherein the first masking portion is connected to a first half-life extension domain; (b) a cytokine or a functional fragment thereof, wherein the cytokine or its functional fragment is connected to a second half-life extension domain; and (c) a second masking portion, wherein the second masking portion is connected to the first masking portion. In some embodiments, the first masking portion is connected to the first half-life extension domain via a first linker. In some embodiments, the second masking portion is connected to the first masking portion via a second linker. In some embodiments, the cytokine or its functional fragment is connected to the second half-life extension 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.

[0083] In some embodiments, the 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, the 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, the 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, the 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, the 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, the masked cytokine comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 567 and 598-601. 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 567 and 598-601. In some embodiments, the 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 the construct according to the teachings herein.

[0084] Also provided herein in some embodiments are masked cytokines comprising (a) a first half-life extension domain and a second half-life extension domain; (b) a masking portion; and (c) a cytokine or a functional fragment thereof, wherein the masking portion is connected to the first half-life extension domain, the cytokine or its functional fragment is connected 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 masking portion is connected to the first half-life extension domain via a first joint, and / or the cytokine or its functional fragment is connected to the second half-life extension domain via a second joint. In some embodiments, the first half-life extension domain is optionally connected to the second half-life extension domain via a third joint. In some embodiments, the masked cytokine comprises (a) a first half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a masking portion 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 portion is linked to the first half-life prolonging domain via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or a functional fragment thereof is linked to the second half-life prolonging domain via a second linker comprising the amino acid sequence of SEQ ID NO: 811, the first half-life prolonging domain and the second half-life prolonging domain contain modifications that promote association of the first and second half-life prolonging domains, and the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 266 and 267. In some embodiments, the masked cytokine comprises (a) a first half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a masking portion 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: 260, wherein the masking portion is linked to the first half-life prolonging domain via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or a functional fragment thereof is linked to the second half-life prolonging domain via a second linker comprising the amino acid sequence of SEQ ID NO: 262, the first half-life prolonging domain and the second half-life prolonging domain contain modifications that promote association of the first and second half-life prolonging domains, and the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 266 and 267. In some embodiments, the masked cytokine comprises 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0085] In some embodiments, the masked cytokine comprises (a) a first half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a masking portion 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 portion is linked to the first half-life prolonging domain via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or a functional fragment thereof is linked to the second half-life prolonging domain via a second linker comprising the amino acid sequence of SEQ ID NO: 807, the first half-life prolonging domain and the second half-life prolonging domain contain modifications that promote association of the first and second half-life prolonging domains, and the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 679 and 267. In some embodiments, the masked cytokine comprises (a) a first half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 156 and a second half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 155; (b) a masking portion 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 portion is linked to the first half-life prolonging domain via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or a functional fragment thereof is linked to the second half-life prolonging domain via a second linker comprising the amino acid sequence of SEQ ID NO: 807, the first half-life prolonging domain and the second half-life prolonging domain contain modifications that promote association of the first and second half-life prolonging domains, and the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 679 and 267.

[0086] In some embodiments, the masked cytokine comprises (a) a first half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a masking portion 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 portion is linked to the first half-life prolonging domain via a first linker, the cytokine or the functional fragment thereof is linked to the second half-life prolonging domain via a second linker, the first half-life prolonging domain and the second half-life prolonging domain contain modifications that promote association of the first and second half-life prolonging domains, 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 prolonging domain comprising the amino acid sequence of SEQ ID NO: 156 and a second half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 155; (b) a masking portion 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 portion is connected to the first half-life prolonging domain via a first linker, the cytokine or the functional fragment thereof is connected to the second half-life prolonging domain via a second linker, the first half-life prolonging domain and the second half-life prolonging domain contain modifications that promote the association of the first and second half-life prolonging domains, wherein the first linker and / or the second linker comprises a cleavable peptide.

[0087] In some embodiments, the masked cytokine comprises (a) a first half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a masking portion 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 portion is linked to the first half-life prolonging domain via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or a functional fragment thereof is linked to the second half-life prolonging domain via a second linker comprising the amino acid sequence of SEQ ID NO: 812, the first half-life prolonging domain and the second half-life prolonging domain contain modifications that promote association of the first and second half-life prolonging domains, and the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 689 and 267. In some embodiments, the masked cytokine comprises (a) a first half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 156 and a second half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 155; (b) a masking portion 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 portion is linked to the first half-life prolonging domain via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or a functional fragment thereof is linked to the second half-life prolonging domain via a second linker comprising the amino acid sequence of SEQ ID NO: 812, the first half-life prolonging domain and the second half-life prolonging domain contain modifications that promote association of the first and second half-life prolonging domains, and the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 689 and 267.

[0088] In some embodiments, the masked cytokine comprises (a) a first half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a masking portion 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 portion is linked to the first half-life prolonging domain via a first linker, the cytokine or the functional fragment thereof is linked to the second half-life prolonging domain via a second linker, the first half-life prolonging domain and the second half-life prolonging domain contain modifications that promote association of the first and second half-life prolonging domains, 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 prolonging domain comprising the amino acid sequence of SEQ ID NO: 156 and a second half-life prolonging domain comprising the amino acid sequence of SEQ ID NO: 155; (b) a masking portion 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 portion is connected to the first half-life prolonging domain via a first linker, and the cytokine or a functional fragment thereof is connected to the second half-life prolonging domain via a second linker, and the first half-life prolonging domain and the second half-life prolonging domain contain modifications that promote the association of the first and second half-life prolonging domains, wherein the first linker and / or the second linker comprises a cleavable peptide.

[0089] 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.

[0090] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0094] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0095] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0096] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0097] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0098] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0099] 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0100] 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0101] 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0103] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0104] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0144] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0145] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0147] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0148] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0174] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0175] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0176] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0177] 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0178] 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0179] 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0180] In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 comprises the amino acid sequences of SEQ ID NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0192] In some embodiments, the masked cytokine is an exemplary masked cytokine construct as described in any of Tables 8-11, or is 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 construct according to the teachings herein.

[0193] Also provided herein in some embodiments are masked cytokines comprising (a) a first half-life extension domain and a second half-life extension domain; (b) a first masking portion and a second masking portion; and (c) a cytokine or a functional fragment thereof, wherein the first masking portion is connected to the first half-life extension domain, the second masking portion is connected to the cytokine or its functional fragment, the second masking portion or the cytokine or its functional fragment is connected 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 portion is connected to the first half-life extension domain via a first linker, and / or the second masking portion or the cytokine or its functional fragment is connected to the second half-life extension domain via a second linker. In some embodiments, the second masking portion is connected to the cytokine or its functional fragment via a third linker. In some embodiments, the first half-life extension domain is optionally connected 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 NOs: 755 and 616. In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 756 and 616. In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 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.

[0194] 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 related to any of the exemplary constructs described in Tables 4-11.

[0195] Each component of the masked cytokines provided herein is discussed in more detail below.

[0196] A. Cytokines

[0197] Provided herein are cytokines or functional fragments thereof. 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 play a role in cell signaling, particularly in cells of the immune system. Examples of cytokines may include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors.

[0198] Cytokines can be classified in a variety of ways, such as based on their three-dimensional structure. Examples include beta-trefoil, short four-helix bundles, and long four-helix bundles.

[0199] 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. 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.

[0200] The short-chain and long-chain four-helix bundle classes are characterized by having a monomeric helical bundle of four amphipathic helices with a unique up-up-down-down topological orientation. The short-chain four-helix bundle class is characterized in that the helix is shorter, such as those with a length of 10-20 amino acid residues, while the long-chain four-helix bundle class is characterized in that the helix is longer, such as those with a length 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).

[0201] In contrast to the short-chain and long-chain four-helix bundles, which are monomers, some cytokines are homodimeric. Examples of dimeric cytokines include IL-10 and IFN-γ (gamma).

[0202] Some cytokines are classified as heterodimeric. Examples of heterodimeric cytokines include IL-12 and IL-23.

[0203] Some cytokines, such as IL-15, also exert their effects by binding to and being presented by membrane-bound cytokine receptors.

[0204] In some embodiments, cytokine or its functional fragment is IL-2, or a functional fragment or variant of IL-2. In some embodiments, cytokine or its functional fragment is IL-15, or a functional fragment or variant of IL-15. In some embodiments, cytokine or its functional fragment 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, cytokine or its functional fragment 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).

[0205] In some embodiments, the cytokine or 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.

[0206] In some embodiments, the cytokine or functional fragment thereof 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).

[0207] In some embodiments, the cytokine or functional fragment thereof 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 functional fragment thereof 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.

[0208] In some embodiments, the cytokine or functional fragment thereof is selected from IFN-α (alpha), IFN-β (beta), IFN-γ (gamma), IFN-ε (epsilon), IFN-κ (kappa), In some embodiments, the cytokine or its functional fragment is IFN-α (alpha), IFN-β (beta), IFN-γ (gamma), IFN-ε (epsilon), IFN-κ (kappa),

[0013] The invention also provides functional fragments or variants of IFN-1 (omega), IFN-tau (omega), IFN-ζ (zeta), IFN-δ (delta), or IFN-λ (lambda).

[0209] In some embodiments, the cytokine or 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 functional fragment thereof is 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-17 A / F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A , functional fragments or variants of IL-28B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36 or IL-37.

[0210] In some embodiments, the cytokine or its functional fragment is selected from the group consisting of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), tumor necrosis factor alpha (TNF-α), transforming growth factor beta (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 its functional fragment is a functional fragment or variant of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), IFN-γ (gamma), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 or IL-12.

[0211] In some embodiments, the cytokine or its functional fragment 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 EDAl. In some embodiments, the cytokine or its functional fragment 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 EDAl.

[0212] The cytokine or its functional fragment comprises an amino terminus and a carboxyl terminus. In some embodiments, the half-life extension domain is connected to the amino terminus or carboxyl terminus of the cytokine or its functional fragment. In some embodiments, the masking portion is connected to the amino terminus or carboxyl terminus of the cytokine or its functional fragment. In some embodiments, the linker is connected to the amino terminus or carboxyl terminus of the cytokine or its functional fragment. In some embodiments, the cleavable peptide of the linker is connected to the amino terminus or carboxyl terminus of the cytokine or its functional fragment. In some embodiments, the N-terminal spacer domain or the C-terminal spacer domain of the linker is connected to the amino terminus or carboxyl terminus of the cytokine or its functional fragment.

[0213] Exemplary embodiments of cytokines or functional fragments thereof in the form of IL-2 polypeptides or functional fragments thereof and IL-15 polypeptides or functional fragments thereof are provided in detail below.

[0214] 1. IL-2 peptide

[0215] In some embodiments, the cytokine or its functional fragment is an IL-2 polypeptide or its functional fragment. In eukaryotic cells, IL-2 is synthesized as a 153 amino acid precursor polypeptide, which is then processed into mature IL-2 by removing amino acid residues 1-20. This produces a mature form of IL-2 consisting of 133 amino acids (amino acid residues 21-153), which is secreted in a mature, active form.

[0216] In some embodiments, the IL-2 polypeptide or functional fragment thereof is any naturally occurring interleukin-2 (IL-2) protein or modified variant thereof that is capable of binding to or otherwise exhibiting affinity for an interleukin-2 receptor (IL-2R) or a component thereof (e.g., an IL-2R α chain). In some embodiments, the IL-2 polypeptide or functional fragment thereof is a 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 functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 160. In some embodiments, the IL-2 polypeptide or 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 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 an 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.

[0217] In some embodiments, the IL-2 polypeptide or its functional fragment comprises an amino acid sequence generated 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 modification can be any amino acid modification, such as a substitution, insertion, or deletion. In some embodiments, the IL-2 polypeptide or its functional fragment comprises an amino acid sequence generated by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten 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 its functional fragment 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 generated 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.

[0218] 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) 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 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 one or more of 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 amino acid residues 38, 42, 45, and 62 are alanine (A), 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 38 and 42 are alanine (A), 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 generated by substituting amino acid residues R38, F42, Y45, and E62 for alanine 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 generated by substituting amino acid residues R38, F42, Y45, and E62 for alanine (A) and substituting amino acid residue C125 for serine (S), glycine (G), or alanine (A) 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 generated by substituting one or more amino acids 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).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 glutamic acid (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).

[0219] 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.

[0220] 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) 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 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 18 is cysteine (C). 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 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 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 16 is isoleucine. 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 29 is leucine (L).

[0221] 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.

[0222] 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 reduce the affinity of the IL-2 polypeptide 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 enhance 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 are 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.

[0223] 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.

[0224] In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence generated by introducing or further introducing a C125S, C125G or C125A substitution into any one of the amino acid sequences for the IL-2 polypeptide or a functional fragment thereof described herein.

[0225] In some embodiments, one or more amino acid residues are removed from the amino acid sequence of the IL-2 polypeptide or its functional fragment for the purpose of removing an O-glycosylation site. In some embodiments, the IL-2 polypeptide or its functional fragment comprises an amino acid sequence generated by deleting the first three amino acid residues (residues 1-3) of the amino acid sequence of any IL-2 polypeptide or its functional fragment disclosed herein. In some embodiments, one or more amino acid residues are substituted into the amino acid sequence of the IL-2 polypeptide or its functional fragment for the purpose of removing an O-glycosylation site. In some embodiments, the IL-2 polypeptide or its functional fragment comprises an amino acid sequence generated by 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 its functional fragment disclosed herein.

[0226] The IL-2 polypeptide or its functional fragment comprises an amino terminus and a carboxyl terminus. In some embodiments, the half-life extension domain is linked to the amino terminus or carboxyl terminus of the IL-2 polypeptide or its functional fragment. In some embodiments, the masking moiety is linked to the amino terminus or carboxyl terminus of the IL-2 polypeptide or its functional fragment. In some embodiments, the linker is linked to the amino terminus or carboxyl terminus of the IL-2 polypeptide or its functional fragment. In some embodiments, the cleavable peptide of the linker is linked to the amino terminus or carboxyl terminus of the IL-2 polypeptide or its functional fragment. In some embodiments, the N-terminal spacer domain or the C-terminal spacer domain of the linker is linked to the amino terminus or carboxyl terminus of the IL-2 polypeptide or its functional fragment.

[0227] 2. IL-15 peptide

[0228] In some embodiments, the cytokine or functional fragment thereof is an IL-15 polypeptide or functional fragment thereof. In eukaryotic cells, IL-15 is synthesized as a 162 amino acid precursor polypeptide, which is then processed into mature IL-15 by removing amino acid residues 1-48. This produces a 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).

[0229] In some embodiments, the IL-15 polypeptide or functional fragment thereof is any naturally occurring interleukin-15 (IL-15) protein or modified variant thereof that is capable of binding to or otherwise exhibiting affinity for the interleukin-15 receptor (IL-15R) or a component thereof (e.g., IL-15Rα, IL-2Rβ, and / or IL-2Rγ chain). In some embodiments, the IL-15 polypeptide or functional fragment thereof is a 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 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.

[0230] In some embodiments, the IL-15 polypeptide or a functional fragment thereof comprises an amino acid sequence generated by making at least one amino acid modification to the amino acid sequence of SEQ ID NO: 167. Each of the at least one amino acid modification 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 substitution of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids in the amino acid sequence of SEQ ID NO: 167.

[0231] The IL-15 polypeptide or its functional fragment comprises an amino terminus and a carboxyl terminus. In some embodiments, the half-life extension domain is linked to the amino terminus or carboxyl terminus of the IL-15 polypeptide or its functional fragment. In some embodiments, the masking moiety is linked to the amino terminus or carboxyl terminus of the IL-15 polypeptide or its functional fragment. In some embodiments, the linker is linked to the amino terminus or carboxyl terminus of the IL-15 polypeptide or its functional fragment. In some embodiments, the cleavable peptide of the linker is linked to the amino terminus or carboxyl terminus of the IL-15 polypeptide or its functional fragment. In some embodiments, the N-terminal spacer domain or the C-terminal spacer domain of the linker is linked to the amino terminus or carboxyl terminus of the IL-15 polypeptide or its functional fragment.

[0232] B. Masked part

[0233] As provided herein, a masking moiety is a moiety that is capable of binding to a cytokine or a functional fragment thereof or otherwise exhibits affinity for a cytokine or a functional fragment thereof, such that, in some embodiments, the binding reduces the affinity of the cytokine or its functional fragment 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 an IL-2 polypeptide or a functional fragment thereof or otherwise exhibits affinity for an IL-2 polypeptide or a functional fragment thereof, such that, in some embodiments, the binding reduces the affinity of the IL-2 polypeptide or its functional fragment for its cognate receptor or protein (e.g., IL-2R or a component thereof, such as IL-2Rα and / or IL-2Rβ chain). When bound to a cytokine or a functional fragment thereof, the masking moiety blocks, occludes, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the activity of the cytokine or its functional fragment or its binding to its cognate receptor or protein. In some embodiments comprising a first masking moiety and a second masking moiety, the presence of one of the masking moieties (e.g., the first masking moiety) blocks, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the activity of the cytokine or its functional fragment 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 its functional fragment after cleavage of the first masking moiety and still allow or potentially even promote the binding of the cytokine or its functional fragment to its cognate receptor or protein at the site where the first masking moiety is bound. See, e.g., Figure 7B Methods for determining the extent of binding of a protein (eg, a cytokine) to a cognate protein (eg, a cytokine receptor) are well known in the art.

[0234] 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 masking moiety 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 a "masking moiety" or "the masking moiety" may refer to a masking moiety in a masked cytokine comprising a single masking moiety, or it may refer to a first masking moiety in a masked cytokine comprising a first masking moiety and a second masking moiety, or it may refer to a second masking moiety in a masked cytokine comprising a first masking moiety and a second masking moiety, or it may refer to a first masking moiety and a second masking moiety in a masked cytokine comprising a first masking moiety and a second masking moiety.

[0235] In some embodiments, the masking portion is connected to a cytokine or a functional fragment thereof. In some embodiments, the masking portion is connected to a cytokine or a functional fragment thereof via a first linker. In some embodiments, the masked cytokine comprises a half-life extension domain connected to the masking portion. In some embodiments, the masked cytokine comprises a half-life extension domain connected to the masking portion via a second linker. In some embodiments, the masking portion is connected to a cytokine or a functional fragment thereof and is connected to the half-life extension domain. In some embodiments, the cytokine or its functional fragment is connected to the amino terminus of the masking peptide, and the half-life extension domain is connected to the carboxyl terminus of the masking peptide. In some embodiments, the cytokine or its functional fragment is connected to the carboxyl terminus of the masking peptide, and the half-life extension domain is connected to the amino terminus of the masking peptide. In some embodiments, the half-life extension domain is connected to the amino terminus or carboxyl terminus of the masking portion. In some embodiments, the second linker is connected to the amino terminus or carboxyl terminus of the masking portion. In some embodiments, the masking portion is connected to the first linker and is connected to the second linker. In some embodiments, the first linker is attached to the amino terminus of the masking peptide and the second linker is attached to the carboxyl terminus of the masking peptide. In some embodiments, the first linker is attached to the carboxyl terminus of the masking peptide and the second linker is attached to the amino terminus of the masking peptide.

[0236] In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety is connected to the cytokine or its functional fragment, and the second masking moiety is connected to the cytokine or its functional fragment. In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety is connected to the cytokine or its functional fragment via a first linker, and the second masking moiety is connected to the cytokine or its functional fragment via a second linker. In some embodiments, the masked cytokine comprises a half-life extension domain connected to the first masking moiety or the second masking moiety. In some embodiments, the masked cytokine comprises a half-life extension domain connected to the first masking moiety or the second masking moiety via a third linker. In some embodiments, the first linker is connected to the amino-terminus or carboxyl-terminus of the first masking moiety. In some embodiments, the second linker is connected to the amino-terminus or carboxyl-terminus of the second masking moiety. In some embodiments, the half-life extension domain is connected to the amino-terminus or carboxyl-terminus of the first masking moiety. In some embodiments, the half-life extension domain is connected to the amino-terminus or carboxyl-terminus of the second masking moiety. In some embodiments, the third linker is attached to the amino terminus or carboxyl terminus of the first masking moiety. In some embodiments, the third linker is attached to the amino terminus or carboxyl terminus of the second masking moiety.

[0237] In some embodiments comprising a first masking portion and a second masking portion, the first masking portion is connected to the first half-life extension domain, the second masking portion is connected to the first masking portion, and the cytokine or its functional fragment is connected to the second half-life extension domain. In some embodiments comprising a first masking portion and a second masking portion, the first masking portion is connected to the first half-life extension domain via a first linker, the second masking portion is connected to the first masking portion via a second linker, and the cytokine or its functional fragment is connected to the second half-life extension domain via a third linker. In some embodiments, the first linker is connected to the amino terminus or carboxyl terminus of the first half-life extension domain. In some embodiments, the second linker is connected to the amino terminus or carboxyl terminus of the second masking portion. In some embodiments, the second half-life extension domain is connected to the amino terminus or carboxyl terminus of the cytokine or its functional fragment. In some embodiments, the first half-life extension domain is connected to the amino terminus or carboxyl terminus of the first masking portion. In some embodiments, the third linker is connected to the amino terminus or carboxyl terminus of the second half-life extension domain. In some embodiments, the third linker is attached to the amino terminus or carboxyl terminus of the cytokine or a functional fragment thereof.

[0238] In some embodiments comprising a first masking portion and a second masking portion, the second masking portion is connected to the first half-life extension domain, the first masking portion is connected to the second masking portion, and the cytokine or its functional fragment is connected to the second half-life extension domain. In some embodiments comprising a first masking portion and a second masking portion, the second masking portion is connected to the first half-life extension domain via a first linker, the first masking portion is connected to the second masking portion via a second linker, and the cytokine or its functional fragment is connected to the second half-life extension domain via a third linker. In some embodiments, the first linker is connected to the amino terminus or carboxyl terminus of the first half-life extension domain. In some embodiments, the second linker is connected to the amino terminus or carboxyl terminus of the first masking portion. In some embodiments, the second half-life extension domain is connected to the amino terminus or carboxyl terminus of the cytokine or its functional fragment. In some embodiments, the first half-life extension domain is connected to the amino terminus or carboxyl terminus of the second masking portion. In some embodiments, the third linker is connected to the amino terminus or carboxyl terminus of the second half-life extension domain. In some embodiments, the third linker is attached to the amino terminus or carboxyl terminus of the cytokine or a functional fragment thereof.

[0239] In some embodiments comprising a first half-life extending domain and a second half-life extending domain, the 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.

[0240] In some embodiments comprising a first half-life extension domain, a second half-life extension domain, a first masking moiety and a second masking moiety, the first masking moiety is connected to the first half-life extension domain, and the second masking moiety is connected to the cytokine or its functional fragment. In some embodiments, the second masking moiety is further connected to the second half-life extension domain. In some embodiments, the first masking moiety is connected to the first half-life extension domain via a first linker, and / or the second masking moiety or the cytokine or its functional fragment is connected to the second half-life extension domain via a second linker. In some embodiments, the second masking moiety is connected to the cytokine or its functional fragment via a third linker.

[0241] In some embodiments, the first half-life extension domain is connected to the amino terminus or carboxyl terminus of the first masking portion. In some embodiments, the cytokine or its functional fragment is connected to the amino terminus or carboxyl terminus of the second masking portion. In some embodiments, the first linker is connected to the amino terminus or carboxyl terminus of the first masking portion. In some embodiments, the second linker is connected to the amino terminus or carboxyl terminus of the second masking portion. In some embodiments, the third linker is connected to the amino terminus or carboxyl terminus of the second masking portion. In some embodiments, the second linker is connected to the amino terminus of the second masking portion, and the third linker is connected to the carboxyl terminus of the second masking portion. In some embodiments, the second linker is connected to the carboxyl terminus of the second masking portion, and the third linker is connected to the amino terminus of the second masking portion.

[0242] 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.

[0243] 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 NO: 10, 161-165, 187-218, 221-229, 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, 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 NO: 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 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, 187-218, 221-229, 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 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.

[0244] 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 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 moiety 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 moiety 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 of 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 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 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 NO: 10, 161-165, 221-226, 261, 826, and 827. 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 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 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, 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 NO: 10, 161-165, 221-226, 261, 826, and 827.

[0245] 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 NO: 187-218. 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 the amino acid sequence of SEQ ID NO: 187-218. In some embodiments, the first masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 187-218, and the second masking moiety comprises an amino acid sequence of SEQ ID NOs: 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: 187-218, 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 of SEQ ID NOs: 9 or 231.

[0246] 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 NO: 227-229. 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 the amino acid sequence of SEQ ID NO: 227-229. In some embodiments, the first masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 227-229, and the second masking moiety comprises an amino acid sequence of SEQ ID NOs: 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: 227-229, 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 of SEQ ID NOs: 9 or 231.

[0247] In some embodiments, the first masking moiety comprises the amino acid sequence of SEQ ID NO: 9, and the second masking moiety comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the first masking moiety comprises the amino acid sequence of SEQ ID NO: 10, and the second masking moiety comprises the amino acid sequence of SEQ ID NO: 9. 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, 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: 10. 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: 10, 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. In some embodiments, the masking moiety comprises an amino acid sequence generated by making one or more of the following amino acid substitutions: C122S, C168S, R42A, K71A, T73A, T74A, V75A, H133A, Y134A, R137D, Q162W, E170A, and Q188A to SEQ ID NO: 10 or 261. In some embodiments, the masking moiety comprising an amino acid sequence generated by one or more amino acid substitutions comprises the amino acid sequence of SEQ ID NO: 826.

[0248] In some embodiments, the masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 NOs: 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.

[0249] 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.

[0250] 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, 261, 826, and 827. In some embodiments, the second masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232-234 and 823-825, and the first masking moiety 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 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, 261, 826, and 827, 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, 261, 826, and 827, 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. 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 of SEQ ID NOs: 826 or 827. In some embodiments, the second masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232-234 and 823-825, and the first masking moiety comprises an amino acid sequence of SEQ ID NOs: 826 or 827.

[0251] 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, 221-226, 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, 221-226, 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, 221-226, 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, 221-226, 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.

[0252] 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: 227- 229. In some embodiments, the first masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 227-229, 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: 227-229, 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: 227-229, 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.

[0253] 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: 219 and 220. In some embodiments, the first masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 219 and 220, 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: 219 and 220, 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: 219 and 220, 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.

[0254] In some embodiments, the masking moiety comprises an amino acid sequence generated by introducing one or more of the following amino acid substitutions into the amino acid sequence of any one 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 the masking moiety comprises an amino acid sequence generated by introducing one or more of the following amino acid substitutions into the amino acid sequence of any one of SEQ ID NOs: 232-234 and 823-825. The amino acid sequences of the group consisting of NOs: 232-234 and 823-825 having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity are further modified by the introduction of one or more of the following amino acid substitutions: R24A, R26A, K34A, S40A, L42A and P67A.

[0255] In some embodiments, the masking moiety comprises IL-2Rα (also known as CD25) or a fragment, portion or variant thereof that maintains 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 maintains 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 portion comprises IL-2Rγ (also known as CD132) or a fragment, portion or variant thereof that maintains or otherwise exhibits affinity for IL-2 and / or IL-15. In some embodiments, the masking portion comprises IL-15Rα (also known as CD215) or a fragment, portion or variant thereof that maintains or otherwise exhibits affinity for IL-15. In some embodiments, the masking portion 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 NOs: 232-234 and 823-825. In some embodiments, the masking portion comprises an antigen binding domain of an antibody or fragment thereof. In some embodiments, the masking portion comprises an antigen binding domain of an anti-IL-2 antibody or fragment thereof. In some embodiments, the masking portion comprises an antigen binding domain of an anti-IL-15 antibody or fragment thereof.

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

[0257] In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety comprises IL-15Rα, or a fragment, portion, or variant thereof, that retains or otherwise exhibits affinity for IL-15, and the second masking moiety comprises IL-2Rβ, or a fragment, portion, or variant thereof, that retains or otherwise exhibits affinity for IL-15. In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety comprises IL-2Rβ, or a fragment, portion, or variant thereof, that retains or otherwise exhibits affinity for IL-15, and the second masking moiety comprises IL-15Rα, or a fragment, portion, or variant thereof, that retains or otherwise exhibits affinity for IL-15. In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety comprises IL-15Rα, or a fragment, portion, or variant thereof, that retains or otherwise exhibits affinity for IL-15, and the second masking moiety comprises IL-2Rγ, or a fragment, portion, or variant thereof, that retains or otherwise exhibits affinity for IL-15. In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety comprises IL-2Rγ or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15, and the second masking moiety comprises IL-15Rα or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15. In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety comprises 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 fragment thereof. In some embodiments comprising 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 fragment thereof, and the second masking moiety comprises IL-15Rα or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15.

[0258] C.Connector

[0259] As provided herein, a linker refers to a peptide of two or more amino acids for connecting two components, such as the two components of any masked cytokine 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 carboxyl-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.

[0260] In some embodiments, the masked cytokine comprises a linker. In some embodiments, the masked cytokine comprises a single linker. In some embodiments, the masked cytokine comprises more than one linker, each of which can be any linker described herein. In some embodiments, the masked cytokine comprises a first linker and a second linker. In some embodiments, the masked cytokine comprises a first linker, a second linker, and a third linker. In some embodiments, the masked cytokine comprises a first linker, a second linker, a third linker, and a fourth linker. For example, reference to a "linker" or "the linker" may refer to a linker in a masked cytokine that comprises a single linker, or it may refer to the first linker in a masked cytokine that comprises a first linker and a second linker, or it may refer to the second linker in a masked cytokine that comprises a first linker and a second linker, or it may refer to the first linker and the second linker in a masked cytokine that comprises a first linker and a second linker, or it may 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 may 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.

[0261] It is to be understood that each of the following terms are considered to be "linkers" as described herein: linker, first linker, second linker, third linker, and fourth linker, as well as any other numbered linkers (e.g., fifth linker, sixth linker, etc.). Thus, in some embodiments, a masked cytokine may comprise a linker, a first linker, a second linker, a third linker, and / or a fourth linker, each of which is considered to be a "linker" as described herein, and any linker described herein may be a linker, a first linker, a second linker, a third linker, and / or a fourth linker as described herein.

[0262] It is also to be understood that two components that are "linked" together include embodiments in which the two components are directly linked together as well as 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 to be interpreted as meaning that the amino acid sequence of the half-life extension domain is directly linked to the amino acid sequence of the masking moiety, or that the amino acid sequence of the half-life extension domain is linked to the amino acid sequence of the masking moiety via a linker, such as any linker described herein.

[0263] The linker comprises an amino terminus and a carboxyl terminus. In some embodiments, the masking portion is connected to the amino terminus or carboxyl terminus of the linker. In some embodiments, the cytokine or its functional fragment is connected to the amino terminus or carboxyl terminus of the linker. In some embodiments, the cytokine or its functional fragment is connected to the amino terminus of the linker, and the masking portion is connected to the carboxyl terminus of the linker. In some embodiments, the cytokine or its functional fragment is connected to the carboxyl terminus of the linker, and the masking portion is connected to the amino terminus of the linker. In some embodiments, the half-life extension domain is connected to the amino terminus or carboxyl terminus of the linker. In some embodiments, the half-life extension domain is connected to the amino terminus of the linker, and the masking portion is connected to the carboxyl terminus of the linker. In some embodiments, the half-life extension domain is connected to the carboxyl terminus of the linker, and the masking portion is connected to the amino terminus of the linker. In some embodiments, the half-life extension domain is connected to the amino terminus of the linker, and the cytokine or its functional fragment is connected to the carboxyl terminus of the linker. In some embodiments, the half-life extension domain is connected to the carboxyl terminus of the linker, and the cytokine or its functional fragment is connected to the amino terminus of the linker.

[0264] In some embodiments comprising a first masking moiety and a second masking moiety, each of which is 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 the linker, and the cytokine or a functional fragment thereof is linked to the carboxyl terminus of the linker. In some embodiments comprising a first masking moiety and a second masking moiety, each of which is 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 the linker, and the cytokine or a functional fragment thereof is linked to the amino terminus of the linker.

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

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

[0267] In some embodiments, the cytokine or a functional fragment thereof comprises 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 the 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.

[0268] In some embodiments, the cytokine or functional fragment thereof comprises 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 functional fragment thereof linked to the 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 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.

[0269] In some embodiments, the cytokine or a functional fragment thereof comprises 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 the 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.

[0270] In some embodiments, the cytokine or a functional fragment thereof comprises 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 the 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.

[0271] In some embodiments comprising a first half-life prolonging domain and a second half-life prolonging domain, the first half-life prolonging domain is linked to the second half-life prolonging domain. In some embodiments comprising a first half-life prolonging domain and a second half-life prolonging domain, the first half-life prolonging domain is linked to the second half-life prolonging domain via a linker.

[0272] 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.

[0273] 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.

[0274] 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 an 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.

[0275] The cleavable peptide and spacer domains of some embodiments of the linker are described in more detail below.

[0276] 1. Cleavable peptides

[0277] In some embodiments, the linker comprises a cleavable peptide. In some embodiments with one or more linkers comprising a cleavable peptide, the cleavable peptide 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 cleavable peptide described herein. For example, reference to a "cleavable peptide" or "the cleavable peptide" may refer to a cleavable peptide in a masked cytokine comprising a single cleavable peptide, or it may refer to a first cleavable peptide in a masked cytokine comprising a first cleavable peptide and a second cleavable peptide, or it may refer to a second cleavable peptide in a masked cytokine comprising a first cleavable peptide and a second cleavable peptide, or it may refer to a first cleavable peptide and a second cleavable peptide in a masked cytokine comprising a first cleavable peptide and a second cleavable peptide, or it may refer to a first cleavable peptide and a second cleavable peptide in a masked cytokine comprising a first cleavable peptide, a second cleavable peptide, and / or a third cleavable peptide in a masked cytokine comprising a first cleavable peptide, a second cleavable peptide, and / or a third cleavable peptide. It is conceivable that in some embodiments, more than three cleavable peptides may be present according to the teachings herein.

[0278] Cleavable peptides are polypeptides that include a cleavage site, such as a protease cleavage site. In some embodiments, a cleavable peptide comprises more than one cleavage site. As used herein, "cleavage site" refers to a recognizable site for cleaving a portion of a cleavable peptide found in any linker comprising a cleavable peptide as described herein. Thus, a cleavage site can be found in the sequence of a cleavable peptide as described herein. In some embodiments, a cleavage site is an amino acid sequence that is recognized and cleaved by a cleavage agent. Exemplary cleavage agents include proteins, enzymes, DNA enzymes, RNA enzymes, metals, acids, and bases.

[0279] 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 whose expression is specific or upregulated for a tumor cell or its 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, ADAMT S18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL 2. ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE , CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, CTRl, 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, MM P24, 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, TMPRSS 11E, 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.

[0280] In several embodiments, the protease cleavage site is a matrix metalloproteinase (MMP) cleavage site, a disintegrin and metalloproteinase domain (ADAM) metalloproteinase 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 matriptase protease cleavage site (ST14), or a leguminase protease cleavage site. In several embodiments, the matrix metalloproteinase (MMP) cleavage site is an MMP9 cleavage site, an MMP13 cleavage site, or an MMP2 cleavage site. In several embodiments, the disintegrin and metalloproteinase domain (ADAM) metalloproteinase cleavage site is an ADAM9 metalloproteinase cleavage site, an ADAM10 metalloproteinase cleavage site, or an ADAM17 metalloproteinase cleavage site. The protease cleavage site can be specified by a specific amino acid sequence.

[0281] 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, CE LA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, CTRl, CTSA, CTSW, CTSB, CTSC, CTSD, E SP1, 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,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 matrices and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of legumin and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of matrices and uPA. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of uPA and legumin. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of matrices and legumin. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of uPA, matrices, and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of uPA, legumin, and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of matrices, legumin, and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of matrices, legumin, and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of matrices, legumin, and uPA.

[0282] In some embodiments, the cleavable peptide is a substrate for a protease that is co-localized in an area 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, I L-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, GMR α(CD116), CD131, GHR, PRLR, EPOR, LIFR(CD118), OSMRβ, TPO-R(CD110), CSF-1R, EDAR, TNFRSF1A, TNFRSF1B, LTBR, TNFRSF 4. 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.

[0283] In some embodiments, the cleavable peptide is a 5-mer (i.e., a peptide of 5 amino acids in length), a 6-mer (i.e., a peptide of 6 amino acids in length), a 7-mer (i.e., a peptide of 7 amino acids in length), an 8-mer (i.e., a peptide of 8 amino acids in length), a 9-mer (i.e., a peptide of 9 amino acids in length), a 10-mer (i.e., a peptide of 10 amino acids in length), a 11-mer (i.e., a peptide of 11 amino acids in length), a 12-mer (i.e., a peptide of 12 amino acids in length), a 13-mer (i.e., a peptide of 13 amino acids in length), a 14-mer (i.e., a peptide of 14 amino acids in length), a 15-mer (i.e., a peptide of 15 amino acids in length), a 16-mer (i.e., a peptide of 16 amino acids in length), a 17-mer (i.e., a peptide of 17 amino acids in length), or an 18-mer (i.e., a peptide of 18 amino acids in length). Exemplary cleavable peptide sequences are shown in Table 1.

[0284] Table 1. Exemplary cleavable peptide sequences

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292] 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 may 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 comprises an N-terminus and a C-terminus. 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, 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.

[0293] 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), non-polar amino acids (e.g., glycine, alanine, valine, proline, leucine, isoleucine, methionine, tryptophan, or phenylalanine), amino acids with aliphatic side chains. Amino acids (e.g., glycine, alanine, valine, leucine, or isoleucine), amino acids with hydroxyl side chains (e.g., serine or threonine), amino acids with sulfur 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.

[0294] The cleavable peptide comprises an amino terminus and a carboxyl terminus. In some embodiments, the cleavable peptide is connected to an N-terminal spacer domain and / or a C-terminal spacer domain. In some embodiments, the N-terminal spacer domain is connected to the amino terminus of the cleavable peptide. In some embodiments, the C-terminal spacer domain is connected to the carboxyl terminus of the cleavable peptide. In some embodiments, the N-terminal spacer domain is connected to the amino terminus of the cleavable peptide, and the C-terminal spacer domain is connected to the carboxyl terminus of the cleavable peptide. In some embodiments, the N-terminal spacer domain is connected to the amino terminus of the cleavable peptide, and the C-terminal spacer domain is connected to the carboxyl terminus of the cleavable peptide.

[0295] In some embodiments, the amino terminus of the cleavable peptide is connected to a component other than the N-terminal spacer domain. In some embodiments, the carboxyl terminus of the cleavable peptide is connected to a component other than the C-terminal spacer domain. In some embodiments, the masking portion is connected to the amino terminus or carboxyl terminus of the cleavable peptide. In some embodiments, a cytokine or a functional fragment thereof is connected to the amino terminus or carboxyl terminus of the cleavable peptide. In some embodiments, a half-life extension domain is connected to the amino terminus or carboxyl terminus of the cleavable peptide. In some embodiments, a first half-life extension domain is connected to the amino terminus or carboxyl terminus of the cleavable peptide. In some embodiments, a second half-life extension domain is connected to the amino terminus or carboxyl terminus of the cleavable peptide.

[0296] 2. Spacer domain

[0297] In some embodiments, the linker comprises a spacer domain. As used herein, in some embodiments, a "spacer domain" may refer to an N-terminal spacer domain and / or a C-terminal spacer domain. In some embodiments, the linker comprises an N-terminal spacer domain and / or a C-terminal spacer domain. In some embodiments, the linker comprises an N-terminal spacer domain. In some embodiments, the linker comprises a C-terminal spacer domain. In some embodiments, the linker comprises an N-terminal spacer domain and a C-terminal spacer domain. In some embodiments, the linker comprises an N-terminal spacer domain and a cleavable peptide. In some embodiments, the linker comprises a cleavable peptide and a C-terminal spacer domain. In some embodiments, the linker comprises an N-terminal spacer domain, a cleavable peptide, and a C-terminal spacer domain.

[0298] In some embodiments, such as those with 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 can be any N-terminal spacer domain described herein. Similarly, in some embodiments, such as those with 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 can 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 an 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.

[0299] The N-terminal spacer domain and the C-terminal spacer domain each comprise an amino terminus and a carboxyl terminus. In some embodiments, the masking moiety is connected to the amino terminus or carboxyl terminus of the N-terminal spacer domain. In some embodiments, a cytokine or a functional fragment thereof is connected to the amino terminus or carboxyl terminus of the N-terminal spacer domain. In some embodiments, a half-life extension domain is connected to the amino terminus or carboxyl terminus of the N-terminal spacer domain. In some embodiments, a first half-life extension domain is connected to the amino terminus or carboxyl terminus of the N-terminal spacer domain. In some embodiments, a second half-life extension domain is connected to the amino terminus or carboxyl terminus of the N-terminal spacer domain.

[0300] In some embodiments, the masking moiety is linked to the amino terminus or carboxyl terminus of the C-terminal spacer domain. In some embodiments, the cytokine or functional fragment thereof is linked to the amino terminus or carboxyl terminus of the C-terminal spacer domain. In some embodiments, the half-life extension domain is linked to the amino terminus or carboxyl terminus of the C-terminal spacer domain. In some embodiments, the first half-life extension domain is linked to the amino terminus or carboxyl terminus of the C-terminal spacer domain. In some embodiments, the second half-life extension domain is linked to the amino terminus or carboxyl terminus of the C-terminal spacer domain.

[0301] In some embodiments, the masking moiety is linked to the amino terminus of the N-terminal spacer domain, and the half-life extension domain is linked to 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 of the N-terminal spacer domain, and the half-life extension domain is linked to the carboxyl terminus of the N-terminal spacer domain. In some embodiments, the masking moiety is linked to the carboxyl terminus of the N-terminal spacer domain, and the half-life extension 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 carboxyl terminus of the N-terminal spacer domain, and the half-life extension domain is linked to the amino terminus of the N-terminal spacer domain.

[0302] In some embodiments, the masking moiety is linked to the amino terminus of the C-terminal spacer domain, and the half-life extension domain is linked to the carboxyl 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 extension domain is linked to the carboxyl terminus of the C-terminal spacer domain. In some embodiments, the masking moiety is linked to the carboxyl terminus of the C-terminal spacer domain, and the half-life extension 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 carboxyl terminus of the C-terminal spacer domain, and the half-life extension domain is linked to the amino terminus of the C-terminal spacer domain.

[0303] In some embodiments comprising 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 the functional fragment thereof is linked to the carboxyl terminus of the N-terminal spacer domain. In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety or the second masking moiety is linked to the carboxyl terminus of the N-terminal spacer domain, and the cytokine or the functional fragment thereof is linked to the amino terminus of the N-terminal spacer domain.

[0304] In some embodiments comprising 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 C-terminal spacer domain, and the cytokine or the functional fragment thereof is linked to the carboxyl terminus of the C-terminal spacer domain. In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety or the second masking moiety is linked to the carboxyl terminus of the C-terminal spacer domain, and the cytokine or the functional fragment thereof is linked to the amino terminus of the C-terminal spacer domain.

[0305] In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety is attached to the amino terminus of the C-terminal spacer domain, and the second masking moiety is attached to the carboxyl terminus of the C-terminal spacer domain. In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety is attached to the carboxyl terminus of the C-terminal spacer domain, and the second masking moiety is attached to the amino terminus of the C-terminal spacer domain.

[0306] In some embodiments comprising a first masking moiety and a second masking moiety, the half-life extension domain is linked to the amino terminus of the N-terminal spacer domain, and the first masking moiety or the second masking moiety is linked to the carboxyl terminus of the N-terminal spacer domain. In some embodiments comprising a first masking moiety and a second masking moiety, the half-life extension domain is linked to the carboxyl terminus of the N-terminal spacer domain, and the first masking moiety or the second masking moiety is linked to the amino terminus of the N-terminal spacer domain.

[0307] In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety is attached to the amino terminus of the N-terminal spacer domain, and the second masking moiety is attached to the carboxyl terminus of the N-terminal spacer domain. In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety is attached to the carboxyl terminus of the N-terminal spacer domain, and the second masking moiety is attached to the amino terminus of the N-terminal spacer domain.

[0308] In some embodiments comprising a first masking moiety and a second masking moiety, the half-life extension domain is linked to the amino terminus of the C-terminal spacer domain, and the first masking moiety or the second masking moiety is linked to the carboxyl terminus of the C-terminal spacer domain. In some embodiments comprising a first masking moiety and a second masking moiety, the half-life extension domain is linked to the carboxyl terminus of the C-terminal spacer domain, and the first masking moiety or the second masking moiety is linked to the amino terminus of the C-terminal spacer domain.

[0309] In some embodiments comprising 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 comprising 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 comprising 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 comprising 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.

[0310] In some embodiments comprising 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 comprising 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 comprising 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 comprising 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.

[0311] In some embodiments comprising 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 carboxyl terminus of the N-terminal spacer domain. In some embodiments comprising 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 carboxyl 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 comprising 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 carboxyl terminus of the C-terminal spacer domain. In some embodiments comprising 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 carboxyl 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.

[0312] In some embodiments comprising 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 carboxyl terminus of the N-terminal spacer domain. In some embodiments comprising 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 carboxyl 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 comprising 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 carboxyl terminus of the C-terminal spacer domain. In some embodiments comprising 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 carboxyl terminus of the C-terminal spacer domain, and the cytokine or functional fragment thereof is linked to the amino terminus of the C-terminal spacer domain.

[0313] In some embodiments comprising 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 a functional fragment thereof or the second masking moiety is linked to the carboxyl terminus of the N-terminal spacer domain. In some embodiments comprising 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 a functional fragment thereof or the second masking moiety is linked to the amino terminus of the N-terminal spacer domain. In some embodiments comprising 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 a functional fragment thereof or the second masking moiety is linked to the carboxyl terminus of the C-terminal spacer domain. In some embodiments comprising 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 functional fragment thereof, or the second masking moiety is linked to the amino terminus of the C-terminal spacer domain.

[0314] In some embodiments comprising a first half-life extending domain and a second half-life extending domain, the first half-life extending domain is connected to the second half-life extending domain via a linker. In some embodiments, the linker connecting the first half-life extending domain and the second half-life extending domain comprises an N-terminal spacer domain and / or a C-terminal spacer domain. In some embodiments, the linker connecting the first half-life extending domain and the second half-life extending domain comprises a cleavable peptide and an N-terminal spacer domain and / or a C-terminal spacer domain. In some embodiments comprising the first half-life extending domain and the second half-life extending domain connected together via a linker, the linker comprises an amino terminus and a carboxyl terminus, the first half-life extending domain is connected to the amino terminus of the linker, and the second half-life extending domain is connected to the carboxyl terminus of the linker. In some embodiments comprising the first half-life extending domain and the second half-life extending domain connected together via a linker, the linker comprises an amino terminus and a carboxyl terminus, the first half-life extending domain is connected to the carboxyl terminus of the linker, and the second half-life extending domain is connected to the amino terminus of the linker.

[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, 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.

[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 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.

[0317] 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 domains and / or C-terminal spacer domains) are shown in Table 2.

[0318] Table 2. Exemplary spacer domains

[0319]

[0320]

[0321]

[0322] In some embodiments, the N-terminal spacer domain comprises an amino acid sequence generated 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 generated 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 generated 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.

[0323] 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. In some embodiments, the N-terminal spacer domain and / or the C-terminal spacer domain are composed of glycine (G). In some embodiments, the N-terminal spacer domain and / or the C-terminal spacer domain are composed of glycine (G) and proline (P), and have an amino acid sequence of GP or PG.

[0324] It is to be understood that modifications to a "spacer domain" as described in some embodiments may refer to modifications to any one or more spacer domains described herein (e.g., any N-terminal spacer domain and / or any C-terminal spacer domain described herein). For example, modifications to a spacer domain may refer to (a) modifications to any N-terminal spacer domain described herein, (b) modifications to any C-terminal spacer domain described herein, or (c) modifications 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 are modified according to the modifications described herein.

[0325] The modification to the sequence of the N-terminal spacer domain and / or the C-terminal spacer domain can be any modification to the amino acid sequence of the spacer domain, including the incorporation of any additional amino acid into the sequence, substitution of any amino acid for a different amino acid and / or removal of any amino acid from the sequence.

[0326] In some embodiments, one or more additional amino acids are incorporated into the amino acid sequence of the N-terminal spacer domain and / or the C-terminal spacer domain by addition. In some embodiments, the one or more amino acids incorporated into the amino acid sequence of the N-terminal spacer domain and / or the C-terminal spacer domain by addition are selected from the group consisting of: a hydrophilic amino acid (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine), a hydrophobic amino acid (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, glycine, or proline), a polar amino acid (e.g., serine, threonine, cysteine, asparagine, glutamine, or tyrosine), a non-polar amino acid (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).

[0327] 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: a hydrophilic amino acid (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine), a hydrophobic amino acid (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, glycine, or proline), a polar amino acid (e.g., serine, threonine, cysteine, asparagine, glutamine, or tyrosine), a non-polar amino acid (e.g., glycine, alanine, valine, proline, leucine, isoleucine, methionine, tryptophan, or phenylalanine), an amino acid with an aliphatic side chain. amino acids (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).

[0328] In some embodiments, modifications to the amino acid sequence of the N-terminal spacer domain and / or the C-terminal spacer domain include substitution of at least one hydrophilic amino acid for a hydrophobic amino acid, substitution of at least one hydrophobic amino acid for a hydrophilic amino acid, substitution of at least one polar amino acid for a non-polar amino acid, substitution of at least one non-polar amino acid for a polar amino acid, substitution of at least one charged amino acid for an uncharged amino acid, substitution of at least one uncharged amino acid for a charged amino acid, substitution of at least one acidic amino acid for a basic amino acid, substitution of at least one basic amino acid for an acidic amino acid, substitution of at least one non-bulky amino acid for a bulky amino acid, substitution of at least one bulky amino acid for a non-bulky amino acid, substitution of at least one amino acid having a hydroxyl-containing side chain or a sulfur-containing side chain for an aliphatic amino acid, substitution of at least one amino acid having a hydroxyl-containing side chain or a sulfur-containing side chain for an aromatic amino acid, or substitution of at least one aromatic amino acid for an amino acid having a hydroxyl-containing side chain or a sulfur-containing side chain.

[0329] In some embodiments, the 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: a hydrophilic amino acid (e.g., lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine), a hydrophobic amino acid (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, glycine, or proline), a polar amino acid (e.g., serine, threonine, cysteine, asparagine, glutamine, or tyrosine), a non-polar amino acid (e.g., glycine, alanine, valine, proline, leucine, isoleucine, methionine, tryptophan, or phenylalanine), a 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).

[0330] Any modification of the spacer domain described herein (e.g., any N-terminal spacer domain and / or any C-terminal spacer domain) can be a modification of 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 of the spacer domain described herein can be a modification for any purpose. For example, the N-terminal spacer domain and / or C-terminal spacer domain of a linker comprising a cleavable peptide can be modified to change the conformation of the linker so that the cleavage efficiency of the cleavage peptide is changed. The N-terminal spacer domain and / or C-terminal spacer domain of a linker comprising a cleavable peptide can also be modified to change the structure of the linker comprising the cleavage peptide so that the cleavage efficiency of the cleavage peptide is changed under certain pH conditions.

[0331] D. Half-life extension domain

[0332] Long half-life in vivo is important for therapeutic proteins. Unfortunately, cytokines administered to subjects typically have short half-lives because they are typically rapidly cleared from the subject's body by mechanisms including renal clearance and endocytic degradation. Therefore, in some embodiments of the masked cytokines provided herein, a half-life extension domain is linked to the masked cytokine in order to extend the half-life of the cytokine in vivo.

[0333] In some embodiments, the masked cytokines provided herein comprise a half-life extension domain selected from the group consisting of antibodies and fragments thereof, albumin, albumin binding proteins, IgG binding proteins, and polyamino acid sequences. It is contemplated that other mechanisms for extending the half-life of masked cytokines available in the art may also be employed. The half-life extension domain comprises an amino terminus and a carboxyl terminus.

[0334] 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 half-life extension domain 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 prolonging domain" or "the half-life prolonging domain" may refer to a half-life prolonging domain in a masked cytokine that comprises a single half-life prolonging domain, or it may refer to a first half-life prolonging domain in a masked cytokine that comprises a first half-life prolonging domain and a second half-life prolonging domain, or it may refer to a second half-life prolonging domain in a masked cytokine that comprises a first half-life prolonging domain and a second half-life prolonging domain, or it may refer to both a first half-life prolonging domain and a second half-life prolonging domain in a masked cytokine that comprises both a first half-life prolonging domain and a second half-life prolonging domain.

[0335] In some embodiments comprising a first half-life extending domain and a second half-life extending domain, the first half-life extending domain is connected 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 connected to the second half-life extending domain via a linker. In some embodiments, the first half-life extending domain and the second half-life extending domain connected 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 first and second half-life extending domains connected are Fc domains or fragments thereof. In some embodiments, the first half-life extending domain and / or the second half-life extending domain of the first and second half-life extending domains connected are antibodies or fragments, variants or derivatives thereof.

[0336] 1. Antibodies and their fragments

[0337] By linking the masked cytokine to an antibody or fragment thereof capable of FcRn-mediated recycling, the clearance of the masked cytokine from the subject can be reduced or otherwise delayed, thereby extending the half-life of the administered masked cytokine.

[0338] In some embodiments of the masked cytokine, the half-life extension domain comprises an antibody or fragment thereof. In some embodiments, the masked cytokine comprises more than one antibody or fragment thereof, each of which can be any antibody or fragment thereof described herein. In some embodiments, the masked cytokine comprises a first half-life extension domain and a second half-life extension domain, each of which comprises an antibody or fragment thereof. It is to be understood that reference to an "antibody or fragment thereof" or "the antibody or fragment thereof" may refer to an antibody or fragment thereof that comprises a half-life prolonging domain in a masked cytokine that comprises a single half-life prolonging domain, or it may refer to an antibody or fragment thereof that comprises a first half-life prolonging domain and a second half-life prolonging domain, or it may refer to an antibody or fragment thereof that comprises a second half-life prolonging domain in a masked cytokine that comprises a first half-life prolonging domain and a second half-life prolonging domain, or it may refer to an antibody or fragment thereof that comprises a first half-life prolonging domain and a second half-life prolonging domain, or it may refer to an antibody or fragment thereof that comprises a first half-life prolonging domain and an antibody or fragment thereof that comprises a second half-life prolonging domain in a masked cytokine that comprises a first half-life prolonging domain and a second half-life prolonging domain.

[0339] The antibody or its fragment can be any antibody or its fragment. In some embodiments, the antibody or its fragment is any antibody or its fragment capable of FcRn-mediated recycling, such as any heavy chain polypeptide or its portion (e.g., Fc domain or its fragment) capable of FcRn-mediated recycling. However, in some embodiments of the masked cytokine comprising a first half-life extension domain and a second half-life extension domain, the first half-life extension domain or the second half-life extension domain may comprise an antibody or its fragment 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 extension domain comprises an antibody or its fragment, and the antibody or its fragment comprises a light chain polypeptide or its portion that does not directly interact with the FcRn receptor, but the masked cytokine has an extended half-life due to comprising a second half-life extension domain (e.g., by comprising a heavy chain polypeptide) that can interact with the FcRn receptor. It is recognized in the art that FcRn-mediated recycling requires the FcRn receptor to bind to the Fc region of the antibody or its fragment. For example, studies have shown that residues I253, S254, H435, and Y436 (numbered according to the Kabat EU index) are important for the interaction between the human Fc region and the human FcRn complex. See, for example, Firan, M. et al., Int. Immunol. 13 (2001) 993-1002; Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604. Various mutants of residues 248-259, 301-317, 376-382, and 424-437 (numbered according to the Kabat EU index) have also been examined and reported. Yeung, YA et al. (J. Immunol. 182 (2009) 7667-7671).

[0340] In some embodiments, the antibody or its fragment comprises a heavy chain polypeptide or a light chain polypeptide. In some embodiments, the antibody or its fragment comprises a portion of a heavy chain polypeptide or a light chain polypeptide. In some embodiments, the antibody or its fragment comprises an Fc domain or a fragment thereof. In some embodiments, the antibody or its fragment comprises a CH2 and CH3 domain or a fragment thereof. In some embodiments, the antibody or its fragment comprises a constant domain of a heavy chain polypeptide. In some embodiments, the antibody or its fragment comprises a constant domain of a light chain polypeptide. In some embodiments, the antibody or its fragment comprises a heavy chain polypeptide or a fragment thereof (e.g., an Fc domain or a fragment thereof). In some embodiments, the antibody or its fragment comprises a light chain polypeptide.

[0341] 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.

[0342] 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.

[0343] 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.

[0344] In some embodiments, the Fc domain or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 NOs: 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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 an amino acid sequence having about or at least about 85%, 86%, 87%, 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.

[0345] In some embodiments comprising 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 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 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 comprising 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, 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, 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: The amino acid sequences of the group consisting of NOs: 154-156, 265, 616, 619, 622, 625, 721, 772-774, 793 and 796 have an amino acid sequence with about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments comprising a first half-life prolonging domain and a second half-life prolonging domain, the first half-life prolonging domain comprises the amino acid sequence of any first half-life prolonging domain listed in Tables 4-11, and the second half-life prolonging domain comprises the amino acid sequence of any first half-life prolonging domain listed in Tables 4-11.

[0346] In some embodiments comprising 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, comprising the amino acid sequence of SEQ ID NO: 155, and the second half-life extending domain comprises an Fc domain, or a fragment thereof, comprising the amino acid sequence of SEQ ID NO: 156. In some embodiments comprising 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, comprising the amino acid sequence of SEQ ID NO: 156, and the second half-life extending domain comprises an Fc domain, or a fragment thereof, comprising the amino acid sequence of SEQ ID NO: 155. In some embodiments comprising 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, 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 the amino acid sequence of SEQ ID NO: 155, and the second half-life extending domain comprises an Fc domain, or a 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 the amino acid sequence of SEQ ID NO: 156. In some embodiments comprising 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, 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 the amino acid sequence of SEQ ID NO: 156, and the second half-life extending domain comprises an Fc domain, or a 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 the amino acid sequence of SEQ ID NO: 155.

[0347] In some embodiments comprising 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, comprising the amino acid sequence of SEQ ID NO: 155, and the second half-life extending domain comprises an Fc domain, or a fragment thereof, comprising the amino acid sequence of SEQ ID NO: 616. In some embodiments comprising 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, comprising the amino acid sequence of SEQ ID NO: 616, and the second half-life extending domain comprises an Fc domain, or a fragment thereof, comprising the amino acid sequence of SEQ ID NO: 155. In some embodiments comprising 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, 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 the amino acid sequence of SEQ ID NO: 155, and the second half-life extending domain comprises an Fc domain, or a 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 the amino acid sequence of SEQ ID NO: 616. In some embodiments comprising 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, 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 the amino acid sequence of SEQ ID NO: 616, and the second half-life extending domain comprises an Fc domain, or a 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 the amino acid sequence of SEQ ID NO: 155.

[0348] In some embodiments comprising a first half-life extending domain and a second half-life extending domain, the first half-life ex...

Claims

1. A nucleic acid encoding a masked cytokine, the masked cytokine comprising: a) a first half-life prolonging domain and a second half-life prolonging domain, wherein the first half-life prolonging domain is a first Fc domain or a fragment thereof, and the second half-life prolonging domain is a second Fc domain or a fragment thereof; b) a masking moiety comprising an amino acid sequence having at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 10; and c) a cytokine, wherein the cytokine is an IL-2 polypeptide, wherein the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 3, wherein the masking moiety is linked to the first half-life prolonging domain via a first linker, wherein the first linker comprises a cleavable peptide; wherein the cytokine is linked to the second half-life prolonging domain via a second linker; and wherein the first half-life prolonging domain and the second half-life prolonging domain contain modifications that promote association of the first and second half-life prolonging domains.

2. A nucleic acid encoding a masked cytokine, the masked cytokine comprising: a) a first polypeptide comprising a first half-life extending domain and a masking moiety; and b) a second polypeptide comprising a second half-life extending domain and a cytokine, wherein the first half-life extending domain is a first Fc domain or a fragment thereof, and the second half-life extending domain is a second Fc domain or a fragment thereof; wherein the masking portion comprises the amino acid sequence of SEQ ID NO: 826; wherein the cytokine is an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 3; wherein the masking moiety is connected to the C-terminus of the first half-life extension domain via a first linker; wherein the cytokine is linked to the C-terminus of the second half-life extension domain via a second linker, wherein the second linker comprises a cleavable peptide; and wherein said first half-life prolonging domain and said second half-life prolonging domain contain modifications that promote association of said first and said second half-life prolonging domains.

3. The nucleic acid of claim 1, wherein the masking moiety comprises the amino acid sequence of SEQ ID NO:

826.

4. The nucleic acid of any one of claims 1 to 3, wherein the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO:

156.

5. The nucleic acid of any one of claims 1 to 3, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO:

96.

6. The nucleic acid of claim 1 or claim 3, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 15 and the second linker comprises the amino acid sequence of SEQ ID NO:

339.

7. The nucleic acid of claim 2, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO:

135.

8. The nucleic acid of claim 2, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 28, and the second linker comprises the amino acid sequence of SEQ ID NO:

35.

9. The nucleic acid of claim 1 , wherein the masking moiety comprises the amino acid sequence of SEQ ID NO: 826; wherein the first Fc domain, or fragment thereof, comprises the amino acid sequence of SEQ ID NO: 155, and the second Fc domain, or fragment thereof, comprises the amino acid sequence of SEQ ID NO: 156; and wherein the first linker comprises the amino acid sequence of SEQ ID NO: 15, and the second linker comprises the amino acid sequence of SEQ ID NO:

339.

10. The nucleic acid of claim 2, wherein the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156; and wherein the first linker comprises the amino acid sequence of SEQ ID NO: 28, and the second linker comprises the amino acid sequence of SEQ ID NO:

35.

11. A vector comprising the nucleic acid of any one of claims 1 to 10.

12. A host cell comprising the nucleic acid of any one of claims 1-10.

13. A method for producing a masked cytokine, comprising culturing the host cell of claim 12 under conditions whereby the masked cytokine is produced.

14. Use of a composition for preparing a medicament for inhibiting tumor growth in a subject, by administering to the subject an effective amount of a masked cytokine comprising: a) a first half-life prolonging domain and a second half-life prolonging domain, wherein the first half-life prolonging domain is a first Fc domain or a fragment thereof, and the second half-life prolonging domain is a second Fc domain or a fragment thereof; b) a masking moiety comprising an amino acid sequence having at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 10; and c) a cytokine, wherein the cytokine is an IL-2 polypeptide, wherein the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 3, wherein the masking moiety is linked to the first half-life extending domain via a first linker, wherein the first linker comprises a cleavable peptide; wherein the cytokine is linked to the second half-life extension domain via a second linker; and wherein said first half-life prolonging domain and said second half-life prolonging domain contain modifications that promote association of said first and said second half-life prolonging domains.

15. Use of a composition for preparing a medicament for inhibiting tumor growth in a subject, by administering to the subject an effective amount of a masked cytokine comprising: a) a first polypeptide comprising a first half-life extending domain and a masking moiety; and b) a second polypeptide comprising a second half-life extending domain and a cytokine, wherein the first half-life extending domain is a first Fc domain or a fragment thereof, and the second half-life extending domain is a second Fc domain or a fragment thereof; wherein the masking portion comprises the amino acid sequence of SEQ ID NO: 826; wherein the cytokine is an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 3; wherein the masking moiety is connected to the C-terminus of the first half-life extension domain via a first linker; wherein the cytokine is linked to the C-terminus of the second half-life extension domain via a second linker, wherein the second linker comprises a cleavable peptide; and wherein said first half-life prolonging domain and said second half-life prolonging domain contain modifications that promote association of said first and said second half-life prolonging domains.

16. The use of claim 14, wherein the masking moiety comprises the amino acid sequence of SEQ ID NO:

826.

17. The use according to any one of claims 14 to 16, wherein the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO:

156.

18. The nucleic acid of any one of claims 14-16, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO:

96.

19. The nucleic acid of claim 14 or claim 16, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 15 and the second linker comprises the amino acid sequence of SEQ ID NO:

339.

20. The use of claim 15, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO:

135.

21. The use of claim 15, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 28, and the second linker comprises the amino acid sequence of SEQ ID NO:

35.

22. The use of claim 14, wherein the masking moiety comprises the amino acid sequence of SEQ ID NO: 826; wherein the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156; and wherein the first linker comprises the amino acid sequence of SEQ ID NO: 15, and the second linker comprises the amino acid sequence of SEQ ID NO:

339.

23. The use of claim 15, wherein the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156; and wherein the first linker comprises the amino acid sequence of SEQ ID NO: 28, and the second linker comprises the amino acid sequence of SEQ ID NO: 35.

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