EA049880B1 - MASKED CYTOKINE POLYPEPTIDES - Google Patents

Abstract

The invention relates to cytokines or functional fragments thereof, which in some embodiments are designed to be masked by a masking moiety at one or more binding sites of a cytokine receptor or a functional fragment thereof. In some embodiments, the cytokines are designed to be activated by a protease at a target site, such as a tumor microenvironment, by incorporating a proteolytically cleavable linker into the design. In some embodiments, the proteolytically cleavable linker links the cytokine to a masking moiety, links the cytokine to a half-life extension domain, and/or links the masking moiety to a half-life extension domain. The masking moiety blocks, hinders, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the activity or binding of the cytokine to its cognate receptor or protein. Following proteolytic cleavage of the cleavable linker at the target site, the cytokine is activated, making it able to bind to its cognate receptor or protein with increased affinity.

Description

Cross-reference to related applications

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 foregoing applications are incorporated herein by reference in their entirety for all purposes.

Inclusion by reference to a sequence listing

This application is submitted together with an electronic sequence listing. The sequence listing is provided as a file named 737762000940SeqList, created on September 26, 2019, and is 1024.111 KB in size. The electronic sequence listing information is incorporated by reference in its entirety.

Field of invention

The invention relates to masked cytokines and methods associated with their use and manufacture.

State of the art

Cancer is the second leading cause of death in the United States, accounting for more deaths than the next five leading causes (chronic respiratory disease, stroke, accidents, Alzheimer's disease, and diabetes). Although great strides have been made, particularly in the area of targeted therapies, much work remains to be done in this area. Immunotherapy and its subfield, immuno-oncology, offer promising and exciting therapeutic options for the treatment of malignancies. In particular, it is now recognized that one of the hallmarks of cancer is its evasion of immune surveillance, and significant efforts have identified targets and developed therapies against these targets to reactivate the immune system to recognize and treat cancer.

Cytokine therapy is an effective strategy for stimulating the immune system to induce antitumor 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, cytokines administered to patients typically have very short half-lives, requiring frequent administration. For example, the label for an aldesleukin product sold under the brand name Proleukin states that patients receiving a 5-minute intravenous (IV) infusion had a half-life of 85 minutes. In addition, high-dose cytokine administration can cause adverse health effects, such as vascular leakage, due to systemic immune activation. These findings illustrate the need to develop cytokine therapeutics that effectively target tumors without the side effects associated with systemic immune activation. This document provides masked cytokines, their compositions and methods of use to address this need.

All references cited herein, including patent applications, patent publications, and scientific literature, are hereby incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference.

Brief summary of the invention

The disclosed invention relates to the design and use of masked cytokines or functional fragments thereof, which in some embodiments are designed to be masked by a masking moiety at one or more binding sites of a receptor of the cytokine or functional fragment thereof. In some embodiments, the cytokines are designed to be activated by a protease at a target site, such as a tumor microenvironment, by including a proteolytically cleavable linker. In some embodiments, the proteolytically cleavable linker links the cytokine to a masking moiety, links the cytokine to a half-life extension domain, and/or links the masking moiety to a half-life extension domain. The masking moiety blocks, hinders, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the activity of or binding of the cytokine to its cognate receptor or protein. Following proteolytic cleavage of the cleavable linker at the target site, the cytokine is activated, rendering it capable of binding to its cognate receptor or protein with increased affinity.

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

In one embodiment, the masked cytokine comprises, in the N- to C-terminal direction

- 1 049880 or from C- to N-terminus: 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 N- to C-terminus or C- to N-terminus 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 N- to C-terminus or C- to N-terminus direction: a) a cytokine or a functional fragment thereof; b) a first linker; c) a masking moiety; and d) a half-life extension domain.

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

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

In one embodiment, the masked cytokine has one or more amino acid substitutions in the amino acid sequence of an IL-2 or IL-15 polypeptide or functional fragment thereof. In one embodiment, the amino acid substitutions reduce the affinity of the IL-2 polypeptide or functional fragment thereof for CD25 (ZE-2Kα).

In one embodiment, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence obtained by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or functional fragment thereof that increases the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rβ or IL-2Ry.

In one embodiment, the half-life extension domain is an antibody or fragment thereof, and in some embodiments has one or more amino acid substitutions that alter effector function.

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

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

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

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

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

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

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

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

In one embodiment, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of: ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, 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,

- 2 049880

KEL, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, KLK5, MMP3, MMP7, MMP8, MMP9, LGMN, LNPEP, MASP1, PAPPA, PAPPA2, PCSK1, NAPSA, PCSK5, PCSK6, MME, MMP1, MMP10, PLAT, PLAU, PLG, PRSS1, PRSS12, PRSS2, PRSS21, PRSS3, PRSS33, PRSS4, PRSS55, PRSS57, MMP12, PRSS8, PRSS9, PRTN3, MMP13, MMP14, ST14, TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1 and TPSG1.

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

In one embodiment, the half-life extension domain is conjugated to an immunostimulant, such as a stimulator of interferon gene agonist (STING), 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 D-dihydro-N-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.

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

In one embodiment, the first half-life extending domain and the second half-life extending domain comprise modifications that facilitate association of the first and second half-life extending domains.

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

In another embodiment, a method for producing a masked cytokine is provided, comprising culturing a host cell under conditions that result in the formation of the masked cytokine. In one embodiment, the masked cytokine produced by the host cell is recovered. One embodiment encompasses a masked cytokine produced by the disclosed methods.

In one embodiment, a composition comprising a disclosed masked cytokine is provided. In another embodiment, the composition further comprises an anti-inflammatory agent or an anti-cancer agent, such as a PD-1 inhibitor, an EGFR inhibitor, a HER2 inhibitor, a VEGFR inhibitor, a CTLA-4 inhibitor, a BTLA inhibitor, a B7H4 inhibitor, a B7H3 inhibitor, a CSFIR inhibitor, an HVEM inhibitor, a CD27 inhibitor, a KIR inhibitor, an NKG2A inhibitor, an NKG2D agonist, a TWEAK inhibitor, an ALK inhibitor, an antibody targeting CD52, an antibody targeting CCR4, a PD-L1 inhibitor, a KIT inhibitor, a PDGFR inhibitor, a BAFF inhibitor, an HDAC inhibitor, a VEGF ligand inhibitor, a molecule targeting CD19, a molecule targeting FOLR1, a molecule targeting DLL3, a molecule targeting DKK1, a molecule targeting MUC1 targeting molecule, MUC16 targeting molecule, PSMA targeting molecule, MSLN targeting molecule, NY-ES0-1 targeting molecule, B7H3 targeting molecule, B7H4 targeting molecule, BCMA targeting molecule, CD29 targeting molecule, targeting molecule on CD151, molecule targeting CD123, molecule targeting CD33, molecule targeting CD37, molecule targeting CDH19, molecule targeting CEA, molecule targeting claudin 18.2, molecule targeting CLEC12A, molecule targeting EGFRVIII, molecule, EPCAM targeting molecule, EPHA2-targeting molecule, FCRH5-targeting molecule, FLT3-targeting molecule, GD2-targeting molecule, glypican 3-targeting molecule, gpA33-targeting molecule, GPRC5D-targeting molecule, IL-23R-targeting molecule, IL-1RAP, MCSP targeting molecule, RON targeting molecule, ROR1 targeting molecule, STEAP2 targeting molecule, TfR targeting molecule, CD166 targeting molecule, TPBG targeting molecule, TROP2 targeting molecule, proteasome inhibitor, ABL inhibitor, inhibitor CD30, FLT3 inhibitor, inhibitor MET, RET inhibitor, ^-1(1) inhibitor, MEK inhibitor, ROS1 inhibitor, BRAF inhibitor, CD38 inhibitor,

- 3 049880

RANKL, B4GALNT1 inhibitor, SLAMF7 inhibitor, IDH2 inhibitor, mTOR inhibitor, CD20 targeting molecule, BTK inhibitor, PI3K inhibitor, FLT3 inhibitor, PARP inhibitor, CDK4 inhibitor, CDK6 inhibitor, FGFR inhibitor, RAF inhibitor, JAK1 inhibitor, JAK2 inhibitor, JAK3 inhibitor, IL-6 inhibitor, IL-17 inhibitor, Smoothened inhibitor, IL-6R inhibitor, BCL2 inhibitor, PTCH inhibitor, PIGF inhibitor, TGFB inhibitor, CD28 agonist, CD3 agonist, CD40 agonist, GITR agonist, OX40 agonist, VISTA agonist, CD137 agonist, LAG3 inhibitor, TIM3 inhibitor, TIGIT inhibitor, and IL-2R inhibitor.

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, mofezolac, TFAP, flurbiprofen, ketoprofen, celecoxib, rofecoxib, meloxicam, piroxicam, deracoxib, parecoxib, valdecoxib, etoricoxib, chromene derivative, chromane derivative, N-(2-cyclohexyloxynitrophenyl)methanesulfonamide, parecoxib, lumiracoxib, RS 57067, T-614, BMS347070, JTE-522, S-2474, SVT-2016, CT-3, ABT-963, SC-58125, nimesulide, flosulide, NS-398, L-745337, RWJ-63556, L-784512, darbufelone, CS-502, LAS-34475, LAS-34555, S-33516, diclofenac, mefenamic acid, SD-8381, ibuprofen, naproxen, ketorolac, indomethacin, aspirin, naproxen, tolmetin, piroxicam, or meclofenamate.

In one embodiment, the anti-inflammatory agent is an NF-kB inhibitor, such as an IKK complex inhibitor, an IkB cleavage inhibitor, an NF-kB nuclear translocation inhibitor, a p65 acetylation inhibitor, an NF-kB DNA binding inhibitor, an NF-kB transactivation inhibitor, or an inhibitor of p53 induction.

In one embodiment, the NF-kB inhibitor is TPCA-1, NF-kB activation inhibitor VI (BOT-64), BMS-345541, amlexanox, SC-514 (GK-01140), IMD-0354, IKK-16, BAY11-7082, MG-115, MG-132, lactacystin, epoxomicin, parthenolide, carfilzomib, MLN-4924 (pevonedistat), JSH-23, rolipram, gallic acid, anacardic acid, GYY-4137, p-XSC, CV-3988, prostaglandin E2 (PGE2), LY-294002, wortmannin, mesalamine, quinacrine, or flavopiridol.

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

Another embodiment includes a kit comprising a disclosed masked cytokine.

Another embodiment includes a method of treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of the disclosed masked cytokine composition for treating a disease or condition, such as a neoplastic disease, for example cancer. In one embodiment, the cancer is leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, bone cancer (e.g., osteosarcoma, chondrosarcoma, Ewing's sarcoma), bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer, testicular cancer, adrenal cancer, adenoid cystic carcinoma, anal cancer, brain cancer, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, oral cancer, thyroid cancer, retinoblastoma, parathyroid cancer, pituitary cancer, 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, renal 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 carcinoma, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, 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 (such as cancer of the head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma. Additional examples include cancer of the thyroid gland, endocrine system, brain, breast, cervix, colon, head and neck, esophagus, liver, kidney, lung, 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, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant insulinoma of the pancreas, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer

- 4,049,880 pathways, malignant hypercalcemia, endometrial cancer, adrenal cortex cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary carcinoma thyroid cancer, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, Paget's disease, phyllodes tumors, lobular carcinoma, ductal carcinoma, pancreatic stellate cell carcinoma, hepatic stellate cell carcinoma, or prostate cancer.

Another embodiment includes a method of treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of the disclosed masked cytokine composition, wherein the inflammatory or autoimmune disease is selected from the group consisting of atherosclerosis, obesity, inflammatory bowel disease (IBD), rheumatoid arthritis, allergic encephalitis, psoriasis, atopic skin disease, osteoporosis, peritonitis, hepatitis, lupus, celiac disease, Sjogren's syndrome, polymyalgia rheumatica, multiple sclerosis (MS), ankylosing spondylitis, type 1 diabetes mellitus, alopecia areata, vasculitis and temporal arteritis, graft versus host disease (GVHD), asthma, COPD, paraneoplastic autoimmune disease, cartilaginous inflammation, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, juvenile rheumatoid arthritis of systemic onset, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative reaction, enthesopathy, articular syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, rheumatoid arthritis of systemic onset, enteropathic arthritis, reactive arthritis, Reiter's syndrome, dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myolitis, polymyolitis, dermatomyolitis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, Crohn's disease, ulcerative colitis, celiac disease, rhinosinusitis, rhinosinusitis with polyps, eosinophilic esophogitis, eosinophilic bronchitis, Guillain-Barré, thyroiditis (eg, Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, transplant rejection, renal injury, hepatitis C-induced vasculitis, and spontaneous pregnancy loss.

Brief description of graphic materials

Fig. 1A and 1B depict the structure of exemplary embodiments of a masked cytokine that comprises a masking moiety, a cytokine or a functional fragment thereof (cytokine), a half-life extension domain, and a first linker that comprises a first cleavable peptide (1CP), a first N-terminal spacer domain (1NSD), and a first C-terminal spacer domain (1CSD). These exemplary embodiments also include a second linker that comprises a second cleavable peptide (2CP), a second N-terminal spacer domain (2NSD), and a second C-terminal spacer domain (2CSD). As shown by the arrows, although the exemplary embodiments show a masking moiety linked to a first linker and a cytokine or functional fragment thereof linked to a first linker and a second linker, the masking moiety and the cytokine or functional fragment thereof may be interchanged such that the cytokine or functional fragment thereof is linked to the first linker and the masking moiety is linked to the first linker and a second linker. Fig. 1A depicts the structure of an exemplary embodiment of a masked cytokine as a monomer. Fig. 1B depicts the structure of an exemplary embodiment of a masked cytokine as a homodimer formed by disulfide bonds.

Fig. 2A and 2B depict the structure of exemplary embodiments of a masked cytokine that comprises a first masking fragment (mask 1), a cytokine or a functional fragment thereof (cytokine), a second masking fragment (mask 2), a half-life extension domain, a first linker that comprises a first cleavable peptide (1CP), a first N-terminal spacer domain (1NSD) and a first C-terminal spacer domain (1CSD) and a second linker that comprises a second cleavable peptide (2CP), a second N-terminal spacer domain (2NSD) and a second C-terminal spacer domain (2CSD). These exemplary embodiments also include a third linker that comprises a third cleavable peptide (3CP), a third N-terminal spacer domain (3NSD) and a third C-terminal spacer domain (3CSD). Fig. 2A shows the structure of an exemplary embodiment of the masked cytokine as a monomer. Fig. 2B shows the structure of an exemplary embodiment of the masked cytokine as a disulfide-bonded homodimer.

Fig. 3A shows the structure of an illustrative embodiment of a masked cytokine that

- 5 049880 comprises a masked fragment, a cytokine or a functional fragment thereof (cytokine), a first half-life extension domain and a second half-life extension domain. The exemplary embodiment depicted in Fig. 3A also includes a first linker that comprises a first cleavable peptide (1CP), a first N-terminal spacer domain (1NSD) and a first C-terminal spacer domain (1CSD), and a second linker that comprises a second cleavable peptide (2CP), a second N-terminal spacer domain (2NSD) and a second C-terminal spacer domain (2CSD). Exemplary first and second half-life-extending domains comprise knob-to-hole modifications that facilitate association of the first half-life-extending domain with the second half-life-extending domain, as represented by a hole in the first half-life-extending domain and a knob in the second half-life-extending domain. The first half-life-extending domain and the second half-life-extending domain are also depicted as associating, at least in part, through the formation of disulfide bonds. It should be understood that although the cavity is depicted as part of the first half-life extension domain (associated with the masking moiety) and the knob is depicted as part of the second half-life extension domain (associated with the cytokine), the cavity and knob may alternatively be included in the second half-life extension domain and the first half-life extension domain, respectively, such that the cavity is part of the second half-life extension domain (associated with the cytokine) and the knob is part of the first half-life extension domain (associated with the masking moiety). In Fig. 3B depicts the structure of an exemplary embodiment of a masked cytokine that comprises a masking moiety, a cytokine or a functional fragment thereof (cytokine), a first half-life extension domain, a second half-life extension domain, and a third linker that comprises a third N-terminal spacer domain (3NSD) and a third C-terminal spacer domain (3CSD). The third linker connects the first half-life extension domain to the second half-life extension domain. The exemplary embodiment shown in FIG. 3B, also includes a first linker that contains a first cleavable peptide (1CP), a first N-terminal spacer domain (1NSD) and a first C-terminal spacer domain (1CSD), and a second linker that contains a second cleavable peptide (2CP), a second N-terminal spacer domain (2NSD) and a second C-terminal spacer domain (2CSD).

Fig. 4 shows a dimer of the exemplary masked cytokine shown in Fig. 3A formed via disulfide bonds.

Fig. 5A depicts the structure of an exemplary embodiment of a masked cytokine that comprises a first masking moiety (mask 1), a first half-life extending domain, a second masking moiety (mask 2), a cytokine or functional fragment thereof (cytokine), and a second half-life extending domain. The exemplary embodiment depicted in Fig. 5A, also includes a first linker that contains a first cleavable peptide (1CP), a first N-terminal spacer domain (1NSD) and a first C-terminal spacer domain (1CSD), a second linker that contains a second cleavable peptide (2CP), a second N-terminal spacer domain (2NSD) and a second C-terminal spacer domain (2CSD), and a third linker that contains a third cleavable peptide (3CP), a third N-terminal spacer domain (3NSD) and a third C-terminal spacer domain (3CSD). It should be understood that, although the cavity is depicted as part of the first half-life extension domain (associated with the masking moiety) and the knob is depicted as part of the second half-life extension domain (associated with the cytokine), the cavity and knob may alternatively be included in the second half-life extension domain and the first half-life extension domain, respectively, such that the cavity is part of the second half-life extension domain (associated with the cytokine) and the knob is part of the first half-life extension domain (associated with the masking moiety). In Fig. 5B depicts the structure of an exemplary embodiment of a masked cytokine that comprises a first masking fragment (mask 1), a first half-life extension domain, a second masking fragment (mask 2), a cytokine or a functional fragment thereof (cytokine), a second half-life extension domain, and a fourth linker that comprises a fourth N-terminal spacer domain (4NSD) and a fourth C-terminal spacer domain (4CSD). The fourth linker connects the first half-life extension domain to the second half-life extension domain. The exemplary embodiment shown in FIG. 5B also includes a first linker that comprises a first cleavable peptide (1CP), a first N-terminal spacer domain (1NSD), and a first C-terminal spacer domain (1CSD), a second linker that comprises a second cleavable peptide (2CP), a second N-terminal spacer domain (2NSD), and a second C-terminal spacer domain (2CSD), and a third linker that comprises a third cleavable peptide (3CP), a third N-terminal spacer domain (3NSD), and a third C-terminal spacer domain (3CSD). As depicted by the arrows in Figs. 5A and 5B, although in the exemplary embodiment

- 6 049880 a second masking fragment is provided, linked to a third linker, and a cytokine or a functional fragment thereof is linked to a third linker and a second linker, the first masking fragment and the cytokine or a functional fragment thereof can be replaced in such a way that the cytokine or a functional fragment thereof is linked to a third linker, and the first masking fragment is linked to a third linker and a second linker.

Fig. 6 shows a dimer of the exemplary masked cytokine shown in Fig. 5A formed via disulfide bonds.

Figs. 7A-7E depict exemplary embodiments of masked cytokines before (left) and after (right) protease cleavage, such as in a tumor microenvironment. Figs. 7A7D depict exemplary embodiments of a masked IL-2 cytokine, and Fig. 7E depicts an exemplary embodiment of a masked IL-15 cytokine. Protease cleavage releases the masking moiety (e.g., IL-2Rβ, as depicted in Figs. 7A, 7B, and 7D), or releases IL-2 (Fig. 7C), or releases IL-15 (Fig. 7E).

Figure 8 shows SDS-PAGE analysis of flow-through (FT) samples (i.e., proteins that did not bind to the protein A column) and eluted (E) samples (i.e., proteins that bound to the protein A column and were eluted from the column) after preparation and purification of representative constructs (AK304, AK305, AK307, AK308, AK309, AK310, AK311, AK312, AK313, AK314, and AK315).

Figures 9A-9D depict the results of an SPR assay examining the binding of exemplary masked IL-2 polypeptide constructs (AK215 and AK216) or a control rhIL2 to CD25-Fc. Figure 9A depicts the interaction between AK215 and CD25-Fc, Figure 9B depicts the interaction between AK216 and CD25-Fc, and Figure 9C depicts the interaction between a control recombinant human IL2 (rhIL2) and CD25-Fc. Figure 9D is a table summarizing the data obtained for the association constant (ka), dissociation constant (kd), equilibrium dissociation constant (KD), and the chi ² value and U value for each interaction.

Figures 10A-10D depict the results of an SPR assay examining the binding of exemplary masked IL-2 polypeptide constructs (AK216 and AK218) or an rhIL2 control to CD122-Fc. Figure 10A depicts the interaction between AK216 and CD122-Fc, Figure 10B depicts the interaction between AK218 and CD122-Fc, and Figure 10C depicts the interaction between a recombinant human IL2 control (rhIL2) and CD122-Fc. Figure 10D is a table summarizing the data obtained for the association constant (ka), dissociation constant (kd), equilibrium dissociation constant (KD), and the chi ² value and U value for each interaction.

Fig. 11A depicts an exemplary embodiment of masked cytokines before (left) and after (right) protease cleavage, such as in a tumor microenvironment. Fig. 11B depicts SDS-PAGE analysis of an exemplary masked IL-2 polypeptide construct that was incubated in the absence (left lane) or presence (right lane) of MMP10 protease, demonstrating release of IL-2 from the Fc region.

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

Figures 13A-13C show STAT5 activation (%) in PBMCs by the AK081 or AK032 construct. The AK081 construct was tested with and without MMP10 pretreatment. An isotype control as well as a negative control other than IL-2 were also tested. STAT5 activation levels (%) are shown for NK cells (Figure 13A), CD8+ T cells (Figure 13C), and CD4+ T cells (Figure 13B).

Figures 14A-14D depict the results of STAT5 activation studies in PBMCs using the AK081 and AK111 constructs, as well as controls that included rhIL-2 and anti-RSV antibody. An untreated control was also tested. _EC50 (pM) is also shown for rhIL-2, AK081, and AK111 treatments. STAT5 activation (%) is shown for CD4 ⁺ FoxP3 ⁺ CD25 ⁺ cells (Figure 14A), CD8 ⁺ cells (Figure 14B), and CD4+FoxP3 ^- CD25 ^- cells (Figure 14C). Figure 14D depicts the _EC50 (pM) and fold change data for the AK081, AK111 constructs, and the rhIL-2 control.

Figures 15A-15D depict the results of STAT5 activation studies in PBMC using the AK167 and AK168 constructs, as well as controls that included rhIL-2 and anti-RSV antibody. An untreated control was also tested. _EC50 (pM) is also shown for rhIL-2, AK167, and AK168 treatments. STAT5 activation (%) is shown for CD4'FoxP3'CD25' cells (Figure 15A), CD8 ⁺ cells (Figure 15B), and CD4'FoxP3 ^- CD25 ^- cells (Figure 15C). Figure 15D depicts _EC50 (pM) and fold change data for the AK167 and AK168 constructs, as well as the rhIL-2 control.

Figures 16A-16D show STAT5 activation (%) in PBMCs treated with AK165 or AK166 construct, or isotype control, or IL-2-Fc control that were previously exposed (+ MMP10) or not exposed to MMP10 protease. The legend shown in Figure 16A,

- 7 049880 also refers to Fig. 16B, and the legend shown in Fig. 16C also refers to Fig. 16D. STAT5 activation (%) is depicted for CD4'FoxP3' T regulatory cells (Fig. 16A), CD4'FoxP3 ^- T helper cells (Fig. 16B), CD8+ cytotoxic T cells (Fig. 16C), and CD56+ NK cells (Fig. 16D).

Figures 17A-17C show STAT5 activation (%) in PBMCs treated with AK109 or AK110 construct or isotype control or IL-2-Fc control that were previously exposed (+MMP10) or not exposed to MMP10 protease. The legend shown in Figure 17B also refers to Figure 17A. STAT5 activation (%) is shown for NK cells (Figure 17A), CD8 cells (Figure 17B), and CD4 cells (Figure 17C).

Figures 18A-18D depict the results of STAT5 activation studies in PBMC using the AK211, AK235, AK253, AK306, AK310, AK314, and AK316 constructs, as well as the rhIL-2 control. STAT5 activation (%) is also depicted for CD3'CD4'FoxP3' cells (Figure 18A), CD3⁺CD4⁺FoxP3 cells (Fig. 18B) and CD3⁺CD8⁺cells (Fig. 18C). Fig. 18D depicts the EC data₅₀for each of the studied constructs, as well as for rhIL-2 control.

Figures 19A-19D depict the results of STAT5 activation studies in PBMCs using the protease-activated constructs AK081, AK167, AK216, AK218, AK219, AK220, and AK223, as well as the rhIL-2 control. STAT5 activation (%) is depicted for CD4'FoxP3'CD25' regulatory T cells (Figure 19A), CD4'FoxP3 ^- CD25 ^- cells (Figure 19B), and CD8 ⁺ cells (Figure 19C). Figure 19D depicts the _EC50 data for each of the constructs studied as well as the rhIL-2 control.

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

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

Fig. 22A and 22B depict the results of reporter bioassays on an exemplary IL-15 masked polypeptide construct, AK248, with or without pretreatment (+MMP) with an activating protease or rhIL-15 as a control. Fig. 22A depicts the results of a reporter bioassay using the HEKBlue IL2 reporter cell line, and Fig. 22B depicts the results of a reporter bioassay using an IL-15 bioassay using the mouse CTLL2 cell line.

Figs. 23A-23D depict the results of pharmacokinetic studies conducted in tumor-bearing mice using construct AK032, AK081, AK111, AK167, or AK168 or a control RSV antibody. Fig. 23A is a simplified representation of the structure of each of the constructs studied. Fig. 23B depicts plasma Fc levels (μg/mL) when human IgG is detected, Fig. 23C depicts plasma Fc-CD122 levels (μg/mL) when human CD122 is detected, and Fig. 23D depicts plasma Fc-IL2 levels (μg/mL) when human IL-2 is detected. Anti-human IG was used as the capture antibody prior to the detection step.

Fig. 24A-24D depict the results of pharmacokinetic studies conducted in tumor-bearing mice using the AK167, AK191, AK197, AK203, AK209, or AK211 construct, or an anti-RSV antibody control. Fig. 24A is a simplified representation of the structure of each of the constructs studied. Fig. 24B depicts plasma Fc levels (μg/mL) when human IgG is detected, Fig. 24C depicts plasma Fc-IL2 levels (μg/mL) when human IL-2 is detected, and Fig. 24D depicts plasma Fc-CD122 levels (μg/mL) when human CD122 is detected. Anti-human IG was used as a capture antibody prior to the detection step.

Figures 25A-25L show the results of in vivo studies examining the responses of CD4, CD8, NK, and Treg percentages in spleen, blood, and tumor using construct AK032, AK081, AK111, AK167, or AK168 or RSV IgG control. For spleen tissue, % CD8 cells of CD3 cells (Figure 25A), % CD4 of CD3 cells (Figure 25B), % NK cells of CD3 cells (Figure 25C), % FoxP3 of CD4 cells (Figure 25D) are shown. For blood, % CD8 cells of CD3 cells (Figure 25E), % CD4 of CD3 cells (Figure 25F), % Nk cells of CD3 cells (Figure 25G), % FoxP3 of CD4 cells (Figure 25H) are shown. For tumor tissue, % CD8 cells from CD3 cells (Fig. 25I), % CD4 from CD3 cells (Fig. 25J), % NK cells from CD3 cells (Fig. 25K), % FoxP3 from CD4 cells (Fig. 25L) are shown.

Figures 26A-26L depict the results of in vivo studies examining the responses of CD4, CD8, NK, and Treg percentages in spleen, blood, and tumor using the AK167, AK168, AK191, AK197, AK203, AK209, or AK211 construct or an RSV IgG control. For spleen tissue, % CD8 cells of CD3 cells are shown (Figure 26A), % CD4 of CD3 cells (Figure 26B), % NK cells of CD3 cells (Figure 26C), % FoxP3 of CD4 cells (Figure 26D). For blood, % CD8 cells of CD3 cells are shown (Figure 26B).

- 8 049880

26E), % CD4 cells from CD3 cells (Fig. 26F), % Nk cells from CD3 cells (Fig. 26G), % FoxP3 from CD4 cells (Fig. 26H). For tumor tissue, % CD8 cells from CD3 cells (Fig. 26I), % CD4 from CD3 cells (Fig. 26J), % NK cells from CD3 cells (Fig. 26K), % FoxP3 from CD4 cells (Fig. 26L) are shown.

Figures 27A-27L show the results of in vivo studies examining the responses of percentages of CD4, CD8, NK, and Tregs in spleen, blood, and tumor using the AK235, AK191, AK192, AK193, AK210, AK189, AK190, or AK211 construct or an RSV IgG control. For spleen tissue, % of CD8 cells of CD3 cells (Figure 27A), % of CD4 of CD3 cells (Figure 27B), % of NK cells of CD3 cells (Figure 27C), % of FoxP3 of CD4 cells (Figure 27D) are shown. For blood, % CD8 cells are shown from CD3 cells (Fig. 27E), % CD4 from CD3 cells (Fig. 27F), % Nk cells from CD3 cells (Fig. 27G), % FoxP3 from CD4 cells (Fig. 27H). For tumor tissue, % CD8 cells are shown from CD3 cells (Fig. 27I), % CD4 from CD3 cells (Fig. 27J), % NK cells from CD3 cells (Fig. 27K), % FoxP3 from CD4 cells (Fig. 27L).

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

Figs. 29A-29D depict the results of in vivo cleavage studies 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). Fig. 29E depicts the results of a pharmacokinetic study of total plasma IgG concentration (μg/mL) for total levels of constructs AK167, AK168, and AK209, as well as for levels of the non-cleavable forms of each construct.

Fig. 30A-30D depict the results of an in vivo study assessing fluid leakage through vessels using an exemplary AK111 or AK168 masked IL-2 polypeptide construct, or an AK081 or AK167 unmasked IL-2 polypeptide construct, or a control RSV antibody. Fig. 30A depicts the percentage (%) of body weight loss, and Fig. 30B, 30C, and 30D depict the body weight in grams of liver, lung, and spleen, respectively, for each.

Figures 31A and 31B show the results of an in vivo study that assessed vascular fluid leakage as assessed by measuring the extent of dye leakage into liver and lung tissue following administration of the AK081, AK111, AK167, or AK168 construct or a control RSV antibody. The extent of dye leakage into the liver (Figure 31A) and lung (Figure 31B) was measured based on absorbance at 650 nm.

Figures 32A and 32B depict the results of an in vivo study that assessed vascular leakage as assessed by measuring the degree of perivascular mononuclear cell invasion in liver and lung tissue following administration of the AK081, AK111, AK167, or AK168 construct or a control RSV antibody. The mean liver mononuclear cell count (Figure 32A) and the mean lung mononuclear cell count (Figure 32B) are shown for each.

Fig. 33A and 33B depict the results of a syngeneic tumor model study in which tumor volume and body weight were assessed during treatment with construct AK032, AK081, AK111, AK167, or AK168 or a control RSV antibody. Fig. 33A depicts data regarding tumor volume during treatment, and Fig. 33B depicts data regarding percent (%) change in body weight during treatment.

Detailed description of the essence of the invention

Provided herein are cytokines or functional fragments thereof that, in some embodiments, are engineered to be masked by a masking moiety at one or more receptor binding sites of the cytokine or functional fragment thereof. In some embodiments, the cytokines are engineered to be activated by a protease at a target site, such as a tumor microenvironment, by including a proteolytically cleavable linker. In some embodiments, the proteolytically cleavable linker links the cytokine to a masking moiety, links the cytokine to a half-life extension domain, and/or links the masking moiety to a half-life extension domain. The masking moiety blocks, hinders, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the activity of or binding of the cytokine to its cognate receptor or protein. Following proteolytic cleavage of the cleavable linker at the target site, the cytokine is activated, rendering it capable of binding to its cognate receptor or protein with increased affinity.

By using a masking moiety, the systemic side effects of the administered cytokine may be reduced by disrupting the binding capacity of the cytokine. For example,

- 9 049880 High-dose recombinant IL-2 (aldesleukin) has been approved by the FDA for the treatment of metastatic renal cell carcinoma and melanoma, but has been associated with severe cardiovascular, hepatic, pulmonary, gastrointestinal, neurologic, and hematologic side effects. Preclinical studies have demonstrated, for example, that IL-2-induced pulmonary edema is caused by an interaction between IL-2 and the IL-2Ra (CD25) subunit of the IL-2 receptor (IL-2R) on pulmonary endothelial cells, and that this IL-2-mediated pulmonary edema can be abolished by interfering with the ability of IL-2 to bind IL-2Rcl See Krieg et al. (2010) PNAS, 107(26): 1190611911. Thus, in some embodiments in which the cytokine or functional fragment thereof is an IL-2 polypeptide, a masking moiety is used that blocks, hinders, inhibits (e.g., attenuates), or otherwise prevents (e.g., masks) the activity of or binding of the IL-2 cytokine to IL-2Ra. To further reduce the systemic effects of the masked IL-2 polypeptide, the masked IL-2 polypeptide can further comprise a masking moiety that blocks, hinders, inhibits (e.g., attenuates), or otherwise prevents (e.g., masks) the activity of or binding of the IL-2 cytokine to the IL-2Re and/or IL-2Ry subunits of IL-2R. Similar strategies have also been used for other cytokines by interfering with their ability to bind certain proteins (e.g., receptor subunit(s)) that are associated with causing adverse systemic side effects. Moreover, by masking the cytokine with a linker that contains a cleavable peptide, the binding ability that is impaired by the masking moiety can be restored by cleavage of the cleavable peptide in the tumor microenvironment. Thus, in some embodiments, the masked cytokines provided herein are designed to precisely target pharmacological activity to the tumor microenvironment by exploiting one of the hallmarks of cancer, namely, high local concentrations of active protease. This feature of the tumor microenvironment is exploited to convert a systemically inert molecule into a locally active cytokine. Activation of the cytokine in the tumor microenvironment significantly reduces the systemic toxicity that may be associated with drugs that are administered to a subject in an active form.

All publications, including patent documents, scientific articles and databases, referenced in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. If a definition set forth herein conflicts with or is otherwise inconsistent with a definition set forth in patents, applications, published applications and other publications that are incorporated herein by reference, the definition set forth herein controls over the definition that is incorporated herein by reference.

The section headings used in this document are for organizational purposes only and should not be construed as limiting the subject matter described.

Masked cytokines.

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

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

- 10 049880 in an N- to C-terminal direction: (a) a cytokine or functional fragment thereof; (b) a first linker; (c) a masking moiety; (d) a second linker; and (e) a half-life extension domain. In some embodiments, the masked cytokine comprises, in a C- to N-terminal direction: (a) a cytokine or functional fragment thereof; (b) a first linker; (c) a masking moiety; (d) a second linker; and (e) a half-life extension domain. In some embodiments, the masked cytokine comprises, in an 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 functional fragment thereof. In some embodiments, the masked cytokine comprises, in an 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 fragment. 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 a variant created by modifying any of the illustrative designs described in Table 4 or 5, such as by incorporating one or more additional components into the structure of the design in accordance with the teachings set forth herein.

In some embodiments, provided herein is also a masked cytokine comprising (a) a first masking moiety; (b) a cytokine or a functional fragment thereof, wherein the first masking moiety is linked to the cytokine or a functional fragment thereof via a first linker; and (c) a second masking moiety, wherein the second masking moiety is linked to the cytokine or a functional fragment thereof via a second linker. In some embodiments, the masked cytokine further comprises a half-life extension domain that is linked to either the first masking moiety or the second masking moiety. In some embodiments, the half-life extension domain is linked to either the first masking moiety or the second masking moiety via a third linker.

Also provided herein in some embodiments is a masked cytokine comprising: (a) a first masking moiety, wherein the first masking moiety is linked to a first half-life extension domain; (b) a cytokine or functional fragment thereof, wherein the cytokine or functional fragment thereof is linked to a second half-life extension domain; and (c) a second masking moiety, wherein the second masking moiety is linked to the first masking moiety. In some embodiments, the first masking moiety is linked to the first half-life extension domain via a first linker. In some embodiments, the second masking moiety is linked to the first masking moiety via a second linker. In some embodiments, the cytokine or functional fragment thereof is linked 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.

In some embodiments, a masked cytokine comprises, in N- to C-terminal direction: (a) a first masking moiety; (b) a first linker; (c) a cytokine or functional fragment thereof; (d) a second linker; (e) a second masking moiety; and (f) a half-life extension domain. In some embodiments, a masked cytokine comprises, in C- to N-terminal direction: (a) a first masking moiety; (b) a first linker; (c) a cytokine or functional fragment thereof; (d) a second linker; (e) a second masking moiety; and (f) a half-life extension domain. In some embodiments, a masked cytokine comprises, in N- to C-terminal direction: (a) a first masking moiety; (b) a first linker; (c) a cytokine or functional fragment thereof; (d) a second linker; (e) a second masking moiety; (f) a third linker; and (g) a half-life extension domain. In some embodiments, a masked cytokine comprises, in C- to N-terminal direction: a) a first masking moiety; (b) a first linker; (c) a cytokine or functional fragment thereof; (d) a second linker; (e) a second masking moiety; (f) a third linker; and (g) a half-life extension domain. In some embodiments, a masked cytokine comprises, in 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 functional fragment thereof; (f) a third linker; and (g) a second masking moiety. In some embodiments, a masked cytokine comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 567 and 598-601. In some embodiments, a masked cytokine comprises an amino acid sequence having about or

- 11 049880 at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 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 created 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 in accordance with the teachings set forth herein.

Also provided herein, in some embodiments, is a masked cytokine comprising (a) a first half-life-extending domain and a second half-life-extending domain; (b) a masking moiety; and (c) a cytokine or functional fragment thereof, wherein the masking moiety is linked to the first half-life-extending domain, the cytokine or functional fragment thereof is linked to the second half-life-extending domain, and the first half-life-extending domain and the second half-life-extending domain comprise modifications that facilitate association of the first and second half-life-extending domain. In some embodiments, the masking moiety is linked to the first half-life-extending domain via a first linker and/or the cytokine or functional fragment thereof is linked to the second half-life-extending domain via a second linker. In some embodiments, the first half-life-extending domain is linked to the second half-life-extending domain via a third linker. In some embodiments, the masked cytokine comprises (a) a first half-life extending domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life extending domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a masking fragment comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 3, wherein the masking fragment is linked to the first half-life-extending domain via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or functional fragment thereof is linked to the second half-life-extending domain via a second linker comprising the amino acid sequence of SEQ ID NO: 811, the first half-life-extending domain and the second half-life-extending domain comprise modifications that facilitate association of the first and second half-life-extending 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 extended half-life domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life-extending domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a masking fragment comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 260, wherein the masking fragment is linked to the first half-life-extending domain via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or functional fragment thereof is linked to the second half-life-extending domain via a second linker comprising the amino acid sequence of SEQ ID NO: 262, the first half-life-extending domain and the second half-life-extending domain comprise modifications that facilitate association of the first and second half-life-extending 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 NO: 266 and 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 266. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267. In some embodiments, the masked cytokine comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 266, and comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

In some embodiments, the masked cytokine comprises (a) a first extended half-life domain comprising the amino acid sequence of SEQ ID NO: 155, and

- 12 049880 a second half-life extension domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a mask fragment comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 3, wherein the masking fragment is linked to the first half-life-extending domain via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or functional fragment thereof is linked to the second half-life-extending domain via a second linker comprising the amino acid sequence of SEQ ID NO: 807, the first half-life-extending domain and the second half-life-extending domain comprise modifications that facilitate association of the first and second half-life-extending 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 extended half-life domain comprising the amino acid sequence of SEQ ID NO: 156 and a second half-life-extending domain comprising the amino acid sequence of SEQ ID NO: 155; (b) a masking moiety comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or a functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 3, wherein the masking moiety is linked to the first half-life-extending 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-extending domain via a second linker comprising the amino acid sequence of SEQ ID NO: 807, the first half-life-extending domain and the second half-life-extending domain comprise modifications that facilitate the association of the first and second half-life-extending domains, and the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 679 and 267.

In some embodiments, a masked cytokine comprises (a) a first extended half-life domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life extension domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a masking moiety comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 3, wherein the masking moiety is linked to the first half-life extension domain via a first linker, the cytokine or functional fragment thereof is linked to the second half-life extension domain via a second linker, the first half-life extension domain and the second half-life extension domain comprise modifications that facilitate association of the first and second half-life extension domains, wherein the first linker and/or the second linker comprises a cleavable peptide. In some embodiments, a masked cytokine comprises (a) a first extended half-life domain comprising the amino acid sequence of SEQ ID NO: 156 and a second half-life extension domain comprising the amino acid sequence of SEQ ID NO: 155; (b) a masking moiety comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 3, wherein the masking moiety is linked to the first half-life extension domain via a first linker, the cytokine or functional fragment thereof is linked to the second half-life extension domain via a second linker, the first half-life extension domain and the second half-life extension domain comprise modifications that facilitate association of the first and second half-life extension 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 extended domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life extended domain comprising the amino acid sequence of SEQ ID NO: 156; (b) a masking fragment comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 1, wherein the masking fragment is linked to the first half-life-extending domain via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or functional fragment thereof is linked to the second half-life-extending domain via a second linker comprising the amino acid sequence of SEQ ID NO: 812, the first half-life-extending domain and the second half-life-extending domain comprise modifications that facilitate association of the first and second half-life-extending 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 extended half-life domain comprising the amino acid sequence of SEQ ID NO: 156 and a second half-life-extending domain comprising the amino acid sequence of SEQ ID NO: 155; (b) a masking fragment comprising the amino acid sequence of SEQ ID NO: 261; and (c) a cytokine or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 1, wherein the masking fragment is linked to the first period extension domain

- 13 049880 half-life via a first linker comprising the amino acid sequence of SEQ ID NO: 28, the cytokine or a functional fragment thereof is linked to a second half-life extension domain via a second linker comprising the amino acid sequence of SEQ ID NO: 812, the first half-life extension domain and the second half-life extension domain contain modifications that facilitate the association of the first and second half-life extension domains, and the masked cytokine contains the amino acid sequences of SEQ ID NO: 689 and 267.

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

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 606 and 603. In some embodiments, the masked

- 14 049880 cytokine comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 606, and comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 603.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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

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

- 15 049880

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

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about

- 16 049880

85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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% identity

- 17 049880 sequence with the amino acid sequence of SEQ ID NO: 684, and contains an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 the amino acid sequences of SEQ ID NOs: 692 and 267. In some embodiments, the masked

- 18 049880 cytokine comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 692, and comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

- 19 049880

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about

- 20 049880

85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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% identity

- 21 049880 sequence with the amino acid sequence of SEQ ID NO: 716, and contains an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 the amino acid sequences of SEQ ID NOs: 728 and 267. In some embodiments, the masked

- 22 049880 cytokine comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 728, and comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 267.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

- 23 049880

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about

- 24 049880

85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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 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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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

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% identity

- 25 049880 sequence with the amino acid sequence of SEQ ID NO: 747, and contains an amino acid sequence having about or at least about 85, 86, 87, 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.

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

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 753 and 671. In some embodiments, the masked

- 26 049880 cytokine comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 753, and comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 671.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least about 85, 86, 87, 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.

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 comprises an amino acid sequence having about or at least 85, 86, 87, 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.

In some embodiments, the masked cytokine is an exemplary masked cytokine construct as described in any of Tables 8-11, or a variant created 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 in accordance with the teachings herein.

Also provided herein, in some embodiments, is a masked cytokine comprising (a) a first half-life-extending domain and a second half-life-extending domain; (b) a first masking moiety and a second masking moiety; and (c) a cytokine or functional fragment thereof, wherein the first masking moiety is linked to the first half-life-extending domain, the second masking moiety is linked to the cytokine or functional fragment thereof, or the second masking moiety or the cytokine or functional fragment thereof is linked to the second half-life-extending domain, and the first half-life-extending domain and the second half-life-extending domain comprise modifications that facilitate association of the first and second half-life-extending domain. In some embodiments, the first masking moiety is linked to the first half-life-extending domain via a first linker, and/or either the second masking moiety or the cytokine or

- 27 049880 its functional fragment is linked to the second half-life extension domain via a second linker. In some embodiments, the second masking fragment is linked to the cytokine or its functional fragment via a third linker. In some embodiments, the first half-life extension domain is linked to the second half-life extension domain via a fourth linker. In some embodiments, the masked cytokine comprises the amino acid sequences of SEQ ID 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.

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 amino acid sequences associated with any of the exemplary constructs as described in Tables 4-11.

Each component of the masked cytokines proposed in this paper is discussed in more detail below.

Cytokines.

Provided herein is a cytokine or a functional fragment thereof. The cytokine or functional fragment thereof may be any cytokine, any functional fragment of any cytokine, or any natural or non-natural variant of any cytokine. A cytokine is a small polypeptide that plays a role in cell signaling, particularly in immune system cells. Examples of cytokines may include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors.

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

The β-trefoil fold class includes cytokines that are characterized by three βββ-loop-β units (12 β-strands in total) that form a cylindrical structure with a hairpin cap to the cylinder. Examples of cytokines in the β-trefoil fold class include IL-Ια, IL-Ιβ, IL-1 receptor antagonist (IL-1RA), IL-18, IL-33, IL-36a, IL-36e, IL-36y, IL-36 receptor antagonist (IL-36RA), IL37, and IL-38.

The short-chain and long-chain four-helix bundle classes are characterized by a monomeric helical bundle having four amphipathic helices oriented in a unique up-up-down-down topology. The short-chain four-helix bundle class is characterized by shorter helices, such as those that are 10-20 amino acid residues in length, while the long-chain four-helix bundle class is characterized by longer helices, such as those that are 20-30 amino acid residues in length. Examples of cytokines from the short-chain four-helix bundle class include IL-2, IL-3, IL-4, IL-5, IL7, 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).

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

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

Some cytokines, such as IL-15, also function by binding to and presenting to a membrane-bound cytokine receptor.

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

In some embodiments, the cytokine or functional fragment thereof is selected from the group consisting of IL-1a, IL-1 receptor antagonist (IL-1RA), IL-18, IL-33, IL-36a, IL-36 β, IL36γ, IL-36 receptor antagonist (IL-36RA), IL-37, and IL-38.

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).

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.

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

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, IL15, 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 a functional fragment or variant of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17A, IL-17C, IL17D, IL-17F, IL-17A/F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A, IL-28B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36 or IL-37.

In some embodiments, the cytokine or functional fragment thereof is selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor 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 functional fragment thereof is a functional fragment or variant of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor 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.

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

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4-1BB-L, TRAIL, RANKL, APO3L, CD256, CD257, CD258, TL1, AITRL and EDA1.

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

Illustrative embodiments of a cytokine or functional fragment thereof in the form of IL-2 polypeptides or functional fragments thereof and IL-15 polypeptides or functional fragments thereof are detailed below.

1. IL-2 polypeptides.

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

In some embodiments, the IL-2 polypeptide or functional fragment thereof is any naturally occurring interleukin-2 (IL-2) protein or a 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-2Rα chain). In some embodiments, the IL-2 polypeptide or functional fragment thereof is a mature form of IL-2 that consists 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 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 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 functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 230. In some embodiments, the IL-2 polypeptide or 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 functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the IL-2 polypeptide or 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.

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

- 30 049880 or a functional fragment thereof comprises an amino acid sequence that comprises 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 obtained by introducing an amino acid substitution T3A 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.

In some embodiments, the IL-2 peptide or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions compared to the amino acid sequence of wild-type IL-2 that reduces the affinity of the IL-2 peptide or functional fragment thereof for IL-2R<c (CD25). In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822 such that one or more amino acid residues 38, 42, 45, and 62 are alanine (A). In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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). In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813822 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 functional fragment thereof comprises an amino acid sequence formed by substituting amino acid residues R38, F42, Y45, and E62 with alanine in the amino acid sequence of SEQ ID NO: 160 or 251. In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by substituting amino acid residues R38, F42, Y45, and E62 with alanine (A) and substituting amino acid residue C125 with 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792 and 813-822 such that amino acid residue 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813822 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 functional fragment thereof comprises an amino acid sequence formed by

- 31 049880 by one or more amino acid substitutions in the amino acid sequence of any 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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).

In some embodiments, the IL-2 peptide or functional fragment thereof comprises an amino acid sequence obtained by introducing one or more of the following amino acid substitutions into any 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.

In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions compared to the amino acid sequence of wild-type IL-2 that increases the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rβ (CD122). In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 combinations thereof. In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 combinations thereof, and amino acid residue 125 is serine (S), glycine (G), or alanine (A). In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813822 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

- 32 049880 IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822 such that amino acid residue 29 is leucine (L).

In some embodiments, the IL-2 peptide or functional fragment thereof comprises an amino acid sequence obtained by introducing one or more of the following amino acid substitutions into any 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.

In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions compared to the amino acid sequence of wild-type IL-2 that decrease the affinity of the IL-2 peptide or functional fragment thereof for IL-2Ra (CD25), and one or more amino acid substitutions compared to the amino acid sequence of wild-type IL-2 that increase the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Re (CD122). In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822 such that one or more 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 combinations thereof. Thus, in some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence that comprises one or more of the following amino acid substitutions compared to a mature wild-type form of 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.

In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 combinations thereof. In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 combinations thereof. In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any 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 combinations thereof. In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by

- 33 049880 by one or more amino acid substitutions in the amino acid sequence of any 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 combinations thereof. In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813822 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 combinations thereof. In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by one or more amino acid substitutions in the amino acid sequence of any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813822 such that amino acid residue amino acid residue 42 is isoleucine (I), amino acid residue 45 is glutamic acid (E), or amino acid residue 68 is valine (V), or combinations thereof.

In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence obtained by introducing or subsequently introducing a C125S, C125G, or C125A substitution into any of the amino acid sequences of the IL-2 polypeptide or functional fragment thereof described herein.

In some embodiments, one or more amino acid residues are deleted from the amino acid sequence of the IL-2 polypeptide or functional fragment thereof to remove an O-glycosylation site. In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence formed by deleting the first three amino acid residues (residues 1-3) of the amino acid sequence of any of the IL-2 polypeptides or functional fragments thereof disclosed herein. In some embodiments, one or more amino acid residues are substituted in the amino acid sequence of the IL-2 polypeptide or functional fragment thereof to remove an O-glycosylation site. In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence obtained by introducing amino acid substitutions into one or more of the first three amino acid residues (residues 1-3) of the amino acid sequence of any of the IL-2 polypeptides or functional fragments thereof disclosed herein.

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

2. IL-15 polypeptides.

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 converted to mature IL-15 by removing amino acid residues 1-48. This results in 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).

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

- 34 049880

NO: 166 or 167.

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

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

Masking fragments.

A masking moiety, as used herein, refers to a moiety that is capable of binding to or otherwise exhibiting affinity for a cytokine or a functional fragment thereof, such that in some embodiments, the binding reduces the affinity of the cytokine or a functional fragment thereof for its cognate receptor or protein. For example, a masking moiety for an IL-2 polypeptide or a functional fragment thereof is capable of binding to or otherwise exhibiting 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 a functional fragment thereof for its cognate receptor or protein (e.g., IL-2R or a component thereof, such as the IL-2Rα and/or IL-2Rβ chains). When bound to a cytokine or a functional fragment thereof, the masking moiety blocks, hinders, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the activity or binding of the cytokine or functional fragment thereof to its cognate receptor or protein. In some embodiments that include a first masking moiety and a second masking moiety, the presence of one of the masking moieties (e.g., the first masking moiety) blocks, hinders, inhibits (e.g., attenuates), or otherwise prevents (e.g., masks) the activity or binding of the cytokine or functional fragment thereof to its cognate receptor or protein, while the other masking moiety (e.g., the second masking moiety) may, in some embodiments, remain bound to the cytokine or functional fragment thereof after cleavage of the first masking moiety and still allows or perhaps even promotes binding of the cytokine or functional fragment thereof to its cognate receptor or protein at the site at which the first masking moiety was bound. See, for example, Fig. 7B. Methods for determining the extent of binding of a protein (e.g., a cytokine) to a cognate protein (e.g., a cytokine receptor) are well known in the art.

The masked cytokines provided herein comprise a masking moiety. The masking moiety comprises an amino terminus and a carboxy terminus. In some embodiments, the masked cytokine comprises one masking moiety. In some embodiments, the masked cytokine comprises more than one masking moiety, each of which can be any of the masking moieties described herein. In some embodiments, the masked cytokine comprises a first masking moiety and a second masking moiety. It should be understood, for example, that a reference to a masking fragment or a given masking fragment may refer to a masking fragment in a masked cytokine containing one masking fragment, or may refer to a first masking fragment in a masked cytokine containing a first masking fragment and a second masking fragment, or it may refer to a second masking fragment in a masked cytokine containing a first masking fragment and a second masking fragment, or it may refer to a first masking fragment and a second masking fragment in a masked cytokine containing a first masking fragment and a second masking fragment.

In some embodiments, the masking moiety is linked to a cytokine or a functional fragment thereof. In some embodiments, the masking moiety is linked to

- 35 049880 cytokine or a functional fragment thereof via a first linker. In some embodiments, the masked cytokine comprises a half-life extension domain that is linked to the masking moiety. In some embodiments, the masked cytokine comprises a half-life extension domain that is linked to the masking moiety via a second linker. In some embodiments, the masking moiety is linked to the cytokine or a functional fragment thereof and linked to the half-life extension domain. In some embodiments, the cytokine or a functional fragment thereof is linked to the amino terminus of the masking peptide and the half-life extension domain is linked to the carboxy terminus of the masking peptide. In some embodiments, the cytokine or a functional fragment thereof is linked to the carboxy terminus of the masking peptide and the half-life extension domain is linked to the amino terminus of the masking peptide. In some embodiments, the half-life extension domain is linked to the amino terminus or carboxy terminus of the masking moiety. In some embodiments, the second linker is linked to the amino terminus or carboxy terminus of the masking moiety. In some embodiments, the masking moiety is linked to a first linker and linked to a second linker. In some embodiments, the first linker is linked to the amino terminus of the masking peptide and the second linker is linked to the carboxy terminus of the masking peptide. In some embodiments, the first linker is linked to the carboxy terminus of the masking peptide and the second linker is linked to the amino terminus of the masking peptide.

In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety is linked to a cytokine or a functional fragment thereof, and the second masking moiety is linked to the cytokine or a functional fragment thereof. In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety is linked to the cytokine or a functional fragment thereof via a first linker, and the second masking moiety is linked to the cytokine or a functional fragment thereof via a second linker. In some embodiments, the masked cytokine comprises a half-life extension domain that is linked to either the first masking moiety or the second masking moiety. In some embodiments, the masked cytokine comprises a half-life extension domain that is linked to either the first masking moiety or the second masking moiety via a third linker. In some embodiments, the first linker is linked to the amino terminus or the carboxy terminus of the first masking moiety. In some embodiments, the second linker is linked to the amino terminus or carboxy terminus of the second masking moiety. In some embodiments, the half-life extension domain is linked to the amino terminus or carboxy terminus of the first masking moiety. In some embodiments, the half-life extension domain is linked to the amino terminus or carboxy terminus of the second masking moiety. In some embodiments, the third linker is linked to the amino terminus or carboxy terminus of the first masking moiety. In some embodiments, the third linker is linked to the amino terminus or carboxy terminus of the second masking moiety.

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

In some embodiments comprising a first masking moiety and a second masking moiety, the second masking moiety is linked to a first half-life extension domain, the first masking moiety is linked to a second masking moiety, and the cytokine or functional fragment thereof is linked to a second half-life extension domain. In some embodiments comprising a first masking moiety and a second masking moiety, the second masking moiety is linked to a first half-life extension domain.

- 36 049 880 via a first linker, the first masking moiety is linked to the second masking moiety via a second linker, and the cytokine or functional fragment thereof is linked to the second half-life extension domain via a third linker. In some embodiments, the first linker is linked to the amino terminus or carboxy terminus of the first half-life extension domain. In some embodiments, the second linker is linked to the amino terminus or carboxy terminus of the first masking moiety. In some embodiments, the second half-life extension domain is linked to the amino terminus or carboxy terminus of the cytokine or functional fragment thereof. In some embodiments, the first half-life extension domain is linked to the amino terminus or carboxy terminus of the second masking moiety. In some embodiments, the third linker is linked to the amino terminus or carboxy terminus of the second half-life extension domain. In some embodiments, the third linker is linked to the amino terminus or carboxyl terminus of the cytokine or functional fragment thereof.

In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the masking moiety is linked to the first half-life extension domain. In some embodiments, the first half-life extension domain is linked to the amino terminus or carboxy terminus of the masking moiety. In some embodiments, the masking moiety is linked to the first half-life extension domain via a first linker. In some embodiments, the first linker is linked to the amino terminus or carboxy terminus of the masking moiety.

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 linked to the first half-life extension domain and the second masking moiety is linked to a cytokine or a functional fragment thereof. In some embodiments, the second masking moiety is further linked to the second half-life extension domain. In some embodiments, the first masking moiety is linked to the first half-life extension domain via a first linker, and/or either the second masking moiety or the cytokine or a functional fragment thereof is linked to the second half-life extension domain via a second linker. In some embodiments, the second masking moiety is linked to the cytokine or a functional fragment thereof via a third linker.

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

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.

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

- 37 049 880 about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 187-218, 221-229, and 261. In some embodiments, the first masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 187-218, 221-229, and 261, and the second masking moiety comprises the amino acid sequence of SEQ ID NO: 9 or 231. In some embodiments, the first masking moiety comprises an amino acid sequence having about or at least about at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 187-218, 221-229, and 261, and the second masking moiety comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231. In some embodiments, the first masking moiety comprises the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking moiety comprises the amino acid sequence of SEQ ID NO: 261. In some embodiments, the second masking moiety comprises the amino acid sequence of SEQ ID NOs: 9 and 231, and the first masking moiety comprises the amino acid sequence of SEQ ID NO: 261. In some embodiments, the first masking moiety comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 261, and the second masking moiety comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231. In some embodiments, the 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: 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 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 NOs: 10, 161-165, 221-226, and 261. In some embodiments, the first masking moiety comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking moiety comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 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 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 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 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 NOs: 10, 161-165, 221-226, 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 the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking moiety comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 221-226, 261, 826, and 827. In some embodiments, the second masking moiety

- 38 049 880 fragment comprises the amino acid sequence of SEQ ID NO: 9 or 231, and the first mask fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 221-226, 261, 826 and 827. In some embodiments, the second mask fragment comprises an amino acid sequence having about or at least about 85, 86, 87, 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 mask fragment comprises an amino acid sequence having 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 first masking fragment comprises an amino acid sequence of SEQ ID NO: 9 or 231, and the second masking fragment 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 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 selected from the group consisting of SEQ ID NOs: 187-218 and the second masking moiety comprises the 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 the amino acid sequence of SEQ ID NO: 9 or 231.

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

- 39 049880 a sequence having about or at least about 85, 86, 87, 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 formed by one or more amino acid substitutions of SEQ ID NO: 10 or 261: C122S, C168S, R42A, K71A, T73A, T74A, V75A, H133A, Y134A, R137D, Q162W, E170A, and Q188A. In some embodiments, a masking fragment comprising an amino acid sequence that is formed by one or more amino acid substitutions comprises the amino acid sequence of SEQ ID NO: 826.

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.

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, 219229, 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, 219229, 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.

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 fragment comprises an amino acid

- 40 049880 a sequence having 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 NO: 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 NO: 232-234 and 823-825, and the second masking moiety comprises the amino acid sequence of SEQ ID NO: 826 or 827. In some embodiments, the second masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 232-234 and 823-825, and the first masking moiety comprises the amino acid sequence of SEQ ID NO: 826 or 827.

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 fragment comprises an amino acid sequence having 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.

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 823825. 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.

In some embodiments, the first masking moiety comprises an amino acid

- 41 049880 a sequence selected from the group consisting of SEQ ID NOs: 232-234 and 823-825, and the second masking fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 219 and 220. In some embodiments, the first masking fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 219 and 220, and the second masking fragment 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 823825. 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.

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

In some embodiments, the masking moiety comprises IL-2Ra (also referred to as CD25) or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-2. In some embodiments, the masking moiety comprises IL-2Rc (also referred to 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-2Re (also referred to as CD122) or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-2 and/or IL15. In some embodiments, the masking moiety comprises IL-2Re (also referred to as CD122) or a fragment, portion or variant thereof, and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 221-226, 261, 826 and 827. In some embodiments, the masking moiety comprises IL-2Ry (also referred to as CD132) or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-2 and/or IL-15. In some embodiments, the masking moiety comprises IL-15Rc (also referred to as CD215) or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-15. In some embodiments, the masking moiety comprises IL-15Rc (also referred to 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 moiety comprises an antigen-binding domain of an antibody or a fragment thereof. In some embodiments, the masking moiety comprises an antigen-binding domain of an anti-IL-2 antibody or a fragment thereof. In some embodiments, the masking moiety comprises an antigen-binding domain of an anti-IL-15 antibody or a fragment thereof.

In some embodiments comprising a first masking moiety and a second masking moiety, the first masking moiety comprises IL-2Rc or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-2, and the second masking moiety comprises IL-2Re or a fragment, portion or variant thereof that retains 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-2Re or a fragment, portion or variant thereof that retains or otherwise

- 42 049880 exhibits affinity for IL-2, and the second masking fragment comprises IL-2Ra or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-2. In some embodiments comprising a first masking fragment and a second masking fragment, the first masking fragment comprises IL-2Ra or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-2, and the second masking fragment comprises IL-2Ry or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-2. In some embodiments comprising a first masking fragment and a second masking fragment, the first masking fragment comprises IL-2Ry or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-2, and the second masking fragment comprises IL-2Ra or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-2. In some embodiments comprising a first masking fragment and a second masking fragment, the first masking fragment comprises IL2Ra or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-2, and the second masking fragment comprises an antigen-binding domain of an IL-2 antibody or fragment thereof. In some embodiments comprising a first masking fragment and a second masking fragment, the first masking fragment comprises an antigen-binding domain of an IL-2 antibody or fragment thereof, and the second masking fragment comprises IL-2Ra or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-2.

In some embodiments comprising a first masking fragment and a second masking fragment, the first masking fragment comprises IL-15Ra or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-15, and the second masking fragment comprises IL-2Re or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-15. In some embodiments comprising a first masking fragment and a second masking fragment, the first masking fragment comprises IL-2Re or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-15, and the second masking fragment comprises IL-15Ra 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-15Ra or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15, and the second masking moiety comprises IL-2Ry 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-2Ry or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15, and the second masking moiety comprises IL-15Ra 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-15Ra 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 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 IL-15 antibody or fragment thereof, and the second masking moiety comprises IL-15Ra or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-15.

Linkers.

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

- 43 049880 cleavable peptide and a C-terminal spacer domain. In some embodiments, the linker consists of a cleavable peptide.

In some embodiments, the masked cytokine comprises a linker. In some embodiments, the masked cytokine comprises one linker. In some embodiments, the masked cytokine comprises more than one linker, each of which can be any of the linkers 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, a reference to a linker or a given linker may refer to a linker in a masked cytokine comprising one linker, or may refer to a first linker in a masked cytokine comprising a first linker and a second linker, or it may refer to a second linker in a masked cytokine comprising a first linker and a second linker, or it may refer to a first linker and a second linker in a masked cytokine comprising a first linker and a second linker, or may refer to a first linker, a second linker and/or a third linker in a masked cytokine comprising a first linker, a second linker and a third linker, or it may refer to a first linker, a second linker, a third linker and/or a fourth linker in a masked cytokine comprising a first linker, a second linker, third linker and fourth linker.

It should be understood that each of the following terms is considered a linker as described herein: linker, first linker, second linker, third linker, and fourth linker, as well as any other numbered linker (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 a linker as described herein, and any of the linkers described herein may be a linker, a first linker, a second linker, a third linker, and/or a fourth linker as described herein.

It should also be understood that two components 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 by a linker, as described herein. For example, a half-life extension domain that is linked to a masking moiety is interpreted to mean either that the amino acid sequence of the half-life extension domain is linked directly 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 by a linker, such as any of the linkers described herein.

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

In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the masking moiety is linked to the amino terminus of the linker, and either the first half-life extension domain or the second half-life extension domain

- 44 049880 is linked to the carboxy terminus of the linker. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the masking moiety is linked to the carboxy terminus of the linker, and either the first half-life extension domain or the second half-life extension domain is linked to the amino terminus of the linker. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the cytokine or functional fragment thereof is linked to the amino terminus of the linker, and either the first half-life extension domain or the second half-life extension domain is linked to the carboxy terminus of the linker. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the cytokine or functional fragment thereof is linked to the carboxy terminus of the linker, and either the first half-life extension domain or the second half-life extension domain is linked to the amino terminus of the linker.

In some embodiments comprising a first masking moiety linked to a first half-life extension domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein either the second masking moiety or the cytokine or a functional fragment thereof is linked to a second half-life extension domain, the first masking moiety is linked to the amino terminus of a linker, and the first half-life extension domain is linked to the carboxy terminus of the linker. In some embodiments comprising a first masking moiety linked to a first half-life extension domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein either the second masking moiety or the cytokine or a functional fragment thereof is linked to a second half-life extension domain, the first masking moiety is linked to the carboxy terminus of the linker, and the first half-life extension domain is linked to the amino terminus of the linker. In some embodiments comprising a first masking moiety linked to a first half-life extension domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein either the second masking moiety or the cytokine or a functional fragment thereof is linked to a second half-life extension domain, the second masking moiety is linked to the amino terminus of a linker, and the cytokine or a functional fragment thereof is linked to the carboxy terminus of the linker. In some embodiments comprising a first masking moiety linked to a first half-life extension domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein either the second masking moiety or the cytokine or a functional fragment thereof is linked to a second half-life extension domain, the second masking moiety is linked to the carboxy 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 extension domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein either the second masking moiety or the cytokine or a functional fragment thereof is linked to a second half-life extension domain, the second masking moiety is linked to the amino terminus of a linker, and the second half-life extension domain is linked to the carboxy terminus of the linker. In some embodiments comprising a first masking moiety linked to a first half-life extension domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein either the second masking moiety or the cytokine or a functional fragment thereof is linked to a second half-life extension domain, the second masking moiety is linked to the carboxy terminus of the linker, and the second half-life extension domain is linked to the amino terminus of the linker. In some embodiments comprising a first masking moiety linked to a first half-life extension domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein either the second masking moiety or the cytokine or a functional fragment thereof is linked to the second half-life extension domain, the cytokine or a functional fragment thereof is linked to the amino terminus of the linker, and the second half-life extension domain is linked to the carboxy terminus of the linker. In some embodiments comprising a first masking moiety linked to a first half-life extension domain, a second masking moiety linked to a cytokine or a functional fragment thereof, and wherein either the second masking moiety or the cytokine or a functional fragment thereof is linked to the second half-life extension domain, the cytokine or a functional fragment thereof is linked to the carboxy terminus of the linker, and the second half-life extension domain is linked to the amino terminus of the linker.

In some embodiments comprising a first masking moiety linked to a first half-life extension 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 a second half-life extension domain via a third linker, the first masking moiety is linked to the carboxy terminus of the first linker and the first half-life extension domain is linked to the amino terminus of the first linker, the second masking moiety is linked to the carboxy terminus of the second linker, and the first masking moiety is linked to

- 45 049880 amino terminus of the second linker, and the cytokine or functional fragment thereof is linked to the carboxyl terminus of the third linker and the second half-life extension domain is linked to the amino terminus of the third linker.

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

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

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

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

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: 20153, 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.

In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 794, 795, 797-812. In some embodiments,

- 46 049880 embodiment, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535 and 538-555.

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 NO: 45-95. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 235. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 262. In some embodiments, the linker comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 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 NO: 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 NO: 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

- 47 049880 a sequence having 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: 800-812.

The cleavable peptides and spacer domains of some embodiments of the linker are described in more detail below.

1. Cleavable peptides.

In some embodiments, the linker comprises a cleavable peptide. In some embodiments having more than one linker that comprises 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 of the cleavable peptides described herein. For example, a reference to a cleavable peptide or a given cleavable peptide may refer to a cleavable peptide in a masked cytokine comprising a single cleavable peptide, or may refer to a first cleavable peptide in a masked cytokine comprising a first cleavable peptide and a second cleavable peptide, or may refer to a second cleavable peptide in a masked cytokine comprising a first cleavable peptide and a second cleavable peptide, or 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 may refer to 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 a third cleavable peptide. It is possible that in some embodiments more than three cleavable peptides may be present in accordance with the teachings set forth herein.

A cleavable peptide is a polypeptide that contains a cleavage site, such as a protease cleavage site. In some embodiments, the cleavable peptide contains more than one cleavage site. The term cleavage site as used herein refers to a recognizable site for cleavage of a portion of the cleavable peptide that occurs in any of the linkers that comprise the cleavable peptide described herein. Thus, the cleavage site may occur in a sequence of the cleavable peptide as described herein. In some embodiments, the cleavage site is an amino acid sequence that is recognized and cleaved by a cleavage agent. Examples of cleavage agents include proteins, enzymes, DNAzymes, RNAzymes, metals, acids and bases.

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

In embodiments, the protease cleavage site is a protease cleavage site.

- 48 049880 matrix metalloprotease (MMP), a metalloprotease cleavage site containing a disintegrin and a metalloprotease domain (ADAM), 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) cleavage site, a matriptase (ST14) protease cleavage site, or a legumain protease cleavage site. In embodiments, the matrix metalloprotease (MMP) cleavage site is an MMP9 cleavage site, an MMP13 cleavage site, or an MMP2 cleavage site. In embodiments, the disintegrin and metalloprotease domain (ADAM) metalloprotease cleavage site is an ADAM9 metalloprotease cleavage site, an ADAM10 metalloprotease cleavage site, or an ADAM17 metalloprotease cleavage site. Protease cleavage sites may be designated by a particular amino acid sequence.

In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of: ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, 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, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1 and TPSG1. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of: ADAM17, HTRA1, PRSS1, FAP, GZMK, NAPSA, MMP1, MMP2, MMP9, MMP10, MMP7, MMP12, MMP28, ADAMTS9, HGFAC, and HTRA3. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of uPA and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of matriptase and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of legumain and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of matriptase and uPA. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of uPA and legumain. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of matriptase and legumain. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of uPA, matriptase, and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of uPA, legumain, and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of matriptase, legumain, and MMP14. In some embodiments, the cleavable peptide is cleaved by one or more enzymes selected from the group consisting of matriptase, legumain, and uPA.

In some embodiments, the cleavable peptide is a substrate for a protease that is colocalized in a region or tissue expressing a cytokine receptor. The cytokine receptor can be any cytokine receptor. In some embodiments, the cytokine receptor is selected from the group consisting of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCR1, CX3CR1, IL-1RAP, IL-1RAPL1, IL-1RAPL2, IL-1RL1, IL-1RL2, IL-1R1, IL1R2, IL-2R, IL-2Ra, IL-2Re, IL-2Ry, IL-3Ra, IL-4R, IL-5Ra, IL-6R, IL-6ST, IL-7R, IL-9R, IL-10Ro, IL-10Re, IL-11RO, IL-12Re1, IL-12Re2, IL-13Ra1, IL-13Ro2, IL-15Ro, IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL17RE, IL-18RAP, IL-18R1, IL-20Ro, IL-20Re, IL-21R, IL-22Ra1, IL-22Ro2, IL-23R, IL-27Ro, IL-28Ro, IL31RA, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, GMRa (CD116), CD131, GHR, PRLR, EPOR, LIFR (CD118), OSMRe, TPO-R (CD110), CSF-1R, EDAR, TNFRSF1A, TNFRSF1B, LTBR, TNFRSF4, CD40, FAS, TNFRSF6B, CD27, TNFRSF8, TNFRSF9, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D,

- 49 049880

TNFRSF11A, TNFRSF11B, TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF14, NGFR, TNFRSF17, TNFRSF18, TNFRSF19, RELT, TNFRSF21, TNFRSF25 and EDA2R.

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), an 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). Illustrative cleavable peptide sequences are presented in Table 1.

Table 1

Illustrative cleavable peptide sequences

Illustrative cleavable peptide sequences MPYDLYHP (SEQ ID NO: 96) GGIGQLTSVLMAAP (SEQ ID NO: 129) GGIGQLTA (SEQ ID NO: 97) DSGGFMLTLVLPVLP (SEQ ID NO: 130) DLGRFQTF (SEQ ID NO: 98) TSEFVFAPDLGRFQTF (SEQ ID NO: 131) DSGGFMLT (SEQ ID NO: 99) TSTSGRSANPR (SEQ ID NO: 132) TSVLMAAP (SEQ ID NO: 100) TSTSGRSANPG (SEQ ID NO: 133) TSEFVFAPDQ (SEQ ID NO: 101) TSTSGRSANPH (SEQ ID NO: 134) KLVLPVLP (SEQ ID NO: 102) VPLSLY (SEQ ID NO: 135) KPILFFRL (SEQ ID NO: 103) TSASGASASAA (SEQ ID NO: 136) ANQLKG (SEQ ID NO: 104) PSSSPGGGSSP (SEQ ID NO: 137) QSQLKE (SEQ ID NO: 105) ISSGLLSGRSDNH (SEQ ID NO: 138) HEQLTV (SEQ ID NO: 106) ISSGLLSGRSDDH (SEQ ID NO: 139) PANLVAPDP (SEQ ID NO: 107) ISSGLLSGRSDIH (SEQ ID NO: 140) PAPGVYPGP (SEQ ID NO: 108) ISSGLLSGRSDQH (SEQ ID NO: 141) APAGLIVPYN (SEQ ID NO: 109) ISSGLLSGRSDTH (SEQ ID NO: 142) PQALVA (SEQ ID NO: 110) ISSGLLSGRSANP (SEQ ID NO: 143) VGNLNF (SEQ ID NO: 111) ISSGLLSGRSDNP (SEQ ID NO: 144) VANLYE (SEQ ID NO: 112) ISSGLLSGRSANPRG (SEQ ID NO: 145) VYNLMD (SEQ ID NO: 113) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 146) TFNIKQ (SEQ ID NO: 114) AVGLLAPPGGLSGRSDDH (SEQ ID NO: 147) DLWKLLP (SEQ ID NO: 115) AVGLLAPPGGLSGRSDIH (SEQ ID NO: 148) PGSTKRA (SEQ ID NO: 116) AVGLLAPPGGLSGRSDQH (SEQ ID NO: 149) QQYRALKS (SEQ ID NO: 117) AVGLLAPPGGLSGRSDTH (SEQ ID NO: 150) YVPRAVL (SEQ ID NO: 118) AVGLLAPPGGLSGRSANP (SEQ ID NO: 151) GVNKWPT (SEQ ID NO: 119) AVGLLAPPGGLSGRSDNP (SEQ ID NO: 152) LAQAVRSS (SEQ ID NO: 120) AVGLLAPPSGRSANPRG (SEQ ID NO: 153) RAAAVKSP (SEQ ID NO: 121) SGRSA (SEQ ID NO: 236) DLLAVVAAS (SEQ ID NO: 122) SGRSANA (SEQ ID NO: 237) VQTVTWPD (SEQ ID NO: 123) SGRNAQ (SEQ ID NO: 238) AIPMSIPP (SEQ ID NO: 124) SGRNAQVR (SEQ ID NO: 239) GYEVHHQK (SEQ ID NO: 125) SGRSDN (SEQ ID NO: 240) VHHQKLVF (SEQ ID NO: 126) SGRSDNPN (SEQ ID NO: 241) IRRVSYSF (SEQ ID NO: 127) GSGKSAA (SEQ ID NO: 242) MPYDLYHPILFFRL (SEQ ID NO: 128) DSGGFMLTS (SEQ ID NO: 264) ISSGLLGGLSGRSDQP (SEQ ID NO: 270) IYDQKT (SEQ ID NO: 342) ISSGLLSGRSDQG (SEQ ID NO: 271) AHNYKT (SEQ ID NO: 343) ISSGLLSGRSDQA (SEQ ID NO: 272) MMDQAN (SEQ ID NO: 344) ISSGLLSGRSDSP (SEQ ID NO: 273) MLGEFVSE (SEQ ID NO: 345) ISSGLLSGRSDTP (SEQ ID NO: 274) GLVALRGA (SEQ ID NO: 346) ISSGLLSGRSDMP (SEQ ID NO: 275) KEHKYKAE (SEQ ID NO: 347) ISSGLLSGRSD (SEQ ID NO: 276) RQARVVG (SEQ ID NO: 356) ISSGLLSGRSDQP (SEQ ID NO: 277) LGGSGRSNAQVRLE (SEQ ID NO: 357) ISSGLLGGLSGRSDNP (SEQ ID NO: 278) LGGSGRKASLSLE (SEQ ID NO: 358) ISSGLLSSGGLSGRSDQP (SEQ ID NO: 279) SGRIGFLRTA (SEQ ID NO: 359) ISSGLLSSGGLSGRSDNP (SEQ ID NO: 280) SGAIGFLRTA (SEQ ID NO: 360) ISSGLLSGRS (SEQ ID NO: 281) RPARSGRSAGGSVA (SEQ ID NO: 361) ISSGLLSGRSESP (SEQ ID NO: 282) VTGRGDSPASS (SEQ ID NO: 362)

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ISSGLLSGRSEQP (SEQ ID NO: 283) PRFKIIGG (SEQ ID NO: 363) ISSGLLSGRSEQH (SEQ ID NO: 284) LSGRIGFLRTA (SEQ ID NO: 364) LSSGLLSGRSDQP (SEQ ID NO: 285) LSGRSNAMPYDLYHP (SEQ ID NO: 365) LSSGLLGGLSGRSDQP (SEQ ID NO: 286) LSGRSNAGGIGQLTA (SEQ ID NO: 366) LSSGLLSGRSDQG (SEQ ID NO: 287) LSGRSNAVPLSLY (SEQ ID NO: 367) LSSGLLSGRSDQA (SEQ ID NO: 288) LSGRSNADSGGFMLT (SEQ ID NO: 368) LSSGLLSGRSDSP (SEQ ID NO: 289) LSGRSNAHEQLTA (SEQ ID NO: 369) LSSGLLSGRSDTP (SEQ ID NO: 290) LSGRSNARAAAVKSP (SEQ ID NO: 370) LSSGLLSGRSDMP (SEQ ID NO: 291) LSGRSNATSVLMAAP (SEQ ID NO: 371) LSSGLSGRSD (SEQ ID NO: 292) VPLSLYLSGRSNA (SEQ ID NO: 372) GKQLRVVNEYSSMDNMLLG (SEQ ID NO: 293) DSGGFMLTLSGRSNA (SEQ ID NO: 373) LSSGLLGGLSGRSDNP (SEQ ID NO: 294) GGIGQLTALSGRSNA (SEQ ID NO: 374) LSSGLLSSGGLSGRSDQP (SEQ ID NO: 295) MPYDLYHPLSGRSNA (SEQ ID NO: 375) LSSGLLSSGGLSGRSDNP (SEQ ID NO: 296) HEQLTVLSGRSNA (SEQ ID NO: 376) GKQLRVVNEYSSEDNMLLG (SEQ ID NO: 297) RAAAVKSPLSGRSNA (SEQ ID NO: 377) LSSGLLSGRSESP (SEQ ID NO: 298) TSVLMAAPLSGRSNA (SEQ ID NO: 378) LSSGLLSGRSEQP (SEQ ID NO: 299) IPVSLRSGRSNAQRLE (SEQ ID NO: 379) LSSGLLSGRSEQH (SEQ ID NO: 300) VPLSLYRQARVVG (SEQ ID NO: 380) MPYDLYH (SEQ ID NO: 301) DSGGFMLTRQARVVG (SEQ ID NO: 381) LSGRSDNH (SEQ ID NO: 302) GGIGQLTARQARVVG (SEQ ID NO: 382) GSIPVSLRSG (SEQ ID NO: 306) MPYDLYHPRQARVVG (SEQ ID NO: 383) GPSGPAGLKGAPG (SEQ ID NO: 307) HEQLTVRQARVVG (SEQ ID NO: 384) GPPGPAGMKGLPG (SEQ ID NO: 308) RAAAVKSPRQARVVG (SEQ ID NO: 385) GYVADAPK (SEQ ID NO: 309) TSVLMAAPRQARVVG (SEQ ID NO: 386) KKLADEPE (SEQ ID NO: 310) KQLRVVNEYSSMDNMLLG (SEQ ID NO: 387) GGSRPAHLRDSGK (SEQ ID NO: 311) KQLRVVNEYSSEDNMLLG (SEQ ID NO: 388) SFTQARVVGG (SEQ ID NO: 312) KQLRVVNGYSSEDNMLLG (SEQ ID NO: 389) VHMPLGFLGPRQARVVN (SEQ ID NO: 313) KQLRVVGGLVHLKNTMET (SEQ ID NO: 390) LSGRSDNHSPLGLAGGS (SEQ ID NO: 314) TRDRLDEVNFKQLRVVNG (SEQ ID NO: 391) VPLSLYSG (SEQ ID NO: 315) TRDRLDEVNFKLLRVVNG (SEQ ID NO: 392) IPESLRAG (SEQ ID NO: 316) TRDRLDPVNFKQLRVVNG (SEQ ID NO: 393) IPVSLRSG (SEQ ID NO: 317) TRDRLDPVNFKLLRVVNG (SEQ ID NO: 394) TYSRSKYLATA (SEQ ID NO: 399) NPMGSEPVNFKQLRVVNG (SEQ ID NO: 395) TYSRSRYLATA (SEQ ID NO: 400) NPMGSEPVNFKLLRVVNG (SEQ ID NO: 396) KQLRVVNEYSSE (SEQ ID NO: 401) NPMGSDPVNFKQLRVVNG (SEQ ID NO: 397) KQLRVVNGYSSE (SEQ ID NO: 402) NPMGSDPVNFKLLRVVNG (SEQ ID NO: 398) KQLRVVGGLVAL (SEQ ID NO: 403) AGQPKQLRVVNG (SEQ ID NO: 494) KQLRVVNGLVAL (SEQ ID NO: 404) AGQPLQLRVVNG (SEQ ID NO: 495) SPGRVVGGLVAL (SEQ ID NO: 405) AGQPLQERVVNG (SEQ ID NO: 496) PQPRTYSRSRYL (SEQ ID NO: 406) AGQPKQERVVNG (SEQ ID NO: 497) PQPRTTSRSRYL (SEQ ID NO: 407) GTANKQLRVVNG (SEQ ID NO: 498) VVNEYSSSRGPYH (SEQ ID NO: 408) GTANKQLHVVNG (SEQ ID NO: 499) VVNEYSSERGPYH (SEQ ID NO: 409) GTANIQLRVVNG (SEQ ID NO: 500) NKVSMSSSRGPYH (SEQ ID NO: 410) GTANIQLHVVNG (SEQ ID NO: 501) NKVSMSSTRGPYH (SEQ ID NO: 411) KQLRTVAGLAGK (SEQ ID NO: 504) APAMMRGSVILTV (SEQ ID NO: 412) KQLRTVNGLAGK (SEQ ID NO: 505) APAMMEGSVILTV (SEQ ID NO: 413) KQLRVVAGLAGK (SEQ ID NO: 506) RGSVIITVQTVTW (SEQ ID NO: 414) KQLRVVNGLAGK (SEQ ID NO: 507) RGSVILTVQTVTW (SEQ ID NO: 415) GIKYKQLRVVNG (SEQ ID NO: 508) RKGKALAAYRLE (SEQ ID NO: 420) GIKYKYLRVVNG (SEQ ID NO: 509) RKGKAGAAYRLE (SEQ ID NO: 421) GIKYLQLRVVNG (SEQ ID NO: 510) RQARVVGGLVAL (SEQ ID NO: 422) GIKYLYLRVVNG (SEQ ID NO: 511) GGVRGPRFKIIGG (SEQ ID NO: 423) THLDLTYSRSKYLATA (SEQ ID NO: 512) GGVRGPRVKIIGG (SEQ ID NO: 424) THLDLTPSRSKYLATA (SEQ ID NO: 513) VTGRGDSHSLTTN (SEQ ID NO: 425) THLDLTYSRSRYLATA (SEQ ID NO: 514) VTGRGDSPSLTTN (SEQ ID NO: 426) THLDLTPSRSRYLATA (SEQ ID NO: 515) TGHGQASQGLLDR (SEQ ID NO:427) TYSRSKYLAPANGNAE (SEQ ID NO: 516)

- 51 049880

TGHGQASSGLLDR (SEQ ID NO: 428) TYSRSKYLATANGNAE (SEQ ID NO: 517) KQLRVVNENLENY (SEQ ID NO: 429) TYSRSRYLAPANGNAE (SEQ ID NO: 518) KQLRVVNGNLENY (SEQ ID NO: 430) TYSRSRYLATANGNAE (SEQ ID NO: 519) SNVNDVANYNFF (SEQ ID NO: 431) DPVNFKQLRVVNEYSSE (SEQ ID NO: 520) SNVNDVSNYNFF (SEQ ID NO: 432) DPVNFKQLRVVNGYSSE (SEQ ID NO: 521) IDFNAAQNLYEK (SEQ ID NO: 433) DPVNFKKLRVVNEYSSE (SEQ ID NO: 522) IDFNAAYNLYEK (SEQ ID NO: 434) DPVNFKKLRVVNGYSSE (SEQ ID NO: 523) IQWNAGQPLQER (SEQ ID NO: 435) RKGKAGAAKNLNEKDY (SEQ ID NO: 524) IQWNAPQPLQER (SEQ ID NO: 436) RKGKAGAAKNLYEKDY (SEQ ID NO: 525) SMDNRLLGLFGE (SEQ ID NO: 437) RKGKAGAAQNLNEKDY (SEQ ID NO: 526) SMDNMLLGLFGE (SEQ ID NO: 438) RKGKAGAAQNLYEKDY (SEQ ID NO: 527) VPIDDPQDLLEG (SEQ ID NO: 439) VTGRGDSHSLTKNQVSL (SEQ ID NO: 528) VPIDDPEDLLEG (SEQ ID NO: 440) VTGRGDSHSLTTNQVSL (SEQ ID NO: 529) IPENLPPGLPLT (SEQ ID NO: 441) VTGRGDSPSLTKNQVSL (SEQ ID NO: 530) IPENLPPLLPLT (SEQ ID NO: 442) VTGRGDSPSLTTNQVSL (SEQ ID NO: 531) QPPSLTKNQVSL (SEQ ID NO: 443) TGHGQASSERSSNIRTS (SEQ ID NO: 532) QPPSLTRNQVSL (SEQ ID NO: 444) TGHGQASSERSSNSRTS (SEQ ID NO: 533) DSHSLTKNQVSL (SEQ ID NO: 445) TGHGQASSERSSTIRTS (SEQ ID NO: 534) DSHSLTTNQVSL (SEQ ID NO: 446) TGHGQASSERSSTSRTS (SEQ ID NO: 535) KAIQLTKNQVSL (SEQ ID NO: 447) DPVNFKLLRVVNEYSSE (SEQ ID NO: 538) KAIQLTYNQVSL (SEQ ID NO: 448) DPVNFKLLRVVNGYSSE (SEQ ID NO: 539) AEPWTNRNTDGS (SEQ ID NO: 449) DPVNFKQLRVVGGLVAL (SEQ ID NO: 540) AEPWTVRNTDGS (SEQ ID NO: 450) DPVNFKQLRVVNGLVAL (SEQ ID NO: 541) KQLRVVNG (SEQ ID NO: 451) DPVNFKLLRVVGGLVAL (SEQ ID NO: 542) KQLRVVTGRGDSP (SEQ ID NO: 452) DPVNFKLLRVVNGLVAL (SEQ ID NO: 543) KQLRVVNGRGDSP (SEQ ID NO: 453) KQLRVQNGDSTE (SEQ ID NO: 544) PSSRRRVVRKGVS (SEQ ID NO: 454) KQLRVVNNDATE (SEQ ID NO: 545) PSSRRRVNRKGVS (SEQ ID NO: 455) KQLRVVNGDSTE (SEQ ID NO: 546) SPGRVVTGRGDSP (SEQ ID NO: 456) ISNNKQLRVVNG (SEQ ID NO: 547) SPGRVVGGRGDSP (SEQ ID NO: 457) ISNNKQLPVVNG (SEQ ID NO: 548) NSGRAVTGRGDSP (SEQ ID NO: 458) ISNNEQLRVVNG (SEQ ID NO: 549) NSGRAVTYRGDSP (SEQ ID NO: 459) ISNNEQLPVVNG (SEQ ID NO: 550) TGHGQPSSRRRVN (SEQ ID NO: 460) KVSNKQLRVVNG (SEQ ID NO: 551) TGHGQASSRRRVN (SEQ ID NO: 461) KVSNKQLPVVNG (SEQ ID NO: 552) TGHGQSSSRGPYH (SEQ ID NO: 462) KVSNKALRVVNG (SEQ ID NO: 553) TGHGQASSRGPYH (SEQ ID NO: 463) KVSNKALPVVNG (SEQ ID NO: 554) RGSVILTKNQVSL (SEQ ID NO: 464) KQLRVQNNDATE (SEQ ID NO: 555) RGSVILTVNQVSL (SEQ ID NO: 465) TGHGQRSSNIRTS (SEQ ID NO: 480) SPGRVVGINYWLA (SEQ ID NO: 466) TGHGQASSNIRTS (SEQ ID NO: 481) SPGRVVGGNYWLA (SEQ ID NO: 467) TGHGQHSSNIANI (SEQ ID NO: 482) SPGRVVGSNKGAI (SEQ ID NO: 468) TGHGQASSNIANI (SEQ ID NO: 483) SPGRVVGGNKGAI (SEQ ID NO: 469) TGHGQASRNDYSY (SEQ ID NO: 484) PGARGRAPNHAVV (SEQ ID NO: 470) TGHGQASSNDYSY (SEQ ID NO: 485) PGARGRAFNHAVV (SEQ ID NO: 471) KALHVTNRNTDGS (SEQ ID NO:486) PGARGNAFNNLDR (SEQ ID NO: 472) KALHVTNINTDGS (SEQ ID NO: 487) PGARGRAFNNLDR (SEQ ID NO: 473) RVVRKKVSNKALP (SEQ ID NO: 488) VSNKYISNNEQLP (SEQ ID NO: 474) RVVRKGVSNKALP (SEQ ID NO: 489) VSNKYFSNNEQLP (SEQ ID NO: 475) RQARVVGINYWLA (SEQ ID NO: 490) KVSNKALHVTNI (SEQ ID NO: 476) RQARVVGGNYWLA (SEQ ID NO: 491) KVSNKALPVTNI (SEQ ID NO: 477) VTGRGPSPDVPLT (SEQ ID NO: 478) VTGRGDSPDVPLT (SEQ ID NO: 479)

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 538-52 049880

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 wherein 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 wherein 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, 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, comprises an N-terminus and a C-terminus, and an amino acid sequence selected from the group consisting of SEQ ID NO: 236-242 is linked to the N-terminus or C-terminus of an amino acid sequence selected from the group consisting of SEQ ID NO: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535 and 538-555. In some embodiments, wherein 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 the C-terminus of the amino acid sequence selected from the group consisting of SEQ ID NOs: 96-131 and 264.

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

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

- 53 049880 the spacer domain is linked to the amino terminus of the cleavage peptide, and the C-terminal spacer domain is linked to the carboxy terminus of the cleavage peptide.

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

2. Spacer domains.

In some embodiments, the linker comprises a spacer domain. The term spacer domain as used herein may, in some embodiments, 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.

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

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

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

- 54 049 880 embodiments, the first half-life extension domain is linked to the amino terminus or the C-terminal spacer domain. In some embodiments, the second half-life extension domain is linked to the amino terminus or the C-terminal spacer domain.

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

In some embodiments comprising 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 second masking moiety is linked to the carboxy 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 linked to the carboxy terminus of the C-terminal spacer domain, and the second masking moiety is linked to the amino 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 amino terminus of the N-terminal spacer domain, and either the first masking moiety or the second masking moiety is linked to the carboxy 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 carboxy terminus of the N-terminal spacer domain, and either the first masking moiety or the second masking moiety is linked to the amino 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 linked to the amino terminus of the N-terminal spacer domain, and the second masking moiety is linked to the carboxy 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 linked to the carboxy terminus of the N-terminal spacer domain, and the second masking moiety is linked to the amino 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 amino terminus of the C-terminal spacer domain, and either the first masking moiety or the second masking moiety is linked to the carboxy terminus of the C-terminal spacer domain. In some embodiments,

- 55 049880 comprising a first masking fragment and a second masking fragment, the half-life extension domain is linked to the carboxy terminus of the C-terminal spacer domain, and either the first masking fragment or the second masking fragment is linked to the amino terminus of the C-terminal spacer domain.

In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the masking moiety is linked to the amino terminus of the N-terminal spacer domain, and either the first half-life extension domain or the second half-life extension domain is linked to the carboxy terminus of the N-terminal spacer domain. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the masking moiety is linked to the carboxy terminus of the N-terminal spacer domain, and either the first half-life extension domain or the second half-life extension domain is linked to the amino terminus of the N-terminal spacer domain. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the cytokine or functional fragment thereof is linked to the amino terminus of the N-terminal spacer domain, and either the first half-life extension domain or the second half-life extension domain is linked to the carboxy terminus of the N-terminal spacer domain. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the cytokine or functional fragment thereof is linked to the carboxy terminus of the N-terminal spacer domain, and either the first half-life extension domain or the second half-life extension domain is linked to the amino terminus of the N-terminal spacer domain.

In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the masking moiety is linked to the amino terminus of the C-terminal spacer domain, and either the first half-life extension domain or the second half-life extension domain is linked to the carboxy terminus of the C-terminal spacer domain. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the masking moiety is linked to the carboxy terminus of the C-terminal spacer domain, and either the first half-life extension domain or the second half-life extension domain is linked to the amino terminus of the C-terminal spacer domain. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the cytokine or functional fragment thereof is linked to the amino terminus of the C-terminal spacer domain, and either the first half-life extension domain or the second half-life extension domain is linked to the carboxy terminus of the C-terminal spacer domain. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the cytokine or functional fragment thereof is linked to the carboxy terminus of the C-terminal spacer domain, and either the first half-life extension domain or the second half-life extension domain is linked to the amino terminus of the C-terminal spacer domain.

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 linked to the amino terminus of the N-terminal spacer domain, and the first half-life extension domain is linked to the carboxy terminus of the N-terminal spacer domain. 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 linked to the carboxy terminus of the N-terminal spacer domain, and the first half-life extension domain is linked to the amino terminus of the N-terminal spacer domain. 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 linked to the amino terminus of the C-terminal spacer domain, and the first half-life extension domain is linked to the carboxy terminus of the C-terminal spacer domain. 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 linked to the carboxy terminus of the C-terminal spacer domain, and the first half-life extension domain is linked to the amino terminus of the C-terminal spacer domain.

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 second masking moiety is linked to the amino terminus of the N-terminal spacer domain, and the cytokine or functional fragment thereof is linked to the carboxy terminus of the N-terminal spacer domain. 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 second masking moiety is linked to the carboxy terminus of the N-terminal spacer domain, and the cytokine or functional fragment thereof is linked to the amino terminus of the N-terminal spacer domain. 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,

- 56 049880 the second masking fragment is linked to the amino terminus of the C-terminal spacer domain, and the cytokine or functional fragment thereof is linked to the carboxy terminus of the C-terminal spacer domain. In some embodiments comprising a first half-life extension domain, a second half-life extension domain, a first masking fragment and a second masking fragment, the second masking fragment is linked to the carboxy 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.

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 second half-life extension domain is linked to the amino terminus of the N-terminal spacer domain, and either the cytokine or a functional fragment thereof or the second masking moiety is linked to the carboxy terminus of the N-terminal spacer domain. 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 second half-life extension domain is linked to the carboxy terminus of the N-terminal spacer domain, and either 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 extension domain, a second half-life extension domain, a first masking moiety and a second masking moiety, the second half-life extension domain is linked to the amino terminus of the C-terminal spacer domain, and either the cytokine or a functional fragment thereof or the second masking moiety is linked to the carboxy terminus of the C-terminal spacer domain. 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 second half-life extension domain is linked to the carboxy terminus of the C-terminal spacer domain, and either the cytokine or a functional fragment thereof or the second masking moiety is linked to the amino terminus of the C-terminal spacer domain.

In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the first half-life extension domain is linked to the second half-life extension domain via a linker. In some embodiments, the linker linking the first half-life extension domain and the second half-life extension domain comprises an N-terminal spacer domain and/or a C-terminal spacer domain. In some embodiments, the linker linking the first half-life extension domain and the second half-life extension domain comprises a cleavable peptide and an N-terminal spacer domain and/or a C-terminal spacer domain. In some embodiments comprising a first half-life extension domain and a second half-life extension domain that are linked together by a linker, the linker comprises an amino terminus and a carboxy terminus, and the first half-life extension domain is linked to the amino terminus of the linker and the second half-life extension domain is linked to the carboxy terminus of the linker. In some embodiments comprising a first half-life extension domain and a second half-life extension domain that are linked together by a linker, the linker comprises an amino terminus and a carboxy terminus, and the first half-life extension domain is linked to the carboxy terminus of the linker and the second half-life extension domain is linked to the amino terminus of the linker.

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 NO: 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, 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

- 57 049 880 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 a 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.

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 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 a sequence having about or at least about 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 of SEQ ID NOs: 20-95, 235, 268, and 269. In some embodiments, the C-terminal spacer 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 any of the amino acid sequences 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 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 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 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 provided in Table 2.

Table 2

Illustrative spacer domains

PA (SEQ ID NO: 20) SPGGSS (SEQ ID NO: 74) GGGGSGGGGSGGGGS (SEQ ID NO: 21) GGPGSSP (SEQ ID NO: 75) PSGPSAGGAA (SEQ ID NO: 22) SGPPGGPSS (SEQ ID NO: 76)

- 58 049880

GGPPASAGS (SEQ ID NO: 23) GPGPGSPPGGSS (SEQ ID NO: 77) GSPPAGGAP (SEQ ID NO: 24) SGPP (SEQ ID NO: 78) GPGSGSGGAA (SEQ ID NO: 25) PGSPSSSS (SEQ ID NO: 79) GGGGSGGGGS (SEQ ID NO: 26) PSPGGPS (SEQ ID NO: 80) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 27) GGPPS (SEQ ID NO: 81) PGSGS (SEQ ID NO: 28) PSPPSS (SEQ ID NO: 82) GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 29) SGGPGP (SEQ ID NO: 83) GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 30) GPSPGS (SEQ ID NO: 84) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 31) GSPGPSP (SEQ ID NO: 85) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 32) PSSGGSS (SEQ ID NO: 86) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GS (SEQ ID NO: 33) SGSSGP (SEQ ID NO: 87) GGSSPP (SEQ ID NO: 34) GGSSSPP (SEQ ID NO: 88) SGP (SEQ ID NO: 35) GSPGSP (SEQ ID NO: 89) GSP (SEQ ID NO: 36) PPPS (SEQ ID NO: 90) GSPP (SEQ ID NO: 37) APPPS (SEQ ID NO: 91) GSPS (SEQ ID NO: 38) AAPPPS (SEQ ID NO: 92) GPPSGSSP (SEQ ID NO: 39) SAPPS (SEQ ID NO: 93) GSSGGPPGG (SEQ ID NO: 40) SSGP (SEQ ID NO: 94) SGSPSGSGGG (SEQ ID NO: 41) SSPGP (SEQ ID NO: 95) GPPGPPGSSG (SEQ ID NO: 42) SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 235) SGGG (SEQ ID NO: 43) GSGP (SEQ ID NO: 268) GSSSGPPGPPS (SEQ ID NO: 44) GPAP (SEQ ID NO: 269) GGS (SEQ ID NO: 45) SGS (SEQ ID NO: 727) GGGSSGGS (SEQ ID NO: 46) SG (SEQ ID NO: 794) GGSGG (SEQ ID NO: 47) GSG (SEQ ID NO: 799) GGGS (SEQ ID NO: 48) PGPGP (SEQ ID NO: 323) GS (SEQ ID NO: 48) SGGCGGHQYERRGGC (SEQ ID NO: 324) GSGGGSSGGS (SEQ ID NO: 50) SGGCSGHQYERREGC (SEQ ID NO: 325) GSSGGS (SEQ ID NO: 51) SGGCGGHYFERHGGC (SEQ ID NO: 326) GGGSSGGSG (SEQ ID NO: 52) SGGCSGHYFERHEGC (SEQ ID NO: 327) GGSAGGS (SEQ ID NO: 53) SGGCSFHQYERHEGC (SEQ ID NO: 328) GHS (SEQ ID NO: 54) PSGS (SEQ ID NO: 329) GPS (SEQ ID NO: 55) GSPG (SEQ ID NO: 330) GAS (SEQ ID NO: 56) GGSPGG (SEQ ID NO: 331) SGG (SEQ ID NO: 57) GGPGGP (SEQ ID NO: 332) SGGSGG (SEQ ID NO: 58) GGSG (SEQ ID NO: 333) SSG (SEQ ID NO: 59) GSPPGG (SEQ ID NO: 334) GGGSGG (SEQ ID NO: 60) GPGSPG (SEQ ID NO: 335) GG (SEQ ID NO: 61) GSSPPG (SEQ ID NO: 336) GGG (SEQ ID NO: 62) GGP (SEQ ID NO: 337) SHGG (SEQ ID NO: 63) SGPGSGS (SEQ ID NO: 338) HGGG (SEQ ID NO: 64) SGPGSGS (SEQ ID NO: 340) SGAA (SEQ ID NO: 65) SGSGGSP (SEQ ID NO: 341) SGPA (SEQ ID NO: 66) GGGSSP (SEQ ID NO: 303) GGSGGS (SEQ ID NO: 67) SGGP (SEQ ID NO: 304) GGSGGP (SEQ ID NO: 68) SGPSGSPG (SEQ ID NO: 305) GGSGGG (SEQ ID NO: 69) GSGGPGPS (SEQ ID NO: 70) SGPPGSS (SEQ ID NO: 71)

- 59 049880

SSGGSGP (SEQ ID NO: 72) SSPSPSGG (SEQ ID NO: 73)

In some embodiments, the N-terminal spacer domain comprises an amino acid sequence formed by modifying the amino acid sequence of any of the N-terminal spacer domains described herein. In some embodiments, the N-terminal spacer domain comprises an amino acid sequence formed by modifying the amino acid sequence of any 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 formed by modifying the amino acid sequence of any of the C-terminal spacer domains described herein. In some embodiments, the C-terminal spacer domain comprises an amino acid sequence formed 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 formed 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 formed 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.

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

It should be understood that a modification of a spacer domain, as described in some embodiments, may refer to a modification of 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, a modification of a spacer domain may refer to (a) a modification of any N-terminal spacer domain described herein, (b) a modification of any C-terminal spacer domain described herein, or (c) a modification of any N-terminal spacer domain and any C-terminal spacer domain described herein. Thus, any of the linkers described herein that comprise an N-terminal spacer domain includes embodiments in which the N-terminal spacer domain is modified in accordance with the modifications described herein, any of the linkers described herein that comprise a C-terminal spacer domain includes embodiments in which the C-terminal spacer domain is modified in accordance with the modifications described herein, and any of the linkers described herein that comprise an N-terminal spacer domain and a C-terminal spacer domain includes embodiments in which the N-terminal spacer domain and/or the C-terminal spacer domain is/are modified in accordance with the modifications described herein.

Modification of the sequence of the N-terminal spacer domain and/or C-terminal spacer

- 60 049880 spacer domain may be any modification of the amino acid sequence of the spacer domain, including the inclusion of any additional amino acid in the sequence, the replacement of any amino acid with another amino acid and/or the deletion of any amino acid from the sequence.

In some embodiments, one or more additional amino acids are included by adding an N-terminal spacer domain and/or a C-terminal spacer domain to the amino acid sequence. In some embodiments, the one or more amino acids that are included by adding the N-terminal spacer domain and/or the C-terminal spacer domain to the amino acid sequence 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), 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).

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

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

In some embodiments, one or more amino acids that are removed from the amino acid sequence of the N-terminal spacer domain and/or the C-terminal spacer domain are selected from the group consisting of hydrophilic amino acids (e.g., lysine, arginine,

- 61 049880 histidine, aspartic acid, glutamic acid, serine, threonine, asparagine or glutamine), hydrophobic amino acids (e.g. alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, cysteine, glycine or proline), polar amino acids (e.g. serine, threonine, cysteine, asparagine, glutamine or tyrosine), non-polar amino acids (e.g. glycine, alanine, valine, proline, leucine, isoleucine, methionine, tryptophan or phenylalanine), amino acids with aliphatic side chains (e.g. glycine, alanine, valine, leucine or isoleucine), amino acids with hydroxyl-containing side chains (e.g. serine or threonine), amino acids with sulfur-containing side chains (e.g. cysteine or methionine), charged amino acids (e.g. arginine, lysine, aspartic acid, or glutamic acid), uncharged amino acids (e.g. serine, threonine, asparagine, or glutamine), aromatic amino acids (e.g. tyrosine, tryptophan, or phenylalanine), cyclic amino acids (e.g. proline), acidic amino acids (e.g. aspartic acid, asparagine, glutamic acid, or glutamine), basic amino acids (e.g. histidine, lysine, or arginine), and bulky amino acids (e.g. phenylalanine, tyrosine, or tryptophan).

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

Half-life extension domains.

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

In some embodiments, the masked cytokine provided herein comprises 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 available in the art for increasing the half-life of the masked cytokine may also be used. The half-life extension domain comprises an amino terminus and a carboxy terminus.

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

In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the first half-life extension domain is linked to the second half-life extension domain. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the first

- 62 049880 the half-life extension domain is linked to the second half-life extension domain via a linker. The first half-life extension domain and the second half-life extension domain that are linked may, in some embodiments, be any half-life extension domain described herein. For example, in some embodiments, the first half-life extension domain and/or the second half-life extension domain of the linked first and second half-life extension domains is an Fc domain or a fragment thereof. In some embodiments, the first half-life extension domain and/or the second half-life extension domain of the linked first and second half-life extension domains is an antibody or a fragment, variant, or derivative thereof.

1. Antibodies and their fragments.

By binding the masked cytokine to an antibody or fragment thereof that is capable of FcRn-mediated recycling, the clearance of the masked cytokine from the subject's body can be reduced or otherwise slowed, thereby prolonging the half-life of the administered masked cytokine.

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 of the antibodies or fragments 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 should be understood that a reference to an antibody or a fragment thereof or a given antibody or a fragment thereof may refer to an antibody or a fragment thereof of a half-life extension domain in a masked cytokine comprising one half-life extension domain, or it may refer to an antibody or a fragment thereof of a first half-life extension domain in a masked cytokine comprising a first half-life extension domain and a second half-life extension domain, or it may refer to an antibody or a fragment thereof of a second half-life extension domain in a masked cytokine comprising a first half-life extension domain and a second half-life extension domain, or it may refer to an antibody or a fragment thereof of a first half-life extension domain and an antibody or a fragment thereof of a second half-life extension domain in a masked cytokine comprising a first half-life extension domain and a second half-life extension domain.

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

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

- 63 049880 In some embodiments, the antibody or fragment thereof comprises a light chain polypeptide.

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.

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.

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.

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 an amino acid sequence SEQ ID NO: 154. In some embodiments, the Fc domain or fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 154. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the Fc domain or fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 155. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156. In some embodiments, the Fc domain or fragment thereof comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 265. In some embodiments, the Fc domain or fragment thereof comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to with the amino acid sequence of SEQ ID NO: 265. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the Fc domain or fragment thereof comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 155. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 772, or comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 772. In some embodiments, the Fc domain

- 64 049880 or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 773, or comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 773. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 774, or comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 774.

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

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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 155, and the second half-life-extending domain comprises an Fc domain or 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 156, and the second half-life-extending domain comprises an Fc domain or 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 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 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 fragment thereof comprising an amino acid sequence a sequence having about or at least about 85, 86, 87, 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 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.

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

- 65 049 880 comprising a first half-life extension domain and a second half-life extension domain, the first half-life extension domain comprising an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 616, and the second half-life extension domain comprising an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 155. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the first half-life extension domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 155, and the second half-life extension domain half-life extension domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 616. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the first half-life extension domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 616, and the second the half-life extension domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 156, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 265. 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 265, and the second half-life-extending domain comprises an Fc domain or 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 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: 265, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to 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 fragment thereof comprising an amino acid sequence a sequence having about or at least about 85, 86, 87, 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 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: 265.

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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 156, and the second half-life-extending domain comprises an Fc domain or 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 155, and the second half-life-extending domain comprises an Fc domain or 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 fragment thereof comprising an amino acid sequence having approximately or at least approximately 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98

- 66 049 880 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 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 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 extension domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 721, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 619. 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 619, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 721. 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 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: 619, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 721. 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 fragment thereof comprising an amino acid sequence a sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 721, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 619.

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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 721, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 772. 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 772, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 721. 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 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: 772, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 721. 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 fragment thereof comprising an amino acid sequence a sequence having approximately or at least

- 67 049880 approximately 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 721, and the second half-life extension domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having approximately or at least approximately 85, 86, 87, 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 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 793, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 622. 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 622, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 793. 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 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: 622, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 793. 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 fragment thereof comprising an amino acid sequence a sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 793, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 622.

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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 793, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 773. 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 773, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 793. 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 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: 773, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 793. 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 fragment thereof comprising an amino acid sequence a sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 793, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 773.

In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the first half-life extension domain comprises

- 68 049880

An Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 796, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 625. 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 625, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 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 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: 625, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 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 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: 796, and the second half-life extension domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 625.

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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 796, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 774. 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 774, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 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 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: 774, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 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 fragment thereof comprising an amino acid sequence a sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 796, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 774.

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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 793, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 773. 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 774, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 793. In some embodiments comprising a first half-life-extending domain

- 69 049880 half-life and a second half-life extending domain, the first half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 773, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 793. 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 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: 793, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 773.

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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 796, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 774. 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 774, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 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 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: 774, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 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 fragment thereof comprising an amino acid sequence a sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 796, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 774.

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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 156, and the second half-life-extending domain comprises an Fc domain or 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 155, and the second half-life-extending domain comprises an Fc domain or 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 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 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,

- 70 049 880 comprising a first half-life extension domain and a second half-life extension domain, the first half-life extension domain comprising an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 156, and the second half-life extension domain comprising an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 772, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 721. 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 721, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 772. 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 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: 721, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 772. 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 fragment thereof comprising an amino acid sequence a sequence having about or at least about 85, 86, 87, 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, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 721.

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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 156, and the second half-life-extending domain comprises an Fc domain or 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 156, and the second half-life-extending domain comprises an Fc domain or 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 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 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 fragment thereof comprising an amino acid sequence a sequence having about or at least about 85, 86, 87, 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 extension domain comprises an Fc domain or fragment thereof comprising an amino acid ...

- 71 049880 sequence 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 157, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 158. 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 158, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 157. 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 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: 157, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 158. 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 fragment thereof comprising an amino acid sequence a sequence having about or at least about 85, 86, 87, 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, and the second half-life extension domain comprises an Fc domain or fragment thereof comprising an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 157.

In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 168. 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: 168. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 169. 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: 169.

In some embodiments, the half-life extension domain comprises an amino acid sequence obtained by introducing one or more modifications into the amino acid sequence of the half-life extension domain, such as by introducing one or more amino acid substitutions, additions, or deletions into the amino acid sequence of any of SEQ ID NOs: 154-158, 168-170, 265, 616, 619, 622, 625, 721, 772-774, 793, and 796, or any of the amino acid sequences of the half-life extension domain. The one or more modifications can be any modifications or alterations described herein, including, in some embodiments, any modifications or alterations disclosed herein that promote heterodimerization of polypeptide chains and/or suppress homodimerization of polypeptide chains, alter effector function, or enhance effector function.

In some embodiments, the half-life extending domain comprises a heavy chain polypeptide that comprises one or more amino acid substitutions that alter effector function. In some embodiments, the half-life extending domain is an IgG1 heavy chain polypeptide and comprises the amino acid substitution(s): N297A, N297G, or N297Q; L234A and L235A; C220S, C226S, C229S, and P238S; C226S, C229S, E233P, L234V, and L235A; L234F, L235E, and P331S; S267E and L328F; D265A; and/or L234A, L235A, and P329G, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG1 heavy chain polypeptide and comprises one or more amino acid substitutions selected from the group consisting of N297A, N297G, N297Q, L234A, L235A, C220S, C226S, C229S, P238S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, D265A, and P329G, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG2 heavy chain polypeptide and comprises an amino acid substitution

- 72 049880 (amino acid) substitution(s): V234A and G237A; H268Q, V309L, A330S and A331S; or V234A, G237A, P238S, H268A, V309L and A330S, numbered(s) according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG2 heavy chain polypeptide and comprises one or more amino acid substitutions selected from the group consisting of V234A, G237A, H268Q, V309L, A330S, A331S, P238S, H268A and V309L, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG4 heavy chain polypeptide and comprises the amino acid substitutions: L235A, G237A, and E318A; S228P, L234A, and L235A; H268Q, V309L, A330S, and P331S; and/or S228P and L235A, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG2 heavy chain polypeptide and comprises one or more amino acid substitutions selected from the group consisting of L235A, G237A, E318A, S228P, L234A, H268Q, V309L, A330S, and P331S, numbered according to the Kabat EU numbering system.

In some embodiments, the half-life extending domain comprises a heavy chain polypeptide that comprises one or more amino acid substitutions that enhance effector function. In some embodiments, the half-life extending domain is an IgG1 heavy chain polypeptide and comprises the amino acid substitution(s): S298A, E333A, and K334A; S239D and I332E; S239D, A330L, and I332E; P247I and A339D or A339Q; D280H and K290S; D280H, K290S, and either S298D or S298V; F243L, R292P, and Y300L; F243L, R292P, Y300L, and P396L; F243L, R292P, Y300L, V305I and P396L; G236A, S239D and I332E; K326A and E333A; K326W and E333S; K290E, S298G and T299A; K290E, S298G, T299A and K326E; K290N, S298G and T299A; K290N, S298G, T299A and K326E; K334V; L235S, S239D and K334V; K334V and Q331M, S239D, F243V, E294L or S298T; E233L, Q311M and K334V; L234I, Q311M and K334V; K334V and S298T, A330M or A330F; K334V, Q311M and either A330M or A330F; K334V, S298T and either A330M or A330F; K334V, S239D and either A330M or S298T; L234Y, Y296W and K290Y, F243V or E294L; Y296W and either L234Y or K290Y; S239D, A330S and I332E, V264I; F243L and V264I; L328M; I332E; L328M and I332E; V264I and I332E; S239E and I332E; S239Q and I332E; S239E; A330Y; I332D; L328I and I332E; L328Q and I332E; V264T; V240I; V266I; S239D; S239D and I332D; S239D and I332N; S239D and I332Q; S239E and I332D; S239E and I332N; S239E and I332Q; S239N and I332D; S239N and I332E; S239Q and I332D; A330Y and I332E; V264I, A330Y and I332E; A330L and I332E; V264I, A330L and I332E; L234E, L234Y or L234I; L235D, L235S, L235Y or L235I; S239T; V240M; V264Y; A330I; N325T; I332E and L328D, L328V, L328T or L328I; V264I, I332E and either S239E or S239Q; S239E, V264I, A330Y and I332E; A330Y, I332E and either S239D or S239N; A330L, I332E and either S239D or S239N; V264I, S298A and I332E; S298A, I332E and either S239D or S239N; S239D, V264I and I332E; S239D, V264I, S298A and I332E; S239D, V264I, A330L and I332E; S239D, I332E and A330I; P230A; P230A, E233D and I332E; E272Y; K274T, K274E, K274R, K274L, or K274Y; F275W; N276L; Y278T; V302I; E318R; S324D, S324I, or S324V; K326I or K326T; T335D, T335R or T335Y; V240I and V266I; S239D, A330Y, I332E and L234I; S239D, A330Y, I332E and L235D; S239D, A330Y, I332E and V240I; S239D, A330Y, I332E and V264T; and/or S239D, A330Y, I332E and either K326E or K326T, numbered in accordance with the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG1 heavy chain polypeptide and comprises one or more amino acid substitutions selected from the group consisting of: P230A, E233D, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q, S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q, L328D, L328V, L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, T335D, T335R and T335Y.

In some embodiments, the half-life extension domain comprises an Fc domain or fragment thereof that comprises one or more amino acid substitutions that alter effector function. In some embodiments, the half-life extension domain is an IgG1 Fc domain or fragment thereof and comprises the amino acid substitution(s): N297A, N297G, or N297Q; L234A and L235A; C220S, C226S, C229S, and P238S; C226S, C229S, E233P, L234V, and L235A; L234F, L235E, and P331S; S267E and L328F; D265A; and/or L234A, L235A and P329G, numbered(s) according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG1 Fc domain or a fragment thereof and comprises one or more amino acid substitutions selected from the group consisting of N297A, N297G, N297Q, L234A, L235A, C220S, C226S, C229S, P238S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, D265A and P329G, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG2 Fc domain or a fragment thereof and comprises the amino acid substitution(s): V234A and G237A; H268Q, V309L, A330S and A331S; and/or V234A, G237A, P238S, H268A, V309L and A330S, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG2 Fc domain or a fragment thereof and comprises one or more amino acid substitutions selected from the group consisting of

- 73 049 880 of V234A, G237A, H268Q, V309L, A330S, A331S, P238S, H268A and V309L, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG4 Fc domain or a fragment thereof and comprises amino acid substitution(s): L235A, G237A and E318A; S228P, L234A and L235A; H268Q, V309L, A330S and P331S; and/or S228P and L235A, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG2 Fc domain or a fragment thereof and comprises one or more amino acid substitutions selected from the group consisting of L235A, G237A, E318A, S228P, L234A, H268Q, V309L, A330S, and P331S, numbered according to the Kabat EU numbering system.

In some embodiments, the half-life extending domain comprises an Fc domain or fragment thereof that comprises one or more amino acid substitutions that enhance effector function. In some embodiments, the half-life extending domain is an IgG1 Fc domain or fragment thereof and comprises the amino acid substitution(s): S298A, E333A, and K334A; S239D and I332E; S239D, A330L, and I332E; P247I and A339D or A339Q; D280H and K290S; D280H, K290S, and either S298D or S298V; F243L, R292P, and Y300L; F243L, R292P, Y300L, and P396L; F243L, R292P, Y300L, V305I and P396L; G236A, S239D and I332E; K326A and E333A; K326W and E333S; K290E, S298G and T299A; K290E, S298G, T299A and K326E; K290N, S298G and T299A; K290N, S298G, T299A and K326E; K334V; L235S, S239D and K334V; K334V and Q331M, S239D, F243V, E294L or S298T; E233L, Q311M and K334V; L234I, Q311M and K334V; K334V and S298T, A330M or A330F; K334V, Q311M and either A330M or A330F; K334V, S298T and either A330M or A330F; K334V, S239D and either A330M or S298T; L234Y, Y296W and K290Y, F243V or E294L; Y296W and either L234Y or K290Y; S239D, A330S and I332E, V264I; F243L and V264I; L328M; I332E; L328M and I332E; V264I and I332E; S239E and I332E; S239Q and I332E; S239E; A330Y; I332D; L328I and I332E; L328Q and I332E; V264T; V240I; V266I; S239D; S239D and I332D; S239D and I332N; S239D and I332Q; S239E and I332D; S239E and I332N; S239E and I332Q; S239N and I332D; S239N and I332E; S239Q and I332D; A330Y and I332E; V264I, A330Y and I332E; A330L and I332E; V264I, A330L and I332E; L234E, L234Y or L234I; L235D, L235S, L235Y or L235I; S239T; V240M; V264Y; A330I; N325T; I332E and L328D, L328V, L328T or L328I; V264I, I332E and either S239E or S239Q; S239E, V264I, A330Y and I332E; A330Y, I332E and either S239D or S239N; A330L, I332E and either S239D or S239N; V264I, S298A and I332E; S298A, I332E and either S239D or S239N; S239D, V264I and I332E; S239D, V264I, S298A and I332E; S239D, V264I, A330L and I332E; S239D, I332E and A330I; P230A; P230A, E233D and I332E; E272Y; K274T, K274E, K274R, K274L, or K274Y; F275W; N276L; Y278T; V302I; E318R; S324D, S324I, or S324V; K326I or K326T; T335D, T335R or T335Y; V240I and V266I; S239D, A330Y, I332E and L234I; S239D, A330Y, I332E and L235D; S239D, A330Y, I332E and V240I; S239D, A330Y, I332E and V264T; and/or S239D, A330Y, I332E and either K326E or K326T, numbered in accordance with the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG1 Fc domain or fragment thereof and comprises one or more amino acid substitutions selected from the group consisting of: P230A, E233D, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q, S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q, L328D, L328V, L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, T335D, T335R and T335Y.

In some embodiments, the half-life extension domain comprises one or more amino acid substitutions that enhance binding of the half-life extension domain to FcRn. In some embodiments, the one or more amino acid substitutions enhance the binding affinity of an Fc-containing polypeptide (e.g., a heavy chain polypeptide or an Fc domain or fragment thereof) to FcRn at acidic pH. In some embodiments, the half-life extension domain comprises one or more amino acid substitutions selected from the group consisting of M428L; T250Q and M428L; M252Y, S254T, and T256E; P257I and N434H; D376V and N434H; P257I and Q3111; N434A; N434W; M428L and N434S; V259I and V308F; M252Y, S254T and T256E; V259I, V308F and M428L; T307Q and N434A; T307Q and N434S; T307Q, E380A and N434A; V308P and N434A; N434H; and V308P.

In some embodiments, the masked cytokine is a dimer that is formed by the half-life extension domain of one copy of the masked cytokine forming a disulfide bond with the corresponding half-life extension domain of a second copy of the masked cytokine.

2. Albumin.

Albumin (also referred to herein as human serum albumin (HSA)) is a natural carrier protein that has an extended serum half-life of approximately three weeks due to its size and its susceptibility to FcRn-mediated recycling, which prevents intracellular degradation. Thus, binding of a masked cytokine to albumin can significantly increase the half-life of the masked cytokine. This approach has been used to increase the half-life of

- 74,049,880 therapeutically useful proteins in blood plasma. See, for example, WO 2001/079271A1 and WO 2003/59934A2, the contents of which are incorporated herein by reference. HSA in its mature form is a 585 amino acid polypeptide as shown in SEQ ID NO: 171.

In some embodiments, the masked cytokine comprises a half-life extension domain that comprises an albumin polypeptide or a fragment or variant thereof (hereinafter referred to as albumin or albumin polypeptide). As used herein, the terms albumin and albumin polypeptide include fragments of albumin as well as variants of albumin. The albumin polypeptide comprises an amino terminus and a carboxy terminus. The albumin polypeptide can be any albumin polypeptide, including any fragment or variant thereof, such as any albumin polypeptide described in WO 2001/079271A1; WO 2003/59934A2; US20160152686A1; WO 2012/059486; WO 2011/124718; US20070048282, the contents of which are incorporated herein by reference. In some embodiments, the albumin polypeptide is HSA.

In some embodiments of the masked cytokine, the half-life extension domain comprises an albumin polypeptide. In some embodiments, the masked cytokine comprises more than one albumin polypeptide, each of which can be any of the albumin polypeptides 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 albumin polypeptide. It should be understood that a reference to an albumin polypeptide or a given albumin polypeptide may refer to an albumin polypeptide of a half-life extension domain in a masked cytokine comprising one half-life extension domain, or it may refer to an albumin polypeptide of a first half-life extension domain in a masked cytokine comprising a first half-life extension domain and a second half-life extension domain, or it may refer to an albumin polypeptide of a second half-life extension domain in a masked cytokine comprising a first half-life extension domain and a second half-life extension domain, or it may refer to an albumin polypeptide of a first half-life extension domain and an albumin polypeptide of a second half-life extension domain in a masked cytokine comprising a first half-life extension domain and a second half-life extension domain.

In some embodiments, the albumin polypeptide is linked to a masking moiety. In some embodiments, the masking moiety is linked to the amino terminus or carboxy terminus of the albumin polypeptide. In some embodiments, the albumin polypeptide is linked to the masking moiety via a linker. In some embodiments, the linker is linked to the amino terminus or carboxy terminus of the albumin polypeptide. In some embodiments, the N-terminal spacer domain or the C-terminal spacer domain of the linker is linked to the amino terminus or carboxy terminus of the albumin polypeptide. In some embodiments, the cleavable peptide of the linker is linked to the amino terminus or carboxy terminus of the albumin polypeptide. In some embodiments, the albumin polypeptide is linked to a cytokine or a functional fragment thereof. In some embodiments, the cytokine or a functional fragment thereof is linked to the amino terminus or carboxy terminus of the albumin polypeptide. In some embodiments, the albumin polypeptide is linked to the cytokine or functional fragment thereof via a linker. In some embodiments, the linker is linked to the amino terminus or carboxy terminus of the albumin polypeptide.

In some embodiments, the albumin polypeptide comprises the amino acid sequence of SEQ ID NO: 171. In some embodiments, the albumin 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: 171.

In some embodiments, the albumin polypeptide is an albumin variant consisting of an HSA fragment (optionally with one or more amino acid modifications). In some embodiments, the HSA fragment is a wild-type HSA fragment. In some embodiments, the albumin polypeptide comprises HSA domain 1 (amino acid residues 1-194 of SEQ ID NO: 171), HSA domain 2 (amino acid residues 195-387 of SEQ ID NO: 171), HSA domain 3 (amino acid residues 388-585), HSA domains 1 and 2, HSA domains 2 and 3, HSA domains 1 and 3, or two copies of HSA domain 3. In some embodiments, the albumin polypeptide consists of HSA domain 1 (amino acid residues 1-194 of SEQ ID NO: 171), HSA domain 2 (amino acid residues 195-387 of SEQ ID NO: 171), HSA domain 3 (amino acid residues 388-585), HSA domains 1 and 2, HSA domains 2 and 3, HSA domains 1 and 3, or two copies of HSA domain 3.

In some embodiments, the albumin polypeptide comprises amino acid residues 1-194 of SEQ ID NO: 171. In some embodiments, the albumin polypeptide comprises amino acid residues 195-387 of SEQ ID NO: 171. In some embodiments, the polypeptide

- 75 049880 albumin comprises amino acid residues 388-585 of SEQ ID NO: 171. In some embodiments, the albumin polypeptide comprises amino acid residues 1-387 of SEQ ID NO: 171. In some embodiments, the albumin polypeptide comprises amino acid residues 195-585 of SEQ ID NO: 171. In some embodiments, the albumin polypeptide comprises amino acid residues 1-105 of SEQ ID NO: 171. In some embodiments, the albumin polypeptide comprises amino acid residues 120-194 of SEQ ID NO: 171. In some embodiments, the albumin polypeptide comprises amino acid residues 195-291 of SEQ ID NO: 171. In some embodiments, the albumin polypeptide comprises amino acid residues 316-387 of SEQ ID NO: 171. In some embodiments, the albumin polypeptide comprises amino acid residues 388-491 of SEQ ID NO: 171. In some embodiments, the albumin polypeptide comprises amino acid residues 512-585 of SEQ ID NO: 171. In some embodiments, the albumin polypeptide comprises amino acid residues 1-194 and 388-585 of SEQ ID NO: 171. In some embodiments, the albumin polypeptide comprises two copies of amino acid residues 388-585 of SEQ ID NO: 171.

In some embodiments, the albumin polypeptide comprises an amino acid sequence formed by one or more amino acid modifications of the amino acid sequence of any of the embodiments of the albumin polypeptide described herein. In some embodiments, the albumin polypeptide comprises an amino acid sequence comprising one or more amino acid modifications that increase the serum half-life of the albumin polypeptide. Exemplary amino acid modifications that increase the serum half-life of the albumin polypeptide include amino acid substitutions E492, N503, D550, and/or K573 of HSA. In some embodiments, the albumin polypeptide comprises one or more amino acid modifications that increase the affinity of the albumin polypeptide for an FcRn receptor. Illustrative amino acid modifications that enhance the affinity of the albumin polypeptide for the FcRn receptor include V418M, T420A, E505G, and V547A. In some embodiments, the albumin polypeptide comprises an amino acid sequence formed by one or more amino acid modifications of the amino acid sequence of SEQ ID NO: 171. In some embodiments, the albumin polypeptide comprises an amino acid sequence formed by one or more amino acid modifications of the amino acid sequence of SEQ ID NO: 171. In some embodiments, the one or more amino acid modifications of the amino acid sequence of any of the albumin polypeptides described herein is selected from the group consisting of Q417A, H440Q, H464Q, A490D, E492G/T/P/H, V493P/L, D494N/Q/A/E/P, E495Q/A, T496A, P499A, K500E/G/D/A/S/C/P/H/F/N/W/T/M/Y/V/Q/L/I/R, E501A/P/Q, N503K/D/H, A504E, E505K/D, T506F/S, H510Q, H535Q, K536A, P537A, K538A/H, T540S, K541A/D/G/N/E, E542P/D, D550N, K573Y/W/P/H/F/V/I/T/N/S/G/M/C/A/E/Q/R/L/D, K574N, Q580K, L575F, A577T/E, A578R/S, S579C/T, Q580K, A581D, A582T and G584A. In some embodiments, one or more amino acid modifications of the amino acid sequence of any of the albumin polypeptides described herein are selected from the group consisting of V418M, T420A, E505G, and V547A.

3. Binding proteins.

Additional strategies for increasing the serum half-life of masked cytokines include binding of the masked cytokine to specific binding proteins, such as albumin-binding proteins or IgG-binding proteins. Binding proteins can be any protein that binds to a serum protein that has a prolonged half-life, such as albumin or IgG. Albumin and IgG are polypeptides that are known to have a long half-life in serum. Because albumin-binding proteins bind or otherwise associate with albumin in serum, masked cytokines that are bound to an albumin-binding protein may exhibit an increased serum half-life. Similarly, because IgG-binding proteins bind or otherwise associate with IgG in serum, masked cytokines that are bound to IgG-binding protein may exhibit an increased half-life in serum.

Albumin binding proteins and methods for linking them to proteins of interest are described, for example, in WO 1991/01743, WO 2001/45746, WO 2002/076489, WO 2004/041865 or US 20070269422 A1, the contents of which are incorporated herein by reference.

In some embodiments, the half-life extension domain comprises an albumin binding protein. The albumin binding protein can be any of the albumin binding proteins described in, for example, WO 1991/01743, WO 2001/45746, WO 2002/076489, WO 2004/041865, US 20070269422 A1; US 20160152686 A1; Dennis et al. (2002), JBC 277(38):35035-35043.

- 76 049880

In some embodiments, the masked cytokine, the half-life extension domain comprises an albumin binding protein. In some embodiments, the masked cytokine comprises more than one albumin binding protein, each of which can be any of the albumin binding proteins 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 albumin binding protein. It should be understood that the reference to the albumin binding protein or this albumin binding protein may refer to the albumin binding protein of the half-life extension domain in the masked cytokine comprising one half-life extension domain, or it may refer to the albumin binding protein of the first half-life extension domain in the masked cytokine comprising a first half-life extension domain and a second half-life extension domain, or it may refer to the albumin binding protein of the second half-life extension domain in the masked cytokine comprising a first half-life extension domain and a second half-life extension domain, or it may refer to the albumin binding protein of the first half-life extension domain and the albumin binding protein of the second half-life extension domain in the masked cytokine comprising a first half-life extension domain and the second half-life extension domain.

In some embodiments, the albumin binding protein comprises the albumin binding domain (ABD) of streptococcal protein G (SPG). See, e.g., Nygren et al., J. Mol. Recogn. (1988) 1(2): 69-74. In some embodiments, the albumin binding protein comprises the ABD of SPG strain G148. In some embodiments, the albumin binding protein comprises the C-terminal albumin binding domain 3 (ABD3) of SPG strain G148. See, e.g., Nilvebrant and Hober (2013), Comput. Struct. Biotechol. J., 6: e201303009. In some embodiments, the albumin binding protein comprises the amino acid sequence of SEQ ID NO: 172. In some embodiments, the albumin binding protein comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 172.

In some embodiments, the albumin binding protein comprises a natural or synthetic peptide. In some embodiments, the albumin binding protein comprises the amino acid sequence of SEQ ID NO: 173 or 174. In some embodiments, the albumin binding protein comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 173 or 174.

In some embodiments, the albumin binding domain comprises an amino acid sequence obtained by introducing one or more modifications into the amino acid sequence of the albumin binding domain, such as by introducing one or more amino acid substitutions, additions, or deletions into the amino acid sequence of any of SEQ ID NOs: 172-174.

In some embodiments, the albumin binding protein is a single domain antibody or fragment thereof, such as a nanobody, that binds to or otherwise associates with albumin. See, for example, WO 2004041865 A2 and US 20070269422 A1, the contents of which are incorporated herein by reference. In some embodiments, the single domain antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 252-259. In some embodiments, a single domain antibody or fragment thereof comprises an amino acid sequence having about or at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 252-259.

In some embodiments, the albumin binding protein is linked to a masking moiety. In some embodiments, the masking moiety is linked to the amino terminus or carboxy terminus of the albumin binding protein. In some embodiments, the albumin binding protein is linked to the masking moiety via a linker. In some embodiments, the linker is linked to the amino terminus or carboxy terminus of the albumin binding protein. In some embodiments, the N-terminal spacer domain or the C-terminal spacer domain of the linker is linked to the amino terminus or carboxy terminus of the albumin binding protein. In some embodiments, the cleavable peptide of the linker is linked to the amino terminus or carboxy terminus of the albumin binding protein. In some embodiments, the albumin binding protein is linked to a cytokine or a functional fragment thereof. In some embodiments, the cytokine or functional fragment thereof is linked to the amino terminus or carboxy terminus of the albumin binding protein. In some embodiments, the albumin binding protein is linked to the cytokine or functional fragment thereof via a linker. In some embodiments,

- 77 049880 the linker is linked to the amino terminus or carboxyl terminus of the albumin binding protein.

Another example of a binding protein is IgG binding protein. IgG binding proteins have been reported. For a review of IgG binding proteins, including specific IgG binding proteins, and their uses, see, e.g., Choe et al. (2016) Materials 9(12):994, the contents of which are incorporated herein by reference.

In some embodiments of the masked cytokine, the half-life extension domain comprises an IgG binding protein. In some embodiments, the masked cytokine comprises more than one IgG binding protein, each of which can be any of the IgG binding proteins 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 IgG binding protein. It should be understood that reference to an IgG binding protein or a given IgG binding protein may refer to an IgG binding protein of a half-life extension domain in a masked cytokine comprising one half-life extension domain, or it may refer to an IgG binding protein of a first half-life extension domain in a masked cytokine comprising a first half-life extension domain and a second half-life extension domain, or it may refer to an IgG binding protein of a second half-life extension domain in a masked cytokine comprising a first half-life extension domain and a second half-life extension domain, or it may refer to an IgG binding protein of a first half-life extension domain and an IgG binding protein of a second half-life extension domain in a masked cytokine comprising a first half-life extension domain and second half-life extension domain.

In some embodiments, the half-life extension domain comprises an IgG binding protein. The IgG binding protein can be any IgG binding protein. The IgG binding protein can be any IgG binding protein described in, e.g., Choe et al. (2016) Materials 9(12): 994; US 20140046037 A1. For example, various bacterial proteins have been demonstrated to bind to mammalian IgG, including protein A, G, L, and Z, as well as fusion proteins of the LG and LA proteins. See, e.g., Choe et al. (2016) Materials 9(12): 994. In some embodiments, the IgG binding protein is the staphylococcal protein A (SpA) protein from the bacterium Staphylococcus aureus, or a fragment or variant thereof. In some embodiments, the IgG binding domain is the SpA IgG binding domain or a fragment or variant thereof. In some embodiments, the IgG binding domain is one or more of the SpA IgG binding domains E, D, A, B, C, and Z, or fragments or variants thereof.

In some embodiments, the IgG binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 175-186. In some embodiments, the IgG binding protein comprises an amino acid sequence having about or at least about 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 of SEQ ID NOs: 175-186.

In some embodiments, the IgG binding domain comprises an amino acid sequence obtained by introducing one or more modifications into the amino acid sequence of the IgG binding domain, such as by introducing one or more amino acid substitutions, additions, or deletions into the amino acid sequence of any of SEQ ID NOs: 175-186.

In some embodiments, the IgG binding protein is linked to a masking moiety. In some embodiments, the masking moiety is linked to the amino terminus or carboxy terminus of the IgG binding protein. In some embodiments, the IgG binding protein is linked to the masking moiety via a linker. In some embodiments, the linker is linked to the amino terminus or carboxy terminus of the IgG binding protein. In some embodiments, the N-terminal spacer domain or the C-terminal spacer domain of the linker is linked to the amino terminus or carboxy terminus of the IgG binding protein. In some embodiments, the cleavable peptide of the linker is linked to the amino terminus or carboxy terminus of the IgG binding protein. In some embodiments, the IgG binding protein is linked to a cytokine or a functional fragment thereof. In some embodiments, the cytokine or functional fragment thereof is linked to the amino terminus or carboxy terminus of the IgG binding protein. In some embodiments, the IgG binding protein is linked to the cytokine or functional fragment thereof via a linker. In some embodiments, the linker is linked to the amino terminus or carboxy terminus of the IgG binding protein.

4. Antibody derivatives.

The masked cytokines described herein may alternatively be linked to various antibody derivatives, including, but not limited to, scFv, scFc, dual

- 78 049880 variable domain (DVD) and antibody derivatives based on the CrossMab approach. See, for example, Klein et al. (2012), MAbs, 4(6): 653-663; US 20070071675 A1. Antibody derivatives include antibody derivatives engineered as bispecific antibodies or fragments thereof. Thus, in some embodiments, the half-life extension domain may comprise any antibody derivative, variant or fusion product thereof, including, but not limited to, scFv, scFc, dual variable domain (DVD), antibody derivatives based on the CrossMab approach and bispecific antibodies or fragments thereof.

5. Polyamino acid sequences.

An additional strategy for increasing the serum half-life of masked cytokines is to bind the masked cytokine to a polyamino acid sequence. Thus, in some embodiments, the half-life extension domain comprises a polyamino acid sequence. The polyamino acid sequence may be any polyamino acid sequence capable of increasing the serum half-life of the masked cytokine when bound to the masked cytokine. Examples of polyamino acid sequences include PAS polypeptides and XTEN polypeptides.

In some embodiments, the polyamino acid sequence is a polypeptide chain comprising small amino acid residues of proline, alanine, and optionally serine (PAS polypeptide). PAS sequences are hydrophilic and uncharged polymers that have biophysical properties similar to those of polyethylene glycol (PEG), which is commonly linked to therapeutic proteins as a strategy to increase the half-life of the therapeutic protein in vivo. See, e.g., Schlapschy et al. (2013) Protein Eng. Des. Sel., 26(8): 489-501; WO 2008155134 A1; WO 2011144756 A1; WO 2016130451 A1, the contents of which are incorporated herein by reference. It has been found that attachment of a PAS polypeptide to a therapeutic protein increases stability in vivo and/or in vitro compared to the therapeutic protein in its native state without the PAS polypeptide attached. In some embodiments, the PAS polypeptide adopts a random coil conformation. In some embodiments, the PAS polypeptide forms a random coil conformation domain.

In some embodiments, the half-life extension domain comprises a PAS polypeptide that comprises the amino acid residues proline and alanine. In some embodiments, the half-life extension domain comprises a PAS polypeptide that comprises the amino acid residues proline, alanine, and serine. In some embodiments, the PAS polypeptide comprises at least 25 amino acid residues, at least 50 amino acid residues, at least 100 amino acid residues, at least 150 amino acid residues, at least 200 amino acid residues, at least 250 amino acid residues, at least 300 amino acid residues, at least 400 amino acid residues, at least 500 amino acid residues, at least 600 amino acid residues, at least 700 amino acid residues, at least 800 amino acid residues, at least 900 amino acid residues, at least 1000 amino acid residues, at least 1100 amino acid residues, at least 1200 amino acid residues, at least 1300 amino acid residues, at least 1500 amino acid residues, at least 2000 amino acid residues, at least 2500 amino acid residues, or at least 3000 amino acid residues. In some embodiments, the PAS polypeptide comprises at least 25 amino acid residues, at least 50 amino acid residues, at least 100 amino acid residues, at least 150 amino acid residues, at least 200 amino acid residues, at least 250 amino acid residues, at least 300 amino acid residues, at least 400 amino acid residues, at least 500 amino acid residues, at least 600 amino acid residues, at least 700 amino acid residues, at least 800 amino acid residues, at least 900 amino acid residues, at least 1000 amino acid residues, at least 1100 amino acid residues, at least 1200 amino acid residues, at least 1300 amino acid residues, at least 1500 amino acid residues, at least 2000 amino acid residues, at least 2500 amino acid residues, or at least 3000 amino acid residues, wherein each amino acid residue is proline or alanine. In some embodiments, the PAS polypeptide comprises at least 25 amino acid residues, at least 50 amino acid residues, at least 100 amino acid residues, at least 150 amino acid residues, at least 200 amino acid residues, at least 250 amino acid residues, at least 300 amino acid residues, at least 400 amino acid residues, at least 500 amino acid residues, at least 600 amino acid residues, at least 700 amino acid residues, at least 800

- 79 049 880 amino acid residues, at least 900 amino acid residues, at least 1000 amino acid residues, at least 1100 amino acid residues, at least 1200 amino acid residues, at least 1300 amino acid residues, at least 1500 amino acid residues, at least 2000 amino acid residues, at least 2500 amino acid residues or at least 3000 amino acid residues, wherein each amino acid residue is an amino acid selected from the group consisting of proline, alanine and serine.

In some embodiments, the polyamino acid sequence is an extended recombinant polypeptide (XTEN polypeptide). XTEN polypeptides are polypeptides with non-naturally occurring, substantially non-repetitive sequences that have little or no secondary or tertiary structure under physiological conditions. XTEN polypeptides typically have from about 36 to about 3,000 amino acid residues, most of which are most or all small hydrophilic amino acids (e.g., arginine, lysine, threonine, alanine, asparagine, glutamine, aspartate, glutamate, serine, and glycine). The physicochemical properties and non-structured characteristics of XTEN polypeptides are the result, in part, of the overall amino acid composition, which is disproportionately limited to 4-6 types of hydrophilic amino acids, the linking of amino acids in a non-repetitive design, and the length of the XTEN polypeptide. It has been found that the attachment of an XTEN polypeptide to a therapeutic protein improves the solubility and stability, and increases the half-life of the therapeutic protein. See, for example, Podust et al. (2016) J. Controlled Release, 240: 52-66; WO 2013130683 A2, the contents of which are incorporated herein by reference.

In some embodiments, the half-life extension domain comprises an XTEN polypeptide that comprises an amino acid sequence consisting of four, five, or six types of amino acid residues selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P).

In some embodiments, the XTEN polypeptide comprises an amino acid sequence of about 25 to about 500 amino acid residues, about 200 to about 1000 amino acid residues, about 500 to about 1500 amino acid residues, about 1000 to about 2000 amino acid residues, or about 1500 to about 3000 amino acid residues. In some embodiments, at least about 70, 75, 80, or 85% of the amino acid sequence consists of non-overlapping sequence motifs. In some embodiments, each of the motifs comprises from 5 to 100 amino acid residues, from 5 to 50 amino acid residues, or from 9 to 36 amino acid residues, and wherein at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of each of the motifs consists of four, five, or six types of amino acid residues selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P). In some embodiments, the content of any one type of amino acid in a full-length XTEN polypeptide does not exceed about 40%, about 35%, about 30%, about 25%, about 15%, about 10%, or about 8%.

In some embodiments, the XTEN polypeptide comprises an amino acid sequence of from about 25 to about 500 amino acid residues, from about 200 to about 1000 amino acid residues, from about 500 to about 1500 amino acid residues, from about 1000 to about 2000 amino acid residues, or from about 1500 to about 3000 amino acid residues, wherein at least about 80% of the amino acid sequence consists of non-overlapping sequence motifs, wherein each of the motifs has from 9 to 36 amino acid residues and wherein at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of each of the motifs consists of four, five, or six types of amino acid residues selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), and wherein the content of one type of amino acid in a full-length XTEN polypeptide does not exceed about 40%, about 35%, about 30%, about 25%, about 15%, about 10%, or about 8%.

6. PEGylation and glycosylation.

Additional strategies for increasing the half-life of masked cytokines proposed herein include PEGylation and engineering of additional glycosylation sites. Each of these strategies is discussed in more detail below.

PEGylation refers to the process of covalent or non-covalent attachment or

- 80 049880 fusions of polyethyleneglycol (PEG) polymer chains with molecules and macrostructures such as a drug, a therapeutic protein, a polypeptide, an antibody, an antibody fragment, an antibody derivative, or with any of the masked cytokines or components thereof provided herein (e.g., a half-life extension domain of a masked cytokine and/or a cytokine or a functional fragment thereof of a masked cytokine). The advantages of PEGylation include, for example, (1) markedly improved half-life in the bloodstream in vivo due to evasion of renal clearance as a result of the polymer increasing the apparent size of the molecule above the glomerular filtration limit and/or by evasion of cellular clearance mechanisms, (2) reduced antigenicity and immunogenicity of the molecule to which the PEG is attached, (3) improved pharmacokinetics, (4) improved solubility, (5) improved formulation and dosing options, (6) improved bioavailability due to reduced loss at subcutaneous injection sites, (7) improved thermal and mechanical stability of the PEGylated molecule.

Methods for PEGylation of various molecules and macrostructures are well known in the art. See, for example, US20140256636A1; Fee and Damodaran (2010) European Pharmaceutical Review, 15(1): 18-26; Chapman et al. (1999) Nature Biotechnol., 17: 780-783; Yang et al. (2003), Protein Eng., 16(10): 761-770; Chapman, Adv. Drug. Deliv. Rev. (2002), 54(4): 531-545, the contents of which are incorporated herein by reference.

In some embodiments, the masked cytokine of any of the embodiments is PEGylated. For example, in some embodiments, this is achieved by functionalizing PEG with group-specific reagents such that conjugation of PEG to a protein (e.g., a half-life extension domain, or a cytokine, or a functional fragment thereof) can target specific groups on the side chain of the protein, such as amino groups, carboxyl, sulfhydryl, or guanidino groups. In some embodiments, cysteine (C) residues are introduced by amino acid substitution into the protein (e.g., a half-life extension domain, or a cytokine, or a functional fragment thereof) in a site-specific manner. The thiol groups of the newly introduced cysteine residues can then be targeted for PEGylation using PEG reagents based on malemide chemistry. Substitution of serine (S) or threonine (T) with cysteine has the advantage that the net charge of the modified protein is not changed by PEGylation. Thus, in some embodiments, a component of a masked cytokine, such as any of the cytokines or functional fragments thereof and/or any of the half-life extension domains, can be modified or further modified by introducing cysteine residues for PEGylation.

Glycosylation refers to the addition of saccharides or glycosyl groups to a polypeptide. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine (N-X-S) and asparagine-X-threonine (N-X-T), where X is any amino acid except proline (P), are the recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide forms a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars (e.g. N-acetylgalactosamine, galactose, or xylose) to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

Naturally occurring glycosylation has been shown to enhance the molecular stability of proteins. See, e.g., Sola et al. (2007), Cell. Mol. Life Sci., 64(16): 21332152. It has also been shown that the formation of additional glycosylation sites can stabilize a variety of therapeutic proteins against most major physicochemical instabilities. See, e.g., Sola and Griebenow (2009), J. Pharm. Sci., 98(4): 1223–1245. Pharmaceutically relevant protein instabilities that have been shown to be ameliorated by glycosylation include, for example, oxidation; cross-linking; pH-, chemical-, thermal-, and cooling-induced denaturation/unfolding; precipitation; kinetic activation; and aggregation. Ibid.

The addition of glycosylation sites to a masked cytokine is typically accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences (for N-linked glycosylation sites) are formed in the amino acid sequence of the masked cytokine (e.g., in the amino acid sequence of the half-life extension domain and/or the cytokine or functional fragment thereof). The alteration can also be accomplished by adding or substituting one or more serine or threonine residues in the amino acid sequence of the masked cytokine (e.g., in the amino acid sequence of the half-life extension domain and/or the cytokine or functional fragment thereof) (for O-linked

- 81 049880 glycosylation).

In some embodiments, the masked cytokine of any of the embodiments is modified or further modified by altering the amino acid sequence of the masked cytokine (e.g., the amino acid sequence of the half-life extension domain and/or the cytokine or functional fragment thereof) such that one or more additional N-X-S and/or N-XT tripeptide sequences are introduced into the amino acid sequence of the masked cytokine or component thereof (to add one or more N-linked glycosylation sites). In some embodiments, the masked cytokine of any of the embodiments is modified or further modified by altering the amino acid sequence of the masked cytokine or component thereof (e.g., the amino acid sequence of the half-life extension domain and/or the cytokine or functional fragment thereof) such that one or more additional serine or threonine residues are introduced into the amino acid sequence of the masked cytokine or component thereof (to add one or more N-linked glycosylation sites). In some embodiments, the amino acid sequence change to add one or more N-linked glycosylation sites and/or to add one or more O-linked glycosylation sites is accomplished by adding one or more amino acids to the amino acid sequence of the masked cytokine or component thereof. In some embodiments, the amino acid sequence change to add one or more N-linked glycosylation sites and/or to add one or more O-linked glycosylation sites is accomplished by replacing one or more amino acids in the amino acid sequence of the masked cytokine or component thereof. In some embodiments, the amino acid sequence change to add one or more N-linked glycosylation sites and/or to add one or more O-linked glycosylation sites is accomplished by adding one or more amino acids to the amino acid sequence and by replacing one or more amino acids in the amino acid sequence of the masked cytokine or component thereof.

7. Modifications of heterodimerization.

The half-life extension domains may comprise one or more modifications that promote heterodimerization of two different half-life extension domains. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, it is desirable to promote heterodimerization of the first and second half-life extension domains so as to efficiently produce the masked cytokine in its proper heterodimeric form. Thus, one or more amino acid modifications may be made in the first half-life extension domain and one or more amino acid modifications may be made in the second half-life extension domain using any strategy available in the art, including any strategy described in Klein et al. (2012) MAbs, 4(6): 653-663. Exemplary strategies and modifications are described in detail below.

The peak-to-valley approach.

One strategy to promote heterodimerization of two different half-life extension domains is an approach called knobs into holes.

In some embodiments, the masked cytokine comprises a first half-life-extending domain and a second half-life-extending domain, each of which comprises a CH3 domain. In some embodiments, the half-life-extending domain comprising the CH3 domain is a heavy chain polypeptide or fragment thereof (e.g., an Fc domain or fragment thereof). The CH3 domains of the two half-life-extending domains can be altered using knob-into-hole technology, which is described in detail with respect to several examples, e.g., in WO 1996/027011; Ridgway, J.B. et al., Protein Eng. (1996) 9(7): 617-621; Merchant, A.M., et al., Nat. Biotechnol. (1998) 16(7): 677-681. See also Klein et al. (2012), MAbs, 4(6): 653–663. Using the knob-into-hole method, the interaction surfaces of two CH3 domains are altered to enhance heterodimerization of two half-life extension domains containing two altered CH3 domains. This is accomplished by introducing a bulky residue into the CH3 domain of one of the half-life extension domains, which acts as a knob. Additionally, to accommodate the bulky residue, a hole is formed in the other half-life extension domain that can accommodate the knob. One of the altered CH3 domains can be a knob, while the other can be a hole. The introduction of a disulfide bridge further stabilizes the heterodimers (Merchant, A.M., et al., Nat. Biotechnol. (1998) 16(7); Atwell, S., et al., J. Mol. Biol. (1997) 270(1): 26-35) and also increases the yield.

It has been reported that heterodimerization yields above 97% can be achieved by introducing S354C and T366W mutations into the heavy chain to form a protrusion and by introducing mutations

- 82 049880

Y349C, T366S, L368A, and Y407V into the heavy chain to form a hole (residue numbering according to the Kabat EU numbering system). Carter et al. (2001), J. Immunol. Methods, 248: 7–15; Klein et al. (2012), MAbs, 4(6): 653–663.

In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the first half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) that comprises one or more amino acid mutations that form a knob, and the second half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) that comprises one or more amino acid mutations that form a hole. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the first half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) that comprises one or more amino acid mutations that form a hole, and the second half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) that comprises one or more amino acid mutations that form a knob. Methods for introducing a hole or a knob into a heavy chain polypeptide or a portion thereof (e.g., into an Fc domain or fragment thereof) are known in the art, such as in WO 1996/027011; Ridgway, J.B. et al., Protein Eng. (1996) 9(7): 617-621; Merchant, A.M., et al., Nat. Biotechnol. (1998) 16(7): 677-681; Klein et al. (2012), MAbs, 4(6): 653-663.

In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the first half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) that comprises the amino acid mutations S354C and T366W (numbered according to the Kabat EU numbering system), and the second half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) that comprises the amino acid mutations Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU numbering system). In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the first half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) that comprises the amino acid mutations Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU numbering system), and the second half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) that comprises the amino acid mutations S354C and T366W (numbered according to the Kabat EU numbering system). 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 155, and the second half-life-extending domain comprises an Fc domain or 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 156, and the second half-life-extending domain comprises an Fc domain or 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 265, and the second half-life-extending domain comprises an Fc domain or 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 156, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 265. 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 fragment thereof comprising the amino acid sequence of SEQ ID NO: 155, and the second half-life-extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 156. In some embodiments comprising a first half-life-extending domain half-life and a second half-life extending domain, the first half-life extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 156, and the second half-life extending domain comprises an Fc domain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 155.

Further examples of substitutions that may be made to form projections and depressions include those described in US 20140302037 A1, the contents of which are incorporated herein by reference.

- 83 049 880 the present disclosure is incorporated by reference. For example, in some embodiments, any of the following amino acid substitutions can be made in a first half-life extending domain (first domain) and a paired second half-life extending domain (second domain), each of which comprises an Fc domain: (a) Y407T in the first domain and T366Y in the second domain; (b) Y407A in the first domain and T366W in the second domain; (c) F405A in the first domain and T394W in the second domain; (d) F405W in the first domain and T394S in the second domain; (e) Y407T in the first domain and T366Y in the second domain; (f) T366Y and F405A in the first domain and T394W and Y407T in the second domain; (g) T366W and F405W in the first domain and T394S and Y407A in the second domain; (h) F405W and Y407A in the first domain and T366W and T394S in the second domain; or (i) T366W in the first domain and T366S, L368A and Y407V in the second domain, numbered in accordance with the Kabat EU numbering system.

In some embodiments, any of the following amino acid substitutions may be made in a first half-life extending domain (first domain) and a paired second half-life extending domain (second domain), each comprising an Fc domain: (a) Y407T in the second domain and T366Y in the first domain; (b) Y407A in the second domain and T366W in the first domain; (c) F405A in the second domain and T394W in the first domain; (d) F405W in the second domain and T394S in the first domain; (e) Y407T in the second domain and T366Y in the first domain; (f) T366Y and F405A in the second domain and T394W and Y407T in the first domain; (g) T366W and F405W in the second domain and T394S and Y407A in the first domain; (h) F405W and Y407A in the second domain and T366W and T394S in the first domain; or (i) T366W in the second domain and T366S, L368A and Y407V in the first domain, numbered in accordance with the Kabat EU numbering system.

In embodiments comprising a first half-life extension domain and a second half-life extension domain, each comprising an Fc domain, any of the heterodimerization alterations described herein can be used in the Fc domains to promote heterodimerization of any of the masked cytokines described herein. For example, any of the heterodimerization alterations described herein, including combinations thereof, can be used to alter an amino acid sequence selected from the group consisting of SEQ ID NOs: 154-156, 158, 168, 169, 265, 616, 619, 622, 625, 721, 772-774, 793, and 796.

Ionic/electrostatic interactions.

Another strategy to promote heterodimerization of two different half-life extension domains is to stabilize ionic interactions that promote heterodimerization by altering charged residues.

In some embodiments, the masked cytokine comprises a first half-life extension domain and a second half-life extension domain, each of which comprises a CH3 domain. In some embodiments, the half-life extension domain comprising the CH3 domain is a heavy chain polypeptide or a fragment thereof (e.g., an Fc domain or a fragment thereof). It has been observed that changing the charge polarities between two different Fc domains can result in ionic interactions such that heterodimerization is favored while homodimerization is suppressed. See, e.g., WO 2006/106905A1; Gunasekaran et al. (2010) 285(25): 19637-19646. For example, it has been observed that negatively charged E356 of an Fc domain binds to positively charged K439 of another Fc domain, negatively charged E357, E357 and D399 of the first Fc domain bind to positively charged K439, K370 and K409, respectively, of the second Fc domain. See WO 2006/106905A1; Gunasekaran et al. (2010) 285(25): 19637-19646. Thus, by introducing at least two mutations E356K, E357K and D399K into the first Fc domain and mutations K370E, K409D and K439E into the second Fc domain, efficient heterodimerization can be achieved while suppressing homodimer formation. Ibid. Efficient heterodimerization was achieved by introducing the K392D and K409D mutations into the first Fc chain and by introducing the D399K and E356K mutations into the second Fc chain. Gunasekaran et al. (2010) 285(25): 19637–19646.

Additional examples of substitutions that can be made to charged amino acid pairs include those described in US20140302037A1, the contents of which are incorporated herein by reference. For example, in some embodiments, any of the following amino acid substitutions can be made in a first half-life extending domain (first domain) and a paired second half-life extending domain (second domain), each of which comprises an Fc domain: (a) K409E in the first domain and D399K in the second domain; (b) K409E in the first domain and D399R in the second domain; (c) K409D in the first domain and D399K in the second domain; (d) K409D in the first domain and D399R in the second domain; (e) K392E in the first domain and D399R in the second domain; (f) K392E in the first domain and D399K in the second domain; (g) K392D in the first domain and D399R in the second domain; (h) K392D in the first domain and D399K in the second domain; (i) K409D and K360D in the first domain and D399K and E356K in the second domain; (j) K409D and K370D in the first domain and D399K and E357K in the second domain; (k) K409D and K392D in the first domain and D399K, E356K and E357K in the second domain; (l) K409D and K392D in the first domain and D399K in the second domain; (m) K409D and K392D in the first

- 84 049880 domain and D399K and E356K in the second domain; (n) K409D and K392D in the first domain and D399K and E357K in the second domain; (o) K409D and K370D in the first domain and D399K and D357K in the second domain; (p) D399K in the first domain and K409D and K360D in the second domain; and/or (q) K409D and K439D in the first domain and D399K and E356K in the second domain, numbered in accordance with the Kabat EU numbering system.

In some embodiments, any of the following amino acid substitutions may be made in a first half-life extending domain (first domain) and a paired second half-life extending domain (second domain), each of which comprises an Fc domain: (a) K409E in the second domain and D399K in the first domain; (b) K409E in the second domain and D399R in the first domain; (c) K409D in the second domain and D399K in the first domain; (d) K409D in the second domain and D399R in the first domain; (e) K392E in the second domain and D399R in the first domain; (f) K392E in the second domain and D399K in the first domain; (g) K392D in the second domain and D399R in the first domain; (h) K392D in the second domain and D399K in the first domain; (i) K409D and K360D in the second domain and D399K and E356K in the first domain; (j) K409D and K370D in the second domain and D399K and E357K in the first domain; (k) K409D and K392D in the second domain and D399K, E356K and E357K in the first domain; (l) K409D and K392D in the second domain and D399K in the first domain; (m) K409D and K392D in the second domain and D399K and E356K in the first domain; (n) K409D and K392D in the second domain and D399K and E357K in the first domain; (o) K409D and K370D in the second domain and D399K and D357K in the first domain; (p) D399K in the second domain and K409D and K360D in the first domain; and/or (q) K409D and K439D in the second domain and D399K and E356K in the first domain, numbered in accordance with the Kabat EU numbering system.

In some embodiments, any of the following amino acid substitutions may be made in a first half-life extending domain (first domain) and a paired second half-life extending domain (second domain), each comprising an Fc domain: (a) K409D or K409E in the first domain and D399K or D399R in the second domain; (b) K392D or K392E in the first domain and D399K or D399R in the second domain; (c) K439D or K439E in the first domain and E356K or E356R in the second domain; and/or (d) K370D or K370E in the first domain and E357K or E357R in the second domain, numbered according to the Kabat EU numbering system.

In some embodiments, any of the following amino acid substitutions may be made in a first half-life extending domain (first domain) and a paired second half-life extending domain (second domain), each comprising an Fc domain: (a) K409D or K409E in the second domain and D399K or D399R in the first domain; (b) K392D or K392E in the second domain and D399K or D399R in the first domain; (c) K439D or K439E in the second domain and E356K or E356R in the first domain; and/or (d) K370D or K370E in the second domain and E357K or E357R in the first domain, numbered according to the Kabat EU numbering system.

In some embodiments comprising a first half-life extension domain and a second half-life extension domain, each comprising an Fc domain, one or more of the following substitutions may be further included in both the first half-life extension domain and the second half-life extension domain to stabilize the heterodimers: R355D, R355E, K360D, and K360R.

In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the first half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or fragment thereof) that comprises one or more mutations that promote stabilization of ionic interactions with the second half-life extension domain while suppressing homodimerization, and the second half-life extension domain comprises one or more mutations that promote stabilization of ionic interactions with the first half-life extension domain while suppressing homodimerization.

In some embodiments comprising a first half-life extension domain and a second half-life extension domain, the first half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) that comprises the amino acid mutations K392D and K409D (numbered according to the Kabat EU numbering system), and the second half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) that comprises the amino acid mutations D399K and E356K (numbered according to the Kabat EU numbering system).

In embodiments comprising a first half-life extension domain and a second half-life extension domain, each comprising an Fc domain, any of the heterodimerization alterations described herein can be used in the Fc domains to promote heterodimerization of any of the masked cytokines described herein. For example, any of the heterodimerization alterations described herein, including combinations thereof, can be used to alter an amino acid sequence selected from the group consisting of SEQ ID NOs: 154-156, 158, 168, 169, 265, 616, 619, 622, 625, 721, 772-774, 793, and 796.

Structural and sequence-based approaches.

Another strategy to promote heterodimerization of two different period-extending domains

- 85 049880 half-life involves the use of structural and sequence-based approaches to identify changes that may promote heterodimerization and/or suppress homodimerization.

Among the methods for identifying changes that promote heterodimerization is the performance of structural calculations to determine the energies of pairwise combinations of variants for residues that interact through the CH3-CH3 dimer interface, as was done in the approach taken in Moore et al. (2011) 3(6): 546–557, the contents of which are incorporated herein by reference. Moore et al. identified pairs that were predicted to have lower energy in the heterodimer form compared to the homodimer form as a starting point for further analysis. It was observed that a heterodimerization yield of 89% could be achieved by introducing the S364H and F405A mutations into the first Fc domain and by introducing the Y349T and T394F mutations into the second Fc domain. Ibid.

In some embodiments comprising a first half-life-extending domain and a second half-life-extending domain, each comprising an Fc domain, the first half-life-extending domain comprises the amino acid mutations S364H and F405A, and the second half-life-extending domain comprises the amino acid mutations Y349T and T394F, numbered according to the Kabat EU numbering system. In some embodiments comprising a first half-life-extending domain and a second half-life-extending domain, each comprising an Fc domain, the second half-life-extending domain comprises the amino acid mutations S364H and F405A, and the first half-life-extending domain comprises the amino acid mutations Y349T and T394F, numbered according to the Kabat EU numbering system.

In embodiments comprising a first half-life extension domain and a second half-life extension domain, each comprising an Fc domain, any of the heterodimerization alterations described herein can be used in the Fc domains to promote heterodimerization of any of the masked cytokines described herein. For example, any of the heterodimerization alterations described herein, including combinations thereof, can be used to alter an amino acid sequence selected from the group consisting of SEQ ID NOs: 154-156, 158, 168, 169, 265, 616, 619, 622, 625, 721, 772-774, 793, and 796.

Binding assays.

The strength or affinity of immunological binding interactions, for example, between a cytokine or a functional fragment thereof and a binding partner (e.g., a target protein, such as a cytokine receptor) for which the cytokine or functional fragment thereof is specific in terms of the dissociation constant (Kd) of the interaction can be expressed, with a lower Kd value representing a higher affinity. For example, binding of the cytokine IL-2 to the cytokine receptor IL-2R (e.g., IL-2R or a component thereof, such as IL-2Ra, IL-2Rβ, IL-2Ry, or combinations thereof) can be expressed by Kd. In some embodiments, the immunological binding interactions occur between a masked cytokine (in the presence or absence of a protease) and a target protein, such as a cytokine receptor. In the context of IL-2 cytokine binding, the target protein can be IL-2R (comprising the IL-2Rcl IL-2Rβ and IL-2Ry chains), the IL-2Rcl chain IL-2Rβ chain, or the dimeric IL-2Ra/e complex. The immunological binding properties of proteins can be quantified using methods well known in the art. For example, one method involves measuring the rates of formation and dissociation of a cytokine receptor (e.g., IL-2R)/cytokine (e.g., IL-2) complex, wherein these rates depend on the concentrations of the components of the complex, the affinity of the interaction, and geometric parameters that equally affect the rate in both directions. Both the association rate constant (K _on ) and the dissociation rate constant (K off ) can be determined by calculating the concentrations and actual rates of association and dissociation. The ratio K _o ff /K _on eliminates all parameters not related to affinity and is equal to the dissociation constant Kd. See Davies et al., Annual Rev Biochem. 59:439473, (1990).

In some aspects, a masked cytokine described herein binds to a target protein with about the same or higher affinity upon protease cleavage compared to a parent cytokine that includes a masking moiety but lacks a cleavable peptide. The target protein can be any cytokine receptor. In some embodiments, the target protein is IL-2R (comprising IL-2Ra, IL-2Re, and IL-2Ry chains). In some embodiments, the target protein is IL-2Ra. In some embodiments, the target protein is IL-2Re. In some embodiments, the target protein is a dimeric IL-2Ra/e complex. In some embodiments, the target protein is IL-15R (comprising IL-15Ra, IL-2Re, and IL-2Ry chains) or a component thereof, such as IL-15Ra, IL-2Re, or IL-2Ry, or a combination thereof.

In some embodiments, the masked cytokine provided herein

- 86 049880 document, which does not contain a cleavable peptide in the linker, has a dissociation constant for the target protein (Kd) of <1 μM, <150 nM, <100 nM, <50 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10-8 M or less, e.g., from 10-8 M to 10-13 M, e.g., from 10-9 M to 1013 M). In some embodiments, a masked cytokine provided herein that comprises a cleavable peptide in a linker has a dissociation constant for a target protein prior to protease cleavage (Kd) of <1 μM, <150 nM, <100 nM, <50 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10-8 M or less, such as from 10-8 M to 10-13 M, such as from 10-9 M to 10-13 M). In some embodiments, a masked cytokine provided herein that comprises a cleavable peptide in a linker has a dissociation constant for a target protein upon protease cleavage (Kd) of <1 μM, <150 nM, <100 nM, <50 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10-8 M or less, such as from 10-8 M to 10-13 M, such as from 10-9 M to 10-13 M). In some embodiments, the cytokine or functional fragment thereof of the masked cytokine provided herein has a dissociation constant with respect to the masked fragment of the masked peptide (Kd) of >500 μM, >250 μM, >200 μM, >150 μM, >100 μM, >50 μM, >10 μM, >1 μM, >500 nM, >250 nM, >150 nM, >100 nM, >50 nM, >10 nM, >1 nM, >0.1 nM, >0.01 nM, or >0.001 nM. In some embodiments, the cytokine or functional fragment thereof, a masked cytokine provided herein has a dissociation constant (Kd) that is from about 200 μM to about 50 nM, such as about or at least about 175 μM, about or at least about 150 μM, about or at least about 125 μM, about or at least about 100 μM, about or at least about 75 μM, about or at least about 50 μM, about or at least about 25 μM, about or at least about 5 μM, about or at least about 1 μm, about or at least about 750 nM, about or at least about 500 nM, about or at least about 250 nM, about or at least about 150 nM, about or at least about 100 nM, about or at least about 75 nM or about or at least about 50 nM. Assays for assessing binding affinity are well known in the art.

In some aspects, masked cytokines are provided that exhibit a desired occlusion ratio. The term occlusion ratio refers to the ratio of (a) the maximum detectable level of a parameter under a first set of conditions to (b) the minimum detectable value of that parameter under a second set of conditions. For example, in the context of a masked IL-2 polypeptide, the occlusion ratio refers to the ratio of (a) the maximum detectable level of binding of a target protein (e.g., an IL-2R protein) to the masked IL-2 polypeptide in the presence of at least one protease capable of cleaving a cleavable peptide of the masked IL-2 polypeptide to (b) the minimum detectable level of binding of a target protein (e.g., an IL-2R protein) to the masked IL-2 polypeptide in the absence of a protease. Thus, the occlusion ratio for a masked cytokine can be calculated by dividing the _EC50 of the masked cytokine before cleavage by the _EC50 of the masked cytokine after cleavage. The occlusion ratio of a masked cytokine can also be calculated as the ratio of the dissociation constant of the masked cytokine before protease cleavage to the dissociation constant of the masked cytokine after protease cleavage. In some embodiments, a higher occlusion ratio for a masked cytokine indicates that the target protein bound by the masked cytokine occurs to a greater extent (e.g., occurs preferentially) in the presence of a protease capable of cleaving the cleavable peptide of the masked cytokine than in the absence of a protease.

In some embodiments, the present invention provides masked cytokines with an optimal occlusion ratio. In some embodiments, the optimal occlusion ratio of the masked cytokine indicates that the masked cytokine has the necessary properties useful for the methods or compositions contemplated herein. In some embodiments, a masked cytokine provided herein exhibits an optimal occlusion ratio of from about 2 to about 10,000, such as from about 80 to about 100. In a further embodiment of any of the masked cytokines provided herein, the occlusion ratio is from about 2 to about 7,500, from about 2 to about 5,000, from about 2 to about 2,500, from about 2 to about 2,000, from about 2 to about 1,000, from about 2 to about 900, from about 2 to about 800, from about 2 to about 700, from about 2 to about 600, from about 2 to about 500, from about 2 to about 400, from about 2 to

- 87 049 880 about 300, about 2 to about 200, about 2 to about 100, about 2 to about 50, about 2 to about 25, about 2 to about 15, about 2 to about 10, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 60 to about 100, about 70 to about 100, about 80 to about 100, or about 100 to about 1000. In some embodiments, a masked cytokine provided herein exhibits an optimal occlusion ratio of about 2 to about 1000. Binding of the masked IL-2 polypeptide to the target protein before and/or after protease cleavage can be determined using techniques well known in the art, such as ELISA.

In some embodiments, a masking moiety described herein binds to a cytokine or functional fragment thereof as described herein with an affinity lower than the affinity between the cytokine or functional fragment thereof and a target protein (e.g., a cytokine receptor). In some embodiments, a masking moiety provided herein binds to a cytokine or functional fragment thereof as described herein with a dissociation constant (Kd) of >500 μM, >250 μM, >200 μM, >150 μM, >100 μM, >50 μM, >10 μM, >1 μM, >500 nM, >250 nM, >150 nM, >100 nM, >50 nM, >10 nM, >1 nM, >0.1 nM, >0.01 nM, or >0.001 nM.

Obtaining masked cytokines.

The masked cytokines described herein are prepared using techniques available in the art, exemplary methods of which are described.

Obtaining antibodies.

Some embodiments of the masked cytokine comprise an antibody or fragment thereof. The following sections provide additional information on the production of antibodies and fragments, variants and derivatives thereof that can be used in some embodiments of the masked cytokine provided herein. In some embodiments, the masked cytokine is in the form of a dimer formed by two copies of the masked cytokine that are associated via disulfide bonds.

1. Antibody fragments.

The present invention encompasses, in some embodiments, antibody fragments. Antibody fragments can be any antibody fragment, such as an Fc domain, a heavy chain portion, a light chain portion, a Fab, Fv, or scFv, among other fragments. Antibody fragments can be produced by conventional methods, such as enzymatic cleavage, or by recombinant techniques. In certain circumstances, there are advantages to linking antibody fragments, rather than whole antibodies, to the masked cytokines described herein. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

Various techniques have been developed for producing antibody fragments. Traditionally, these fragments have been produced by proteolytic cleavage of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, here, these fragments can be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed and secreted from E. coli and other cell types such as HEK293 and CHO cells, allowing the easy production of large quantities of these fragments. Alternatively, Fab'-SH fragments can be directly recovered from the culture medium and chemically coupled to form F(ab') ₂ fragments (Carter et al., Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab') ₂ fragments can be isolated directly from recombinant host cell culture. Fab and F(ab') ₂ fragments with increased in vivo half-life containing remnants of the FcRN/salvage receptor binding epitope are described in U.S. Patent No. 5,869,046. Other techniques for producing antibody fragments for use in masked cytokines will be apparent to the skilled artisan. In certain embodiments, the masked cytokine comprises a single-chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894; and 5,587,458. scFv fusion proteins can be engineered to fusion the effector protein to either the amino or carboxy terminus of the scFv. See Antibody Engineering, ed. Borrebaeck, supra. Additionally, in some embodiments, a bi-scFv comprising two scFvs linked by a polypeptide linker can be used with masked cytokines.

The present invention includes, in some embodiments, a linear antibody (e.g., as described in U.S. Patent No. 5,641,870) or a single-chain immunoglobulin comprising

- 88 049880 heavy and light chain sequences of an antibody linked by an appropriate linker. Such linear antibodies or immunoglobulins may be monospecific or bispecific. Such a single-chain immunoglobulin may be dimerized to thereby retain a structure and activity similar to those of an antibody that is initially a tetramer. Furthermore, in some embodiments, the antibody or fragment thereof may be an antibody that has one variable region of the heavy chain and does not have a light chain sequence. Such an antibody is called a single-domain antibody (sdAb) or a nanobody. These antibodies are also encompassed by the meaning of a functional fragment of an antibody according to the present invention. Antibody fragments may be linked to the masked cytokines described herein in accordance with the teachings provided herein.

2. Humanized antibodies.

In some embodiments, the present invention encompasses humanized antibodies or fragments thereof. In some embodiments, the humanized antibodies may be any antibody, including any antibody fragment. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be accomplished essentially according to the method of Winter (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by replacing the hypervariable region sequence with the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain is replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies. Humanized antibodies can be linked to the masked cytokines described herein in accordance with the guidelines provided herein.

3. Human antibodies.

Human antibodies according to some embodiments can be constructed by combining the variable domain sequence(s) of an Fv clone selected from human-derived phage display libraries with known human constant domain sequence(s). Alternatively, human monoclonal antibodies according to some embodiments can be produced by a hybridoma method, for example, using mouse, rat, bovine (e.g., cow) or rabbit cells, for example, to produce human monoclonal antibodies. In some embodiments, human antibodies and human monoclonal antibodies can be antibodies that bind to any antigen. In some embodiments, human monoclonal antibodies of the present invention can be produced by immunizing a non-human animal that contains human immunoglobulin loci with a target antigen and isolating the antibody from the immunized animal or from cells obtained from the immunized animal. Examples of suitable non-human animals include a transgenic or transchromosomal animal such as the HuMAb Mouse® (Medarex, Inc.), KM Mouse®, TC mice, and Xenomouse™. See, e.g., Lonberg, et al. (1994) Nature 368: 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851; WO 2002/43478; U.S. Patent Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584; 6,162,963; and Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727.

Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies are described, for example, by Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991). Human antibodies can be coupled to the masked cytokines described herein according to the guidelines provided herein.

4. Bispecific antibodies.

Bispecific antibodies are monoclonal antibodies that have binding specificity for at least two different antigens. In some embodiments, the bispecific antibodies are human or humanized antibodies. In some embodiments, one of the binding specificities is for a first antigen and the other binding specificity is for a second antigen, which may

- 89 049880 represent either two different epitopes on the same target protein, or two different epitopes on two different target proteins. Bispecific antibodies can also be used to localize cytotoxic agents to cells that express a first antigen and/or a second antigen. Bispecific antibodies can also be used to recruit cells, such as T cells or natural killer cells, to kill specific cells, such as cancer cells. Bispecific antibodies can be produced as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Bispecific antibodies can be linked to the masked cytokines described herein in accordance with the guidelines provided herein.

Methods for generating bispecific antibodies are known in the art. See Milstein and Cuello, Nature, 305: 537 (1983), WO 93/08829 published May 13, 1993, Traunecker et al., EMBO J., 10: 3655 (1991); Kontermann and Brinkmann, Drug Discovery Today, 20(7):838-847. For additional information on generating bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986). Bispecific antibodies include cross-linked or heteroconjugate antibodies. For example, one of the antibodies in a heteroconjugate can be linked to avidin and the other to biotin. Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980, along with a number of cross-linking techniques.

5. Single-domain antibodies.

In some embodiments, the single domain antibody is linked to a masked cytokine as described herein. The single domain antibody can be any antibody. A single domain antibody is a single polypeptide chain comprising all or a portion of a heavy chain variable domain or all or a portion of a light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass; see, e.g., U.S. Patent No. 6,248,516 B1). In some embodiments, the single domain antibody consists of all or a portion of a heavy chain variable domain of an antibody. In some embodiments, the single domain antibody is a camelid-derived antibody produced by immunizing camelids with a target antigen. In some embodiments, the single-domain antibody is a shark-derived antibody obtained by immunizing a shark with a target antigen. In some embodiments, the single-domain antibody is a nanobody (see, for example, WO 2004041865 A2 and US 20070269422 A1).

6. Antibody variants.

In some embodiments, modifications to the amino acid sequence of the antibodies or fragments thereof described herein are provided. For example, it may be desirable to improve the FcRn binding affinity and/or the pH-dependent FcRn binding affinity of the antibody. It may also be desirable to promote heterodimerization of the heavy chains of the antibodies by introducing certain amino acid modifications. Methods for promoting heterodimerization of antibody chains, including certain modifications that can be made to facilitate heterodimerization, are described by Klein et al. (2012), MAbs, 4(6): 653-663.

Variants of the amino acid sequence of an antibody can be obtained by incorporating appropriate changes into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletion, insertion and substitution can be used to obtain the final construct, provided that the final construct has the desired characteristics. Amino acid changes can be introduced into the amino acid sequence of the antibody in question during the creation of this sequence.

A useful method for identifying specific residues or regions of an antibody that are preferred sites for mutagenesis is called alanine scanning mutagenesis, as described by Cunningham and Wells (1989) Science, 244:1081-1085. Herein, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) in order to affect the interaction of the amino acid with the antigen. Those amino acid positions that exhibit functional sensitivity to substitutions are then refined by introducing additional or different variants at or in place of the substitution sites. Thus, although the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the efficiency of a mutation at a given site, alanine scanning or random mutagenesis is performed at the target codon or region, and the expressed immunoglobulins are screened for the desired activity.

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Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusion of the N- or C-terminus of the antibody to an enzyme or polypeptide, which increases the serum half-life of the antibody.

In some embodiments, the masked cytokine is modified to eliminate, reduce, or otherwise interfere with protease cleavage near the hinge region. The hinge region of IgG is typically defined as comprising E216 and ending at P230 of human IgG1 according to the EU index as in Kabat, however, the flexible portion of the chain can functionally be considered to contain additional residues referred to as the upper and lower hinge regions, such as E216 to G237 (Roux et al., 1998 J Immunol 161:4083), and the lower hinge has been designated as residues 233 to 239 of the Fc region, with FcγR binding typically being attributed. Modifications to any of the masked cytokines described herein can be made, for example, in accordance with the methods described in US 20150139984 A1, which is incorporated herein by reference, as well as by incorporating any modifications described herein.

In some embodiments, FcRn mutations that improve pharmacokinetics include, but are not limited to, M428L, T250Q/M428L, M252Y/S254T/T256E, P257I/N434H, D376V/N434H, P257I/Q3111, N434A, N434W, M428L/N434S, V259I/V308F, M252Y/S254T/T256E, V259I/V308F/M428L, T307Q/N434A, T307Q/N434S, T307Q/E380A/N434A, V308P/N434A, N434H, V308P. In some embodiments, such mutations enhance binding of the antibody to FcRn at low pH, but do not alter the affinity of the antibody at neutral pH.

In some embodiments, the antibody or fragment thereof is altered to increase or decrease the degree of glycosylation of the antibody. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagineX-threonine, wherein X is any amino acid except proline, are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the side chain of asparagine. Thus, the presence of any of these tripeptide sequences in a polypeptide forms a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

Addition or removal of glycosylation sites to the masked cytokine is conveniently accomplished by altering the amino acid sequence such that one or more of the tripeptide sequences described above are formed or removed (for N-linked glycosylation sites). The alteration can also be accomplished by adding, deleting, or substituting one or more serine or threonine residues in the original antibody sequence (for O-linked glycosylation sites).

If the antibody or fragment thereof comprises an Fc region, the carbohydrate attached thereto may be altered. For example, antibodies with a mature carbohydrate structure that lacks fucose attached to the Fc region of the antibody are described in U.S. Patent Application No. US 2003/0157108 (Presta, L.). See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with a bisected N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the Fc region of the antibody are mentioned in WO 2003/011878, Jean-Mairet et al. and U.S. Patent No. 6,602,684, Umana et al. Antibodies with at least one galactose residue in an oligosaccharide attached to the Fc region of the antibody are described in WO 1997/30087, Patel et al., see also WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.) concerning antibodies with an altered carbohydrate attached to their Fc region. See also US 2005/0123546 (Umana et al.) concerning antigen-binding molecules with modified glycosylation.

In certain embodiments, the glycosylation variant comprises an Fc region, wherein the carbohydrate structure attached to the Fc region lacks fucose or has a reduced amount of fucose. Such variants have improved ADCC function. Optionally, the Fc region further comprises one or more amino acid substitutions that further improve ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (Eu residue numbering). Examples of publications relating to defucosylated or fucose-deficient antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Examples of cell lines producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys.

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249:533-545 (1986); U.S. Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially Example 11), and knockout cell lines such as CHO knockout cells for the alpha-1,6-fucosyltransferase gene, FUT8 (Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004)), and cells overexpressing β1,4-N-acetylglycosyltransferase III (GnT-III) and Golgi μ-mannosidase II (ManII).

In any of the embodiments herein, the masked cytokine can be engineered to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, the masked cytokine can be produced in a cell line that has a knockout for alpha-1,6-fucosyltransferase (Fut8). In some embodiments, the host cells have been modified to have reduced alpha1,6-fucosylation activity. Examples of methods for modifying fucosylation pathways in mammalian host cells can be found, for example, in Yamane-Ohnuki and Satoh, MAbs, 1(3): 230-236 (2009), the contents of which are incorporated herein by reference. Examples of methods and compositions for partially or completely inactivating FUT8 gene expression can be found, for example, in U.S. Publ. No. 20160194665 A1; WO 2006133148 A2, the contents of which are incorporated herein by reference. In some embodiments, the masked cytokine is produced in a Lec13 variant of CHO cells (see, e.g., Shields et al., J. Biol. Chem., 277(30):26733-40 (2002)) or a YB2/0 cell line having reduced FUT8 activity (see, e.g., Shinkawa et al., J. Biol. Chem., 278(5):3466-73 (2003)). In some embodiments, small interfering RNA (siRNA) against genes related to alpha-1,6-fucosylation can be administered (see, e.g., Mori et al., Biotechnol. Bioeng. 88(7):901-908 (2004); ImaiNishiya et al., BMC Biotechnol. 7:84 (2007); Omasa et al., J. Biosci. Bioeng., 106(2):168-173 (2008)). In some further embodiments, the masked cytokine can be produced in a cell line overexpressing β1,4-H-acetylglycosyltransferase III (GnT-III). In further embodiments, the cell line further overexpresses Golgi μ-mannosidase II (ManII). In some embodiments of the present invention, a masked cytokine may comprise at least one amino acid substitution in the Fc region that improves ADCC activity.

In some embodiments, the masked cytokine is altered to increase its serum half-life. To increase the serum half-life of the cytokine, an FcRN/salvage receptor binding epitope can be included in the bound antibody (especially an antibody fragment), as described, for example, in U.S. Patent No. 5,739,277. The term salvage receptor binding epitope, as used herein, refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing serum half-life in vivo. IgG molecule (US 2003/0190311, US Patent No. 6,821,505; US Patent No. 6,165,745; US Patent No. 5,624,821; US Patent No. 5,648,260; US Patent No. 6,165,745; US Patent No. 5,834,597).

Another type of variant is an amino acid substitution variant. In these variants, at least one amino acid residue in the antibody molecule is replaced by another residue. Sites of interest for substitution mutagenesis include hypervariable regions, but FR changes are also contemplated. Conservative substitutions are presented in Table 3 under the heading preferred substitutions. If such substitutions result in a desired change in biological activity, then more substantial changes, indicated by the illustrative substitutions in Table 3 or as further described below with respect to amino acid classes, may be introduced and the products screened.

Table 3

Original balance Illustrative Substitutions Preferred Substitutions Ala (A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu;Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn;Glu Asn Glu (E) Asp;Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile

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Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu Significant modis ications of the biological properties of antibodies are achieved by choosing substitutions

which differ significantly in their effect on the maintenance of (a) the structure of the polypeptide backbone at the site of substitution, such as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Amino acids may be grouped according to the similarity of their side chain properties (in A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M);

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q);

(3) acidic: Asp (D), Glu (E);

(4) basic: Lys (K), Arg (R), His (H).

Alternatively, naturally occurring residues can be divided into groups based on common side chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will involve the replacement of a member of one of these classes by another class. Such substituted residues can also be introduced into conservative substitution sites or into the remaining (non-conservative) sites.

Another type of substitution variant involves replacing a naturally occurring amino acid residue with a non-naturally occurring amino acid residue. Non-naturally occurring amino acid residues can be incorporated, for example, by recoding tRNA or by any of the methods described, for example, in WO 2016154675A1, which is incorporated herein by reference.

One type of substitution variant involves substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variants selected for further development will have modified (e.g., improved) biological properties compared to the parent antibody from which they were derived. A convenient method for generating such substitution variants involves affinity maturation using phage display, yeast display, or mammalian display. Briefly, multiple hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibodies thus generated are displayed from filamentous phage particles as fusions to at least a portion of a phage coat protein (e.g., the M13 gene III product) packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity). To identify candidate hypervariable region sites for modification, scanning mutagenesis (e.g., alanine scanning) can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be useful to analyze the crystal structure of the antigen-antibody complex to determine contact points between the antibody and the antigen. Such contact residues and adjacent residues are candidates for substitution according to techniques known in the art, including those developed herein. Once such variants are generated, the panel of variants is screened using techniques known in the art, including those described herein, and antibodies with more effective properties in one or more appropriate assays can be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of masked cytokines are prepared by various methods known in the art.

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These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or production by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, or cassette mutagenesis of an earlier prepared variant or, for example, a non-variant version of an antibody.

It may be desirable to introduce one or more amino acid modifications into the Fc region of the antibodies of the present invention, thereby creating a variant Fc region. A variant Fc region may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions, including a hinge cysteine position.

In some embodiments, a masked cytokine provided herein comprises an antibody or fragment thereof having an IgG1, IgG2, IgG3, or IgG4 isotype with enhanced effector function. In some embodiments, a masked cytokine provided herein comprises an antibody or fragment thereof having an IgG1 isotype with enhanced effector function. In some embodiments, a masked cytokine provided herein (e.g., a masked IL-2 polypeptide or functional fragment thereof, or a masked IL-15 polypeptide or functional fragment thereof) has an IgG1 isotype with enhanced effector function. In some embodiments, the masked cytokine is afucosylated. In some embodiments, the masked cytokine has increased levels of mannose moieties. In some embodiments, the masked cytokine has increased levels of bisected glycan moieties. In some embodiments, the IgG1 comprises amino acid mutations.

In some embodiments, a masked cytokine provided herein comprises an antibody having an IgG1 isotype (e.g., a human IgG1 isotype). In some embodiments, the IgG1 comprises one or more amino acid substitutions that enhance effector function. In one embodiment, the IgG1 comprises the amino acid substitutions S298A, E333A, and K334A, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions S239D and I332E, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions S239D, A330L, and I332E, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions P247I and A339D or A339Q, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions D280H, K290S with or without S298D or S298V, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions F243L, R292P, and Y300L, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions F243L, R292P, Y300L, and P396L, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions F243L, R292P, Y300L, V305I, and P396L, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions G236A, S239D, and I332E, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions K326A and E333A, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions K326W and E333S, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions K290E, S298G, T299A with or without K326E, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions K290N, S298G, T299A with or without K326E, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitution K334V, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions L235S, S239D and K334V, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions K334V and Q331M, S239D, F243V, E294L, or S298T, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions E233L, Q311M, and K334V, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions L234I, Q311M, and K334V, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1

- 94 049880 comprises the amino acid substitutions K334V and S298T, A330M or A330F, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions K334V, Q311M, and either A330M or A330F, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions K334V, S298T, and either A330M or A330F, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions K334V, S239D, and either A330M or S298T, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions L234Y, Y296W, and K290Y, F243V, or E294L, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions Y296W and either L234Y or K290Y, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions S239D, A330S, and I332E, wherein the amino acid residues are numbered according to the EU index as in Kabat.

In some embodiments, the IgG1 comprises one or more amino acid substitutions that reduce or inhibit effector function. In one embodiment, the IgG1 comprises the amino acid substitution N297A, N297G, or N297Q, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitution L234A or L235A, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions C220S, C226S, C229S, and P238S, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions C226S, C229S, E233P, L234V, and L235A, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions L234F, L235E, and P331S, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions S267E and L328F, wherein the amino acid residues are numbered according to the EU index as in Kabat.

In accordance with this disclosure and the teachings in the art, it is contemplated that in some embodiments, an antibody or masked cytokine fragment thereof may comprise one or more alterations compared to a similar wild-type antibody, such as in the Fc region. For example, it is contemplated that certain alterations may be made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and/or complement-dependent cytotoxicity (CDC), such as described in WO 99/51642. See also Duncan & Winter Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 for other examples of Fc region variants. WO 00/42072 (Presta) and WO 2004/056312 (Lowman) describe antibody variants with improved or reduced binding to FcR. The contents of these patent publications are specifically incorporated herein by reference. See also Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US 2005/0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Polypeptide variants with altered amino acid sequences of the Fc region and increased or decreased C1q binding ability are described in U.S. Patent Nos. 6,194,551 B1, WO 99/51642. The contents of these patent publications are specifically incorporated herein by reference. See also Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

Masked cytokine-drug conjugates.

The present invention also provides masked cytokine drug conjugates (MCDCs) comprising a masked cytokine as provided herein, which may be any masked cytokine disclosed herein conjugated to one or more agents. In some embodiments, the one or more agents are a cytotoxic agent, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes. In some embodiments, the one or more agents are an immunostimulant.

In some embodiments, the one or more drugs conjugated to the masked cytokine include, but are not limited to, a maytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent EP 0 425 235 B1); an auristatin, such as the DE and DF drug moieties of monomethyl auristatin (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatin; calicheamicin or a derivative thereof (see U.S. Patent Nos.

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5712374, 5714586, 5739116, 5767285, 5770701, 5770710, 5773001 and 5877296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58: 2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.

In another embodiment, the one or more drugs conjugated to the masked cytokine include, but are not limited to, a tubulin polymerization inhibitor (e.g., maytansinoids and auristatins), DNA damaging agents (e.g., pyrrolobenzodiazepine dimers (PBDs), calicheamicins, duocarmycins, and indolinobenzodiazepine dimers), and DNA synthesis inhibitors (e.g., exatecan derivative Dxd).

In another embodiment, a masked cytokine drug conjugate comprises a masked cytokine as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecenes.

In another embodiment, a masked cytokine drug conjugate comprises a masked cytokine as described herein conjugated to a radioactive atom to form a radioconjugate. Various radioactive isotopes are available for producing radioconjugates. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioactive isotopes of Lu. When a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, such as Tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 re, iodine-131, indium111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

In some embodiments, a masked cytokine drug conjugate comprises a masked cytokine as described herein conjugated to one or more immunostimulants. In some embodiments, the immunostimulant is a stimulating interferon gene agonist (STING) or a toll-like receptor (TLR) agonist.

The STING agonist can be any STING agonist. In some embodiments, the STING agonist is a cyclic dinucleotide (CDN). The CDN can be any CDN or derivative or variant thereof. In some embodiments, the STING agonist is a CDN selected from the group consisting of cGAMP, cdi-AMP, c-di-GMP, cAIMP, and c-di-IMP. In some embodiments, the STING agonist is a derivative or variant of a CDN selected from the group consisting of cGAMP, c-diAMP, c-di-GMP, cAIMP, and c-di-IMP. In some embodiments, the STING agonist is 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide or a derivative or variant thereof. See, e.g., Sali et al. (2015) PloS Pathog., 11(12): e1005324.

A TLR agonist may be an agonist of any TLR, such as TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10. In some embodiments, a TLR agonist is an agonist of a TLR expressed on the cell surface, such as TLR1, TLR2, TLR4, or TLR5. In some embodiments, a TLR agonist is an agonist of a TLR expressed intracellularly, such as TLR3, TLR7, TLR8, TLR9, or TLR10.

Conjugates of a masked cytokine and a cytotoxic agent can be prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, the immunotoxin ricin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating a radionucleotide to an antibody. See WO 94/11026. The linker may be a cleavable linker that facilitates release of the cytotoxic drug in

- 96 049880 cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020).

MCDC herein expressly contemplates, but are not limited to, such conjugates prepared using cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB and SVSB (succinimidyl (4-vinyl sulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

Vectors, host cells and recombinant methods.

For recombinant production of a masked cytokine of the present invention, one or more nucleic acids encoding it are isolated and inserted into a replicable vector for further cloning (DNA amplification) or for expression. DNA encoding the masked cytokine, including its components, is readily isolated and sequenced using standard procedures. A variety of vectors are available. The choice of vector depends in part on the host cell used. Typically, host cells are of prokaryotic or eukaryotic (usually mammalian) origin. It is understood that constant regions of any isotype of antibody or fragment thereof, if desired, can be used for this purpose, including constant regions of IgG, IgM, IgA, IgD and IgE, and that such constant regions can be obtained from humans or any animal species. In some embodiments, a single vector is used to encode the masked cytokine. In some embodiments, more than one vector is used to encode the masked cytokine.

1. Production of masked cytokines using prokaryotic host cells.

Construction of vectors.

Polynucleotide sequences encoding the polypeptide components of the masked cytokines of the present invention can be prepared using standard recombinant techniques. The desired polynucleotide sequences of the antibody or fragment thereof can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, the polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques or obtained from other sources. Once prepared, the sequences encoding the components of the masked cytokine are inserted into a recombinant vector capable of replicating and expressing the heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purposes of the present invention. The selection of a suitable vector will depend primarily on the size of the nucleic acids to be inserted into the vector and the particular host cell being transformed by the vector. Each vector contains various components depending on its function (amplification or expression of a heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides. Vector components typically include, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

Typically, plasmid vectors containing a replicon and control sequences derived from a species compatible with the host cell are used in connection with these hosts. The vector typically carries a replication site as well as marker sequences that are capable of mediating phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides a simple means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain or be modified to contain promoters that can be used by the microbial organism to express endogenous proteins. Examples of pBR322 derivatives useful for expressing specific antibodies are described in detail in Carter et al., U.S. Patent No. 5,648,237.

In addition, phage vectors containing a replicon and control sequences that are compatible with the host microorganism can be used as transformation vectors in connection with these hosts. For example, a bacteriophage such as λGEM TM-11 can be used to create a recombinant vector that can be used to transform susceptible host cells such as E. coli LE392.

The expression vector of the present invention may comprise two or more promotercistron pairs encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence located upstream (5') of a cistron that modulates its expression. Prokaryotic promoters are generally divided into two classes: inducible

- 97 049880 and constitutive. An inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in culture conditions, such as the presence or absence of a nutrient or a change in temperature.

A large number of promoters recognized by a variety of potential host cells are well known. A selected promoter can be operably linked to a cistron DNA encoding any chain of the masked cytokine by removing the promoter from the original DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention. Both the native promoter sequence and many heterologous promoters can be used to drive amplification and/or expression of target genes. In some embodiments, heterologous promoters are used because they typically provide higher transcription and higher yields of expressed target gene compared to the promoter of the native target polypeptide.

Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the β-galactamase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleotide sequences have been published, allowing one skilled in the art to operably link them to cistrons encoding, for example, the light and heavy chains of targeting masked cytokines containing light and heavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors to provide any necessary restriction sites.

In one aspect of the present invention, each cistron in the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across the membrane. Typically, the signal sequence may be a component of the vector or a portion of the DNA of the target polypeptide that is inserted into the vector. The signal sequence selected for the purposes of the present invention must be recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process signal sequences native to heterologous polypeptides, the signal sequence is replaced by a prokaryotic signal sequence selected, for example, from the group consisting of alkaline phosphatase, penicillinase, Ipp or enterotoxin II (STII) heat-stable leader sequences, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the present invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

In another aspect, production of the polypeptide components of the present invention may occur in the cytoplasm of the host cell and, therefore, does not require the presence of secretory signal sequences in each cistron. In this regard, for embodiments comprising immunoglobulin light and heavy chains, for example, the light and heavy chains are expressed with or without masking fragment sequences, linker sequences, etc., folded and assembled to form functional immunoglobulins in the cytoplasm. Certain host strains (e.g., trxB strains of E. coli) provide cytoplasmic conditions favorable for disulfide bond formation, thereby ensuring proper folding and assembly of the expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

The masked cytokines of the present invention can also be produced using an expression system in which the proportion of expressed polypeptide components can be modulated to maximize the yield of secreted and properly assembled antibodies of the present invention. Such modulation is achieved at least in part by simultaneously modulating the translation strength of the polypeptide components.

Prokaryotic host cells suitable for expressing the masked cytokines of the present invention include archaebacteria and eubacteria, such as gram-negative or gram-positive organisms. Examples of suitable bacteria include species of Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negative cells are used. In one embodiment, E. coli cells are used as hosts of the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190–1219; ATCC deposition no. 27325) and its

- 98 049880 derivatives, including strain 33D3 having the genotype W3110 ΔΠπιΛ (AtonA) ptr3 lac Iq lacL8 AompTA(nmpc-fepE) degP41 kanR (U.S. Patent No. 5,639,635). Other strains and derivatives thereof are also suitable, such as E. coli 294 (ATCC, 31446), E. coli B, E. coli λ 1776 (ATCC, 31537), and E. coli RV308 (ATCC, 31608). These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above bacteria having specific genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). It is usually necessary to select suitable bacteria taking into account the reproducibility of the replicon in bacterial cells. For example, E. coli, Serratia or Salmonella species can be suitably used as a host when well-known plasmids such as pBR322, pBR325, pACYC177 or pKN410 are used to deliver the replicon. Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors can be included in the cell culture if necessary.

Obtaining masked cytokines.

Host cells are transformed with the expression vectors described above and cultured in conventional nutrient media modified as required for promoter induction, selection of transformants, or amplification of genes encoding the desired sequences.

Transformation involves introducing DNA into a prokaryotic host in such a way that the DNA can be replicated either as an extrachromosomal element or via a chromosomal integrant. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. Calcium treatment using calcium chloride is commonly used for bacterial cells that contain significant cell wall barriers. Another transformation method uses polyethyleneglycol/DMSO. Electroporation is another technique used.

The prokaryotic cells used to produce the masked cytokines of the present invention are grown in a medium known in the art and suitable for culturing the selected host cells. Examples of suitable media include Luria broth (LB) along with necessary nutrient supplements. In some embodiments, the medium also contains a selection agent selected based on the design of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium to grow cells expressing an ampicillin resistance gene.

Any necessary supplements other than carbon, nitrogen and inorganic phosphorus sources may also be included in appropriate concentrations, administered alone or in admixture with another supplement or medium, such as a complex nitrogen source. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and dithiothreitol.

Prokaryotic host cells are cultured at suitable temperatures. In certain embodiments, for growth of E. coli, the growth temperature is in the range of about 20°C to about 39°C; about 25°C to about 37°C; or about 30°C. The pH of the medium can be any in the range of about 5 to about 9, depending primarily on the host organism. In certain embodiments, for E. coli, the pH is about 6.8 to about 7.4, or about 7.0.

When an inducible promoter is used in an expression vector of the present invention, protein expression is induced under conditions suitable for activation of the promoter. In one aspect of the present invention, PhoA promoters are used to control transcription of polypeptides. Accordingly, transformed host cells are cultured in a phosphate-limiting medium for induction. In certain embodiments, the phosphate-limiting medium is C.R.A.P. medium (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263:133-147). Depending on the vector construct used, a variety of other inducers may be used, as is known in the art.

In one embodiment, the expressed masked cytokines of the present invention are secreted into and recovered from the periplasm of host cells. Protein recovery typically involves disruption of the microorganism, typically by methods such as osmotic shock, sonication, or lysis. Following disruption of the cells, cell debris or whole cells can be removed by centrifugation or filtration. The proteins can be further purified, such as by affinity resin chromatography. Alternatively, the proteins can be transported to and recovered in a culture medium. The cells can be removed from the culture and the culture supernatant filtered and concentrated to further purify the proteins produced. The expressed polypeptides can be further isolated and identified using well-known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blotting.

In one aspect of the present invention, the production of masked cytokines in large

- 99 049 880 quantities are carried out in a fermentation process. Various industrial fed-batch fermentation procedures are available for producing recombinant proteins. Industrial fermenters have a capacity of at least 1000 L, and in some embodiments, from about 1000 to 100,000 L. These fermenters use paddle stirrers to distribute oxygen and nutrients, especially glucose. Limited fermentation generally refers to fermentation in a fermenter with a volumetric capacity of no more than about 100 L, and can be from about 1 liter to about 100 L.

In the fermentation process, the induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, such as an OD550 of approximately 180-220, at which point the cells are in the early stationary phase. Depending on the vector construct used, a variety of inducers can be used, as is known in the art and described above. The cells can be grown for shorter periods of time before induction. The cells are typically induced for approximately 12-50 h, although longer or shorter induction times can be used.

To enhance the production yield and quality of the polypeptides of the present invention, various fermentation conditions can be manipulated. For example, to enhance proper assembly and folding of, for example, secreted antibody polypeptides, additional vectors overexpressing chaperone proteins such as the Dsb proteins (DsbA, DsbB, DsbC, DsbD, and/or DsbG) or FkpA (a peptidylprolyl cis, trans isomerase with chaperone activity) can be used to co-transform prokaryotic host cells. Chaperone proteins have been demonstrated to promote proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., US patent No. 6027888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains deficient in proteolytic enzymes can be used in the present invention. For example, host cell strains can be modified to have genetic mutation(s) in genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Certain protease-deficient E. coli strains are available and are described in, for example, Joly et al. (1998), supra; Georgiou et al., U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

In some embodiments, E. coli strains deficient in proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins are used as host cells in the expression system of the present invention.

Purification of masked cytokines.

In some embodiments, the masked cytokine produced herein is further purified to produce preparations that are substantially homogeneous for further assays and applications. Standard protein purification techniques known in the art may be used. The following procedures are examples of suitable purification procedures: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

In some embodiments, protein A immobilized on a solid phase is used for the immunoaffinity purification of masked cytokines of the present invention. Protein A is a 41 kDa cell wall protein from Staphylococcus aureas that binds with high affinity to the Fc region of antibodies. Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase on which protein A is immobilized can be a column containing a glass or silica surface, or a controlled pore glass column, or a silicic acid column. In some cases, the column is coated with a reagent, such as glycerol, to prevent non-specific attachment of contaminants.

As a first purification step, the cell culture preparation as described above can be applied to a solid phase with immobilized protein A to ensure specific binding of the masked cytokine of interest to protein A. The solid phase should then be washed to remove contaminants non-specifically bound to the solid phase. Finally, the masked cytokine of interest is isolated from the solid phase by elution.

Other purification methods that provide high affinity binding to the component

- 100 049880 masked cytokine, can be used in accordance with standard protein purification methods known in the art.

2. Production of masked cytokines using eukaryotic host cells.

A vector for use in a eukaryotic host cell typically includes one or more of the following non-limiting components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

Signal sequence component.

A vector for use in a eukaryotic host cell may also contain a signal sequence or another polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected may be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences are available, as are viral secretory leader sequences, such as the herpes simplex gD signal. The DNA for such a precursor region is ligated in frame to DNA encoding the masked cytokine.

Origin of replication.

Typically, an origin of replication component is not required for mammalian expression vectors. For example, the SV40 origin of replication can usually be used simply because it contains an early promoter.

Selective gene component.

Expression and cloning vectors may contain a selectable gene, also called a selectable marker. Typical selectable genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies when needed, or (c) supply essential nutrients unavailable from complex environments.

One example of a selection scheme uses a drug to stop the growth of a host cell. Those cells that are successfully transformed with the heterologous gene produce a protein that confers drug resistance and thus survive the selection regime. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

Another example of suitable selectable markers for mammalian cells are those that allow the identification of cells capable of taking up a masked cytokine encoding a nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II, primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

For example, in some embodiments, cells transformed with a selectable DHFR gene are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. In some embodiments, a suitable host cell when using wild-type DHFR is a Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (especially wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding a masked cytokine, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by growing the cells in a medium containing a selection agent for the selectable marker such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199. The host cells can include NS0, including glutamine synthetase (GS)-deficient cell lines. Methods of using GS as a selectable marker for mammalian cells are described in U.S. Patent No. 5,122,464 and U.S. Patent No. 5,891,693.

Promoter component.

Expression and cloning vectors typically contain a promoter that is recognized by the host organism and operably linked to a nucleic acid encoding a masked cytokine of interest, which may be any masked cytokine described herein. Promoter sequences are known for eukaryotes. For example, virtually all eukaryotic genes have a region of high AT content located approximately 25-30 bases upstream of the site where transcription begins. Another sequence found 70-80 bases upstream of the start of transcription of many genes is the CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is the sequence AATAAA, which can be a signal for the addition of a poly-A tail to the 3' end of the coding sequence. In some embodiments, any or all of these sequences can be conveniently inserted into eukaryotic expression vectors.

Transcription of vectors in mammalian host cells is controlled by, for example,

- 101 049880 promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or the immunoglobulin promoter, from heat shock gene promoters, provided that such promoters are compatible with the host cell systems.

The early and late promoters of SV40 virus are conveniently prepared as a restriction fragment of SV40 which also contains the SV40 replication site. The immediate early promoter of human cytomegalovirus is conveniently prepared as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papillomavirus as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982), describing expression of human β-interferon cDNA in murine cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the long terminal repeat of Rous sarcoma virus can be used as a promoter.

Enhancer element component.

Transcription of DNA encoding the masked cytokine of the present invention by higher eukaryotes is often enhanced by insertion of an enhancer sequence into the vector. Many enhancer sequences from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin) are now known. However, an enhancer from a eukaryotic cell virus is usually used. Examples include the SV40 enhancer on the late side of the replication origin (100-270 bp), the human cytomegalovirus early promoter enhancer, the mouse cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) (describing enhancer elements for activating eukaryotic promoters). The enhancer can be spliced into the vector at a position 5' or 3' relative to the masked cytokine coding sequence, but is usually located at a site 5' from the promoter.

Transcription termination component.

Expression vectors for use in eukaryotic host cells may also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are typically available from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the masked cytokine. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vector disclosed therein.

Selection and transformation of host cells.

Suitable host cells for cloning or expressing the DNA in the vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of suitable mammalian host cell lines include the SV40-transformed monkey kidney cell line CV1 (COS-7, ATCC CRL 1651); the human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44–68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2).

Host cells are transformed with the expression or cloning vectors described above for the production of masked cytokines and cultured in conventional nutrient media modified as required for promoter induction, selection of transformants, or amplification of genes encoding the desired sequences.

Cultivation of host cells.

The host cells used to produce the masked cytokines of the present invention can be cultured in a variety of media. Commercially available media such as Ham's F10 medium (Sigma), minimal essential medium (MEM), Sigma, RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM), Sigma are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or

- 102 049880

5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Re. 30,985, can be used as a culture medium for the host cells. Any of these media can be optionally supplemented with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other additives can also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those previously used in connection with the host cell selected for expression and will be apparent to one skilled in the art.

Purification of masked cytokines.

When using recombinant techniques, masked cytokines can be produced intracellularly or directly secreted into the medium. If the masked cytokine is produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration. When the masked cytokine is secreted into the medium, supernatants from such expression systems can first be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor, such as PMSF, can be included at any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants.

The masked cytokine composition obtained from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a convenient technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain, if any, that is present in the masked cytokine. Protein A can be used to purify antibodies based on the heavy chains of human IgG1, IgG2, or IgG4 (Lindmark et al., J. Immunol. Methods 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached can be agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrene divinyl) benzene allow higher flow rates and shorter processing times than can be achieved with agarose. If the masked cytokine contains an OIZ domain, Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, NJ) can be used for purification. Other protein purification methods such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica chromatography, SEPHAROSE™ heparin chromatography, anionic or cation exchange resin chromatography (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the masked cytokine to be extracted.

Following any pre-purification step, the mixture containing the masked cytokine of interest and contaminants may be further purified, such as by low pH hydrophobic interaction chromatography using an elution buffer at a pH of approximately 2.5-4.5, performed at low salt concentrations (e.g., approximately 0-0.25 M salt).

In general, various methodologies for producing masked cytokines for use in research, testing, and clinical applications are well known in the art, consistent with the above-described methodologies, and/or are considered appropriate by those skilled in the art for a particular masked cytokine of interest.

Compositions.

In some aspects, the present document also provides compositions comprising any of the masked cytokines described herein. In some embodiments, the composition comprises any of the exemplary embodiments of a masked cytokine described herein. In some embodiments, the composition comprises a dimer of any of the masked cytokines described herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a masked cytokine and further comprises one or more components as described in detail below. For example, in some embodiments, the composition comprises one or more pharmaceutically acceptable carriers, excipients, stabilizers, buffers, preservatives, tonicity regulators, nonionic surfactants or detergents, or other therapeutic agents or active compounds, or combinations thereof. Various embodiments of the composition are sometimes referred to in

- 103 049880 in this document as compositions.

Therapeutic formulations are prepared for storage by admixing the active ingredient, having the required degree of purity, with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wiklins, Pub., Gennaro Ed., Philadelphia, Pa. 2000). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers; antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives; isotonizers; stabilizers; metal complexes (e.g., Zn-protein complexes); chelating agents such as EDTA; and/or nonionic surfactants.

Buffers can be used to adjust the pH to a range that optimizes therapeutic efficacy, especially if stability is pH dependent. Buffers can be present in concentrations ranging from about 50 to about 250 mM. Suitable buffering agents for use in the present invention include both organic and inorganic acids and their salts. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. In addition, buffers can consist of salts of histidine and trimethylamine, such as Tris.

Preservatives may be added to prevent microbial growth and are typically present in the range of from about 0.2 to 1.0% (w/v). Examples of suitable preservatives commonly used with therapeutic agents include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g. chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, m-cresol, o-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, ethanol, chlorobutanol, thiomersal, bronopol, benzoic acid, imidurea, chlorhexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, and chlorphenesin (3-chlorophenoxypropane-1,2-diol).

Tonicity regulators, sometimes referred to as stabilizers, may be present to regulate or maintain the tonicity of the fluid in the composition. When used with large charged biomolecules such as proteins and antibodies, they are often referred to as stabilizers because they can interact with the charged groups of the amino acid side chains, thereby reducing the possibility of inter- and intramolecular interactions. Tonicity regulators may be present in any amount from about 0.1% to about 25% by weight or from about 1 to about 5% by weight, taking into account the relative amounts of the other ingredients. In some embodiments, tonicity regulators include polyhydric sugar alcohols, trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

Additional excipients include agents which may serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents which prevent denaturation or attachment to the container wall. Such excipients include: polyhydric sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myo-insitose, myo-insitol, galactose, galactitol, glycerol, cyclitols (e.g. inositol), polyethyleneglycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (eg, xylose, mannose, fructose, glucose); disaccharides (eg, lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

Nonionic surfactants or detergents (also known as wetting agents) may be present to aid in the solubilization of the therapeutic agent, as well as to protect the therapeutic protein from aggregation caused by mixing, which also allows the formulation to be subjected to surface shear stress without denaturing the active therapeutic protein or antibody. Nonionic surfactants are present in the range of about 0.05 mg/mL to about 1.0 mg/mL, or about 0.07 mg/mL to about 0.2 mg/mL. In some embodiments, nonionic surfactants are present in the range of about 0.001% to about 0.1% w/v, or about 0.01% to about 0.1% w/v, or about 0.01% to about 0.025% w/v.

Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80

- 104 049880, etc.), polyoxamers (184, 188, etc.), PLURONIC®, TRITON® polyols, polyoxyethylene sorbitan monoesters (TWEEN®-20, TWEEN® -80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methylcellulose and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

In order for the formulations to be used for in vivo administration, they must be sterile. The formulation can be rendered sterile by filtration through sterile filter membranes. Therapeutic compositions herein are typically placed in a container having a port for sterile access, such as a bag of intravenous solution or a vial having a stopper that can be pierced with a hypodermic needle.

The route of administration is according to known and accepted methods, such as single or multiple bolus or infusion over a long period of time by a suitable method, for example, injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intra-articular routes, by topical administration, inhalation or by means of delayed or prolonged release means.

Any of the masked cytokines described herein can be used alone or in combination with other therapeutic agents, such as in the methods described herein. The term "in combination with" encompasses two or more therapeutic agents (e.g., a masked cytokine and a therapeutic agent) that are included in the same or separate formulations. In some embodiments, the term "in combination with" refers to simultaneous administration, in which case administration of a masked cytokine of the present invention occurs simultaneously with administration of one or more additional therapeutic agents (e.g., at the same time or within one hour between administration(s) of the masked cytokine and administration of one or more additional therapeutic agents). In some embodiments, the term "in combination with" refers to sequential administration, in which case administration of a masked cytokine of the present invention occurs before and/or after administration of one or more additional therapeutic agents (e.g., more than one hour between administration(s) of the masked cytokine and administration of one or more additional therapeutic agents). Agents contemplated herein include, but are not limited to, a cytotoxic agent, a cytokine, an agent targeting an immune checkpoint molecule, an agent targeting an immunostimulatory molecule, a growth inhibitory agent, an immunostimulatory agent, an anti-inflammatory agent, or an anticancer agent.

The composition described herein may also contain more than one active compound, if necessary for the particular indication being treated, preferably compounds with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may contain a cytotoxic agent, a cytokine, an agent targeting an immune checkpoint molecule or a stimulatory molecule, a growth inhibitory agent, an immunostimulatory agent, an anti-inflammatory agent, or an anti-cancer agent. Such molecules are typically present in combination in amounts effective for the intended purpose.

The composition may be presented in any suitable state, such as a liquid composition, a solid (lyophilized) composition, or a frozen composition. Approaches to preparing each of these types of compositions for therapeutic use are well known in the art.

Treatment methods.

Provided herein are methods of treating or preventing a disease in a subject comprising administering to the subject an effective amount of any masked cytokine described herein or compositions thereof. In some embodiments, provided are methods of treating or preventing a disease in a subject comprising administering to the subject an effective amount of a masked cytokine. In some embodiments, provided are methods of treating or preventing a disease in a subject comprising administering to the subject an effective amount of any variant of a masked cytokine described herein. In some embodiments, provided are methods of treating or preventing a disease in a subject comprising administering to the subject any composition described herein. In some embodiments, the subject (e.g., a human patient) has been diagnosed with or is at risk of developing a neoplastic disorder (e.g., cancer).

For the prevention or treatment of a disease, the appropriate dose of the active agent will depend on the type of disease being treated, as defined herein, the severity and course of the disease, and whether the agent is administered prophylactically or therapeutically.

- 105 049880 purposes, prior therapy, the subject's medical history and response to the agent, and the discretion of the treating physician. The agent is appropriately administered to the subject in a single treatment or over a series of treatment courses.

In some embodiments of the methods described herein, the interval between administrations of the masked cytokine described herein is about one week or longer. In some embodiments of the methods described herein, the interval between administrations of the masked cytokine described herein is about two days or longer, about three days or longer, about four days or longer, about five days or longer, or about six days or longer. In some embodiments of the methods described herein, the interval between administrations of the masked cytokine described herein is about one week or longer, about two weeks or longer, about three weeks or longer, or about four weeks or longer. In some embodiments of the methods described herein, the interval between administrations of the masked cytokine described herein is about one month or longer, about two months or longer, or about three months or longer. As used herein, the interval between administrations refers to the period of time between one administration of the masked cytokine and the next administration of the masked cytokine. As used herein, an interval of about one month includes four weeks. In some embodiments, the treatment includes multiple administrations of the masked cytokine, wherein the interval between administrations may vary. For example, in some embodiments, the interval between the first administration and the second administration is about one week, and the intervals between subsequent administrations are about two weeks. In some embodiments, the interval between the first administration and the second administration is about two days, three days, four days, or five days or six days, and the intervals between subsequent administrations are about one week.

In some embodiments, the masked cytokine is administered multiple times over a period of time. The dose that is administered to the subject multiple times may, in some embodiments, be the same dose for each administration, or, in some embodiments, the masked cytokine may be administered to the subject in two or more different doses. For example, in some embodiments, the masked cytokine is initially administered in one dose one or more times, and then administered in a second dose one or more times, starting at a later time.

In some embodiments, the masked cytokine described herein is administered in a fixed dose. In some embodiments, the masked cytokine described herein is administered to a subject in a dose of about 25 mg to about 500 mg per dose. In some embodiments, the masked cytokine is administered to the subject at a dose of from about 25 mg to about 50 mg, from about 50 mg to about 75 mg, from about 75 mg to about 100 mg, from about 100 mg to about 125 mg, from about 125 mg to about 150 mg, from about 150 mg to about 175 mg, from about 175 mg to about 200 mg, from about 200 mg to about 225 mg, from about 225 mg to about 250 mg, from about 250 mg to about 275 mg, from about 275 mg to about 300 mg, from about 300 mg to about 325 mg, from about 325 mg to about 350 mg, from about 350 mg to about 375 mg, from about 375 mg to about 400 mg, from about 400 mg up to about 425 mg, from about 425 mg to about 450 mg, from about 450 mg, to about 475 mg, or from about 475 mg to about 500 mg per dose.

In some embodiments, the masked cytokine described herein is administered to a subject at a dose based on the subject's body weight or body surface area (BSA). Depending on the type and severity of the disease, from about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) of the masked cytokine can be the initial dose of the candidate for administration to the patient, such as by one or more separate administrations or by continuous infusion. One typical daily dose can range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. With repeated administrations over several days or longer, depending on the condition, treatment will typically continue until the desired suppression of disease symptoms occurs. One exemplary dose of the masked cytokine can range from about 0.05 mg/kg to about 10 mg/kg. Thus, the patient may be administered one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof). In some embodiments, the masked cytokine described herein is administered to the subject at a dose of about 0.1 mg/kg to about 10 mg/kg, or about 1.0 mg/kg to

- 106 049880 about 10 mg/kg. In some embodiments, a masked cytokine described herein is administered to a subject at any one of about 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, or 10.0 mg/kg. In some embodiments, a masked cytokine described herein is administered to a subject at a dose of about or at least about 0.1 mg/kg, about or at least about 0.5 mg/kg, about or at least about 1.0 mg/kg, about or at least about 1.5 mg/kg, about or at least about 2.0 mg/kg, about or at least about 2.5 mg/kg, about or at least about 3.0 mg/kg, about or at least about 3.5 mg/kg, about or at least about 4.0 mg/kg, about or at least about 4.5 mg/kg, about or at least about 5.0 mg/kg, about or at least about 5.5 mg/kg, about or at least about 6.0 mg/kg, about or at least about 6.5 mg/kg, about or at least about 7.0 mg/kg, about or at least about 7.5 mg/kg, about or at least about 8.0 mg/kg, about or at least about 8.5 mg/kg, about or at least about 9.0 mg/kg, about or at least about 9.5 mg/kg, about or at least about 10.0 mg/kg, about or at least about 15.0 mg/kg, about or at least about 20 mg/mg, about or at least about 30 mg/kg, about or at least about 40 mg/kg, about or at least about 50 mg/mg, about or at least about 60 mg/kg, about or at least about 70 mg/kg, about or at least about 80 mg/kg, about or at least about 90 mg/kg, or about or at least about 100 mg/kg. Any of the above-described dosing frequencies can be used.

In some embodiments, the masked IL-2 polypeptide described herein is administered in a fixed dose. In some embodiments, the masked IL-2 polypeptide described herein is administered to a subject in a dose of about 25 mg to about 500 mg per dose. In some embodiments, the masked IL-2 polypeptide is administered to the subject at a dose of from about 25 mg to about 50 mg, from about 50 mg to about 75 mg, from about 75 mg to about 100 mg, from about 100 mg to about 125 mg, from about 125 mg to about 150 mg, from about 150 mg to about 175 mg, from about 175 mg to about 200 mg, from about 200 mg to about 225 mg, from about 225 mg to about 250 mg, from about 250 mg to about 275 mg, from about 275 mg to about 300 mg, from about 300 mg to about 325 mg, from about 325 mg to about 350 mg, from about 350 mg to about 375 mg, from about 375 mg to about 400 mg, from about 400 mg to about 425 mg, about 425 mg to about 450 mg, about 450 mg to about 475 mg, or about 475 mg to about 500 mg per dose.

In some embodiments, the masked IL-2 polypeptide described herein is administered to a subject at a dose based on the subject's body weight or body surface area (BSA). Depending on the type and severity of the disease, from about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) of the masked IL-2 polypeptide can be the initial dose of the candidate for administration to the patient, such as by one or more separate administrations or by continuous infusion. One typical daily dose can range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. With repeated administrations over several days or longer, depending on the condition, treatment will typically continue until the desired suppression of disease symptoms occurs. One exemplary dose of a masked IL-2 polypeptide can be in the range of about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) can be administered to a patient. In some embodiments, a masked IL-2 polypeptide described herein is administered to a subject at a dose of about 0.1 mg/kg to about 10 mg/kg or about 1.0 mg/kg to about 10 mg/kg. In some embodiments, a masked IL-2 polypeptide as described herein is administered to a subject at any of about 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 mg/kg. In some embodiments, a masked IL-2 polypeptide as described herein is administered to a subject at about or at least about 0.1 mg/kg, about or at least about 0.5 mg/kg,

- 107 049880 about or at least about 1.0 mg/kg, about or at least about 1.5 mg/kg, about or at least about 2.0 mg/kg, about or at least about 2.5 mg/kg, about or at least about 3.0 mg/kg, about or at least about 3.5 mg/kg, about or at least about 4.0 mg/kg, about or at least about 4.5 mg/kg, about or at least about 5.0 mg/kg, about or at least about 5.5 mg/kg, about or at least about 6.0 mg/kg, about or at least about 6.5 mg/kg, about or at least about 7.0 mg/kg, about or at least about 7.5 mg/kg, about or at least about 8.0 mg/kg, about or at least about 8.5 mg/kg, about or at least about 9.0 mg/kg, about or at least about 9.5 mg/kg, about or at least about 10.0 mg/kg, about or at least about 15.0 mg/kg, about or at least about 20 mg/mg, about or at least about 30 mg/kg, about or at least about 40 mg/kg, about or at least about 50 mg/mg, about or at least about 60 mg/kg, about or at least about 70 mg/kg, about or at least about 80 mg/kg, about or at least about 90 mg/kg, or about or at least about 100 mg/kg. Any of the above-described dosing frequencies may be used.

In some embodiments, the masked IL-15 polypeptide described herein is administered in a fixed dose. In some embodiments, the masked IL-15 polypeptide described herein is administered to a subject in a dose of about 25 mg to about 500 mg per dose. In some embodiments, the masked IL-15 polypeptide is administered to the subject at a dose of from about 25 mg to about 50 mg, from about 50 mg to about 75 mg, from about 75 mg to about 100 mg, from about 100 mg to about 125 mg, from about 125 mg to about 150 mg, from about 150 mg to about 175 mg, from about 175 mg to about 200 mg, from about 200 mg to about 225 mg, from about 225 mg to about 250 mg, from about 250 mg to about 275 mg, from about 275 mg to about 300 mg, from about 300 mg to about 325 mg, from about 325 mg to about 350 mg, from about 350 mg to about 375 mg, from about 375 mg to about 400 mg, from about 400 mg to about 425 mg, about 425 mg to about 450 mg, about 450 mg to about 475 mg, or about 475 mg to about 500 mg per dose.

In some embodiments, the masked IL-15 polypeptide described herein is administered to a subject at a dose based on the subject's body weight or body surface area (BSA). Depending on the type and severity of the disease, from about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) of the masked IL-15 polypeptide can be the initial dose of the candidate for administration to the patient, such as by one or more separate administrations or by continuous infusion. One typical daily dose can range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. With repeated administrations over several days or longer, depending on the condition, treatment will typically continue until the desired suppression of disease symptoms occurs. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) may be administered to the subject. In some embodiments, the masked IL-15 polypeptide described herein is administered to the subject at a dose of about 25 mg to about 500 mg per dose. In some embodiments, the masked IL-15 polypeptide described herein is administered to the subject at a dose of about 0.1 mg/kg to about 10 mg/kg or about 1.0 mg/kg to about 10 mg/kg. In some embodiments, the masked IL-15 polypeptide described herein is administered to a subject at any dose of about 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 mg/kg. In some embodiments, a masked IL-15 polypeptide described herein is administered to a subject at a dose of about or at least about 0.1 mg/kg, about or at least about 0.5 mg/kg, about or at least about 1.0 mg/kg, about or at least about 1.5 mg/kg, about or at least about 2.0 mg/kg, about or at least about 2.5 mg/kg, about or at least about 3.0 mg/kg, about or at least about 3.5 mg/kg, about or at least about 4.0 mg/kg, about or at least about 4.5 mg/kg, about or at least

- 108 049880 about 5.0 mg/kg, about or at least about 5.5 mg/kg, about or at least about 6.0 mg/kg, about or at least about 6.5 mg/kg, about or at least about 7.0 mg/kg, about or at least about 7.5 mg/kg, about or at least about 8.0 mg/kg, about or at least about 8.5 mg/kg, about or at least about 9.0 mg/kg, about or at least about 9.5 mg/kg, about or at least about 10.0 mg/kg, about or at least about 15.0 mg/kg, about or at least about 20 mg/mg, about or at least about 30 mg/kg, about or at least about 40 mg/kg, about or at least about 50 mg/mg, about or at least about 60 mg/kg, about or at least about 70 mg/kg, about or at least about 80 mg/kg, about or at least about 90 mg/kg, or about or at least about 100 mg/kg. Any of the above-described dosing frequencies may be used.

The method of treatment contemplated herein is the treatment of a disorder or disease using any of the masked cytokines or compositions described herein. In some embodiments, the method of treatment utilizes any masked IL-2 polypeptide described herein or any masked IL-15 polypeptide described herein or any of the compositions comprising a masked cytokine described herein. Disorders or diseases treatable with the compositions of the present invention include leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), or testicular cancer.

In some embodiments, provided herein is a method of treating or preventing cancer by administering any of the masked cytokines or compositions described herein. In some embodiments, provided herein is a method of treating or preventing cancer by administering a masked IL-2 polypeptide or a masked IL-15 polypeptide, or any of the compositions comprising a masked cytokine described herein. As used herein, the term cancer refers to all types of cancer, neoplasms, or malignancies occurring in mammals, including leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas, and sarcomas. Illustrative cancers that can be treated with the masked cytokine, pharmaceutical composition or method provided herein include 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 carcinoma lung, squamous cell carcinoma of the lung, adenocarcinoma, large cell carcinoma of the lung, small cell carcinoma of the lung, 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 (such as cancer of the head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma. Additional examples include cancer of the thyroid gland, endocrine system, brain, breast, cervix, colon, head and neck, esophagus, liver, kidney, lung, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, gastric cancer, uterine cancer or medulloblastoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant insulinoma of the pancreas, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortex cancer, neoplasms endocrine or exocrine pancreas, medullary thyroid cancer, medullary carcinoma thyroid cancer, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, Paget's disease, phyllodes tumors, lobular carcinoma, ductal carcinoma, pancreatic stellate cell carcinoma, hepatic stellate cell carcinoma

- 109 049880 or prostate cancer.

In some embodiments, provided herein is a method of treating or preventing leukemia by administering any of the masked cytokines or compositions described herein. The term leukemia broadly refers to progressive malignant diseases of the hematopoietic organs and is typically characterized by abnormal proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is typically classified based on (1) the duration and nature of the disease - acute or chronic; (2) the type of cell involved - myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) an increase or lack of increase in the number of abnormal cells in the blood - leukemic or non-leukemic (subleukemic). Illustrative types of leukemia that can be treated with a compound, pharmaceutical composition, or method provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, nonleukemic leukemia, leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, cutaneous leukemia, embryonal leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Reeder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia, or undifferentiated cell leukemia.

In some embodiments, the present invention provides a method for treating or preventing a sarcoma by administering any masked cytokine or composition described herein. The term sarcoma generally refers to a tumor that is composed of a substance similar to embryonic connective tissue and is typically composed of tightly packed cells embedded in a fibrillar or homogeneous substance. Sarcomas that can be treated with the compound, pharmaceutical composition or method provided herein include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemeti's sarcoma, adipose tissue sarcoma, liposarcoma, alveolar soft tissue sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma-type sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma B-cell lymphoma, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant sarcoma of the mesenchymal type, parosteal sarcoma, reticulocyte sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, or telangiectatic sarcoma.

In some embodiments, provided herein is a method of treating or preventing melanoma by administering any masked cytokine or composition described herein. The term melanoma means a tumor arising from the melanocytic system of the skin and other organs. Melanomas that can be treated with a compound, pharmaceutical composition or method provided herein include, for example, acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, Harding-Passy melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma or superficial spreading melanoma.

In some embodiments, the present document provides a method of treating or preventing carcinoma by administering any masked cytokine or composition described herein. The term carcinoma refers to a malignant neoplasm consisting of epithelial cells that tend to invade surrounding tissues and cause metastasis. Examples of carcinomas that can be treated with the compound, pharmaceutical composition or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma, adrenal cortex carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basocellular carcinoma, basaloid carcinoma, basosquamous carcinoma, bronchoalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, carcinoma

- 110 049880 comedo type, carcinoma of corpus uteri, cribriform carcinoma, carcinoma en cuirasse, carcinoma of skin, cylindrical carcinoma, cylindrical cell carcinoma, ductal carcinoma, carcinoma of the ducts, carcinoma of the dura mater, embryonal carcinoma, ductal carcinoma, carcinoma of the dura mater, embryonal carcinoma, encephaloid carcinoma, epidermoid carcinoma, carcinoma of the adenoid epithelium, exophytic carcinoma, carcinoma ex ulcere, fibrous carcinoma, gelatinous carcinoma, gelatinous carcinoma, giant cell carcinoma, giant cell carcinoma, glandular carcinoma, granulosa cell carcinoma, hair matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, carcinoma of the Hurthle cell, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher carcinoma, Kulchitsky cell carcinoma, large cell carcinoma, band carcinoma, lenticular carcinoma, lipomatous carcinoma, lobular carcinoma, lymphoepithelial carcinoma, medullary carcinoma, melanotic carcinoma, Moll carcinoma, mucinous carcinoma, muciform carcinoma, mucocellular carcinoma, mucoepidermoid carcinoma, mucosal carcinoma, mucosal carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, pinpoint cell carcinoma, mucinous carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatoid carcinoma, carcinoma of the nasal mucosa, scirrhous carcinoma, carcinoma of the scrotum, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, spheroid cell carcinoma, spindle cell carcinoma, spongiform carcinoma, squamous cell carcinoma, squamous cell carcinoma, filamentous carcinoma, telangiectatic carcinoma, telangiectoid carcinoma, transitional cell carcinoma, tuberous carcinoma, tubular carcinoma, tuberous carcinoma, verrucous carcinoma or pilar carcinoma.

In some embodiments, provided herein is a method of treating or preventing metastatic cancer by administering any of the masked cytokines or compositions described herein. As used herein, the terms metastasis, metastatic, and metastatic cancer may be used interchangeably and refer to the spread of a neoplastic disease or disorder, such as cancer, from one organ or another non-adjacent organ or part of the body. Cancer arises from a site of origin, such as the breast, which is called a primary tumor, such as primary breast cancer. Some cancer cells in the primary tumor or site of origin acquire the ability to invade and infiltrate surrounding normal tissues in the local area and/or the ability to penetrate the walls of the lymphatic system or vascular system, circulating through this system to other sites and tissues in the body. A second clinically detectable tumor formed from cancer cells of the primary tumor is called a metastatic or secondary tumor. When cancer cells metastasize, it is assumed that the metastatic tumor and its cells are similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor in the breast area consists of abnormal lung cells, not abnormal breast cells. A secondary tumor in the breast is referred to as metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which the subject has or has had a primary tumor and one or more secondary tumors. The phrases nonmetastatic cancer or subjects with nonmetastatic cancer refer to diseases in which the subjects have a primary tumor but no one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and one or more secondary tumors in a second site or sites, such as the breast.

In some embodiments, provided herein is a method of treating or preventing cancer by administering any masked cytokine or composition described herein in combination with an anticancer agent. The anticancer agent may be any agent capable of reducing tumor growth, interfering with the replication of cancer cells, directly or indirectly killing cancer cells, reducing metastasis, reducing tumor blood supply, or reducing cell survival. In some embodiments, the anti-cancer agent is selected from the group consisting of a PD-1 inhibitor, an EGFR inhibitor, a HER2 inhibitor, a VEGFR inhibitor, a CTLA-4 inhibitor, a BTLA inhibitor, a B7H4 inhibitor, a B7H3 inhibitor, a CSFIR inhibitor, an HVEM inhibitor, a CD27 inhibitor, a KIR inhibitor, an NKG2A inhibitor, an NKG2D agonist, a TWEAK inhibitor, an ALK inhibitor, an antibody targeting CD52, an antibody targeting CCR4, a PD-L1 inhibitor, a KIT inhibitor, a PDGFR inhibitor, a BAFF inhibitor, an HDAC inhibitor, a VEGF ligand inhibitor, a molecule targeting CD19, a molecule targeting FOLR1, a molecule targeting DLL3, DKK1 targeting molecule, MUC1 targeting molecule, molecule,

- 111 049880 targeting MUC16, targeting PSMA, targeting MSLN, targeting NY-ES0-1, targeting B7H3, targeting B7H4, targeting BCMA, targeting CD29, targeting CD151, targeting CD123, targeting CD33, targeting CD37, targeting CDH19, targeting CEA, targeting claudin 18.2, targeting CLEC12A, targeting EGFRVIII, targeting EPCAM, EPHA2-targeting molecule, FCRH5-targeting molecule, FLT3-targeting molecule, GD2-targeting molecule, glypican 3-targeting molecule, gpA33-targeting molecule, GPRC5D-targeting molecule, IL-23R-targeting molecule, IL-1RAP-targeting molecule, MCSP-targeting molecule, RON-targeting molecule, ROR1-targeting molecule, STEAP2-targeting molecule, TfR-targeting molecule, CD166-targeting molecule, TPBG-targeting molecule, TROP2-targeting molecule, proteasome inhibitor, ABL inhibitor, CD30 inhibitor, FLT3, MET inhibitor, RET inhibitor, IL-1p inhibitor. MEK inhibitor, ROS1 inhibitor, BRAF inhibitor, CD38 inhibitor, RANKL inhibitor, B4GALNT1 inhibitor, SLAMF7 inhibitor, IDH2 inhibitor, mTOR inhibitor, CD20 targeting molecule, BTK inhibitor, PI3K inhibitor, FLT3 inhibitor, PARP inhibitor, CDK4 inhibitor, CDK6 inhibitor, FGFR inhibitor, RAF inhibitor, JAK1 inhibitor, JAK2 inhibitor, JAK3 inhibitor, IL-6 inhibitor, IL-17 inhibitor, Smoothened inhibitor, IL-6R inhibitor, BCL2 inhibitor, PTCH inhibitor, PIGF inhibitor, TGFB inhibitor, CD28 agonist, CD3 agonist, CD40 agonist, GITR agonist, OX40, VISTA agonist, CD137 agonist, LAG3 inhibitor, TIM3 inhibitor, TIGIT inhibitor and IL-2R inhibitor.

In some embodiments, provided herein is a method of treating or preventing cancer by administering any masked cytokine described herein in combination with an anti-inflammatory agent. The anti-inflammatory agent may be any agent capable of preventing, neutralizing, inhibiting, or otherwise reducing inflammation.

In some embodiments, the anti-inflammatory agent is a cyclooxygenase (COX) inhibitor. The COX inhibitor can be any agent that inhibits COX-1 and/or COX-2 activity. In some embodiments, the COX inhibitor selectively inhibits COX-1 (i.e., the COX inhibitor inhibits COX-1 activity more than it inhibits COX-2 activity). In some embodiments, the COX inhibitor selectively inhibits COX-2 (i.e., the COX inhibitor inhibits COX-2 activity more than it inhibits COX-1 activity). In some embodiments, the COX inhibitor inhibits both COX-1 and COX-2.

In some embodiments, the COX inhibitor is a selective COX-1 inhibitor and is selected from the group consisting of SC-560, FR122047, P6, mofezolac, TFAP, flurbiprofen, and ketoprofen. In some embodiments, the COX inhibitor is a selective COX-2 inhibitor and is selected from the group consisting of celecoxib, rofecoxib, meloxicam, piroxicam, deracoxib, parecoxib, valdecoxib, etoricoxib, chromene derivative, chromane derivative, 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, flosulide, NS-398, L-745337, RWJ-63556, L-784512, darbufelone, CS-502, LAS34475, LAS-34555, S-33516, diclofenac, mefenamic acid, and SD-8381. In some embodiments, the COX inhibitor is selected from the group consisting of ibuprofen, naproxen, ketorolac, indomethacin, aspirin, naproxen, tolmetin, piroxicam, and meclofenamate. In some embodiments, the COX inhibitor is selected from the group consisting of SC-560, FR122047, P6, mofezolac, TFAP, flurbiprofen, ketoprofen, celecoxib, rofecoxib, meloxicam, piroxicam, deracoxib, parecoxib, valdecoxib, etoricoxib, chromene derivative, chromane derivative, 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, flosulide, NS-398, L745337, RWJ-63556, L-784512, darbufelone, CS-502, LAS-34475, LAS-34555, S-33516, diclofenac, mefenamic acid, SD-8381, ibuprofen, naproxen, ketorolac, indomethacin, aspirin, naproxen, tolmetin, piroxicam, and meclofenamate.

In some embodiments, the anti-inflammatory agent is an NF-kB inhibitor. The NF-kB inhibitor can be any agent that inhibits the NF-kB pathway. In some embodiments, the NF-kB inhibitor is selected from the group consisting of an IKK complex inhibitor, an IkB cleavage inhibitor, an NF-kB nuclear translocation inhibitor, a p65 acetylation inhibitor, an NF-kB DNA binding inhibitor, an NF-kB transactivation inhibitor, and an p53 induction inhibitor.

In some embodiments, the IKK complex inhibitor is selected from the group consisting of

- 112 049880 of TPCA-1, NF-κΒ VI activation inhibitor (BOT-64), BMS-345541, amlexanox, SC-514 (GK-01140), IMD-0354, and IKK-16. In some embodiments, the IkB cleavage inhibitor is selected from the group consisting of BAY-11-7082, MG-115, MG-132, lactacystin, epoxomicin, parthenolide, carfilzomib, and MLN-4924 (pevonedistat). In some embodiments, the NF-kB nuclear translocation inhibitor is selected from the group consisting of JSH-23 and rolipram. In some embodiments, the p65 acetylation inhibitor is selected from the group consisting of gallic acid and anacardic acid. In some embodiments, the NF-kB DNA binding inhibitor is selected from the group consisting of GYY-4137, p-XSC, CV-3988, and prostaglandin E2 (PGE2). In some embodiments, the NF-kB transactivation inhibitor is selected from the group consisting of LY-294002, wortmannin, and mesalamine. In some embodiments, the p53 induction inhibitor is selected from the group consisting of quinacrine and flavopiridol. In some embodiments, the NF-kB inhibitor is selected from the group consisting of TPCA-1, NFkB activation inhibitor VI (BOT-64), BMS-345541, amlexanox, SC-514 (GK-01140), IMD-0354, IKK-16, BAY-11-7082, MG115, MG-132, lactacystin, epoxomicin, parthenolide, carfilzomib, MLN-4924 (pevonedistate), JSH-23, rolipram, gallic acid, anacardic acid, GYY-4137, p-XSC, CV-3988, prostaglandin E2 (PGE2), LY-294002, wortmannin, mesalamine, quinacrine, and flavopiridol.

In some embodiments, provided herein is a method of treating or preventing cancer by administering any masked cytokine or composition described herein in combination with an anti-cancer therapeutic protein. The anti-cancer therapeutic protein may be any therapeutic protein capable of reducing tumor growth, interfering with the replication of cancer cells, directly or indirectly killing cancer cells, reducing metastasis, reducing tumor blood supply, or decreasing cell survival. Exemplary anti-cancer therapeutic proteins may be in the form of an antibody or fragment thereof, an antibody derivative, a bispecific antibody, a chimeric antigen receptor (CAR) T cell, a fusion protein, or a bispecific T cell activator (BiTE).

In some embodiments, the present document provides a method of treating or preventing an inflammatory or autoimmune disease by administering any masked cytokine or composition described herein. The term inflammatory disease refers to a disease caused by, resulting from, or leading to inflammation. The term autoimmune disease refers to a disease in which a subject's own immune system attacks its own cells or tissues.

In some embodiments, 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, Sjogren's syndrome, polymyalgia rheumatica, multiple sclerosis (MS), ankylosing spondylitis, type 1 diabetes mellitus, alopecia areata, vasculitis and temporal arteritis, graft-versus-host disease (GVHD), asthma, COPD, paraneoplastic autoimmune disease, inflammatory cartilage, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, juvenile rheumatoid arthritis of systemic onset, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative reaction, enthesopathy, articular syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, rheumatoid arthritis of systemic onset, enteropathic arthritis, reactive arthritis, Reiter's syndrome, dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myolitis, polymyolitis, dermatomyolitis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, Crohn's disease, ulcerative colitis, celiac disease, rhinosinusitis, rhinosinusitis with polyps, eosinophilic esophogitis, eosinophilic bronchitis, Guillain-Barré disease, thyroiditis (eg, Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, rejection transplant, renal injury, hepatitis C-induced vasculitis, and spontaneous pregnancy loss.

Finished products or kits.

In another aspect, there is provided an article of manufacture or a kit that comprises any masked cytokine described herein. The article of manufacture or kit may further comprise instructions for using the cytokines in the methods of the present invention. Thus, in some embodiments, the article of manufacture or kit comprises instructions for

- 113 049880 use of a masked cytokine in methods of treating or preventing a disorder (e.g., cancer) in an individual comprising administering to the individual an effective amount of the masked cytokine. For example, in some embodiments, the article of manufacture or kit comprises instructions for using a masked IL-2 polypeptide in methods of treating or preventing a disorder (e.g., cancer) in an individual comprising administering to the individual an effective amount of a masked IL-2 polypeptide. In some embodiments, the individual is a human. In some embodiments, the individual has a disease selected from the group consisting of leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer, or testicular cancer.

The finished product or kit may further comprise a container. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (such as single- or dual-chamber syringes), tubes, and intravenous (IV) bags. The container may be made of various materials, such as glass or plastic. The container comprises a formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a liquid formulation.

The article or kit may further comprise a label or package insert, which is on or associated with the container, may indicate instructions for dilution and/or use of the composition. The label or package insert may further indicate that the composition is suitable or intended for subcutaneous, intravenous or other routes of administration for the treatment or prevention of a disorder (e.g., cancer) in an individual. The container containing the composition may be a single-use vial or a multi-use vial, allowing for multiple administration of the diluted composition. The finished article or kit may further comprise a second container containing a suitable diluent. The finished article or kit may further comprise other materials necessary from a commercial, therapeutic and consumer standpoint, including other buffers, diluents, filters, needles, syringes and package inserts with instructions for use.

In a particular embodiment, the present invention provides single dose administration kits. Such kits comprise a container with an aqueous formulation of a therapeutic cytokine, including both single and multi-chamber pre-filled syringes. Exemplary pre-filled syringes are available from Vetter GmbH, Ravensburg, Germany.

The finished article or kit of the present invention optionally further comprises a container containing a second drug, wherein the masked cytokine is the first drug, and the finished article or kit further comprises instructions on a label or package insert for treating a subject with the second drug in an effective amount.

In another embodiment, the present document provides a finished article or kit containing the compositions described herein for administration in an autoinjector device. An autoinjector can be described as an injection device that, once activated, will deliver its contents without further required action by the patient or administrator. They are particularly suitable for self-medication with therapeutic compositions where the delivery rate must be constant and the delivery time exceeds a few seconds.

Definitions

Unless otherwise specified, all terms of art, designations and other technical and scientific terms or terminology used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or convenience, and the inclusion of such definitions herein should not necessarily be construed as representing a material difference from what is commonly understood in the art.

It should be understood that the present invention is not limited to particular compositions or biological systems, which, of course, may vary. It should also be understood that the terminology used herein is intended to describe particular embodiments only and is not intended to be limiting. In the context of the present specification and the appended claims, singular forms include reference to plural forms unless otherwise clearly indicated in the content. Thus, for example, a reference to an IL-2 polypeptide does not necessarily include a combination of two or more such polypeptides, and the like.

The term "approximately" in the context of this document refers to a typical error range for the corresponding value, well known to one skilled in the art.

- 114 049880

Reference to approximately a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter as such.

It should be understood that the aspects and embodiments of the present invention described herein include those that contain, consist of, and consist essentially of them.

As used herein, the term and/or refers to any of the elements, any combination of elements, or all of the elements with which the term is associated. For example, the phrase A, B, and/or C is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A and B or C; B and A or C; C and A or B; A (only); B (only); and C (only).

The term antibody includes polyclonal antibodies, monoclonal antibodies (including full-length antibodies that have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab')2, and Fv). The term immunoglobulin (Ig) is used herein interchangeably with antibody.

The term diabodies refers to small antibody fragments with two antigen-binding sites that contain a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL).

The basic 4-chain unit of an antibody is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic heterotetrameric units along with an additional polypeptide called the J chain and contain 10 antigen-binding sites, while IgA antibodies include 2 to 5 basic 4-chain units that can polymerize to form multivalent complexes in combination with the J chain. In the case of IgG, the size of the 4-chain unit is typically approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for each of the α and γ chains and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain at the other end. VL is aligned with VH and CL is aligned with the first constant domain of the heavy chain (CH1). Certain amino acid residues are thought to form the interface between the variable domains of the light chain and heavy chain. The pairing of VH and VL together forms a single antigen-binding site. For information on the structure and properties of the various classes of antibodies, see, for example, Basic and Clinical Immunology, ^8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

The L chain of any vertebrate species can be classified into one of two distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be classified into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies can exist in a variety of polymorphic variants called allotypes (see review in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7), any of which is suitable for use in the present invention. Common allotypic variants in human populations are designated by the letters a, f, n, z.

An isolated antibody is an antibody that has been identified, separated, and/or separated from a component of its production medium (e.g., naturally or recombinantly). In some embodiments, the isolated polypeptide is not in association with all other components of its production medium. Contaminating components of its production medium, such as those obtained from recombinant transfected cells, are materials that typically interfere with the assay, diagnosis, or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide is purified: (1) to greater than 95% by weight of the antibody, as determined, for example, by the Lowry method, and in some embodiments to greater than 99% by weight; (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequencer, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver stain. Isolated antibody includes antibody in situ in recombinant cells since at least one component of the antibody's natural environment will not be present. However, typically an isolated polypeptide or

- 115 049880 the antibody is obtained by at least one purification step.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. In some embodiments, the monoclonal antibodies have a cleavage at the C-terminus of the heavy chain and/or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved from the C-terminus of the heavy chain and/or light chain. In some embodiments, the C-terminal cleavage removes the C-terminal lysine from the heavy chain. In some embodiments, the monoclonal antibodies have a cleavage at the N-terminus of the heavy chain and/or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved from the N-terminus of the heavy chain and/or light chain. In some embodiments, truncated forms of monoclonal antibodies can be produced by recombinant techniques. In some embodiments, monoclonal antibodies are highly specific, directed against a single antigenic site. In some embodiments, monoclonal antibodies are highly specific, directed against multiple antigenic sites (such as a bispecific antibody or a multispecific antibody). The modifier monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, for example, the hybridoma technique, recombinant DNA methods, phage display technologies, and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

The terms full-length antibody, intact antibody, or whole antibody are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. In particular, whole antibodies include antibodies with heavy and light chains, including the Fc region. The constant domains may be native sequence constant domains (e.g., native human sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody may perform one or more effector functions.

An antibody fragment comprises a portion of an intact antibody, such as an antigen-binding region and/or a variable region of an intact antibody and/or a constant region of an intact antibody. Examples of an antibody fragment include an Fc region of an antibody, a portion of an Fc region, or a portion of an antibody comprising an Fc region. Examples of antigen-binding antibody fragments include domain antibodies (dAbs), Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Single heavy chain antibodies or single light chain antibodies can be engineered, or, in the case of the heavy chain, isolated from camel, shark, or mouse libraries engineered to produce single heavy chain molecules.

Papain digestion of antibodies yielded two identical antigen-binding fragments, termed Fab" fragments, and a residual Fc" fragment, named for its ability to crystallize readily. The Fab fragment consists of the complete L chain together with the variable region domain of the H chain (VH) and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of the antibody yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments that have different antigen-binding activities and are still capable of antigen cross-linking. Fab' fragments differ from Fab fragments by the presence of several additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the hinge region of the antibody. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab') ₂ antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical combinations of antibody fragments are also known. The Fc fragment contains the carboxyl-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences and glycan in the Fc region, a region that is also recognized by Fc receptors (FcRs) found on certain cell types.

The term Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the Fc region of the human IgG heavy chain is generally defined as being located from the amino acid residue

- 116 049880 at position Cys226 or from Pro230 to the carboxyl terminus thereof. Suitable native sequence Fc regions for use in the antibodies of the present invention include human IgG1, IgG2, IgG3 and IgG4.

The term cytokine refers to a secreted polypeptide or an active fragment or mutant thereof that modulates the activity of cells, especially cells of the immune system. Examples of cytokines include, for example, chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. The term encompasses any cytokine protein or a functional fragment or variant thereof. The term encompasses any naturally occurring cytokine from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., rats and mice), unless otherwise specified. The term encompasses the unprocessed form of the cytokine as well as any form of the cytokine that results from processing within the cell. The term also includes naturally occurring variants of the cytokine. The term also encompasses non-naturally occurring variants of the cytokine, such as truncations, deletions, forms in which the cytokine is linked to another molecule, and variants caused by at least one amino acid change in the amino acid sequence (e.g., substitution, addition, or deletion). In some aspects, variants or homologs have at least 90, 95, 96, 97, 98, 99, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 125, or 150 or more contiguous amino acid residues) compared to a naturally occurring cytokine polypeptide.

The term IL-2 or IL-2 polypeptide, as used herein, refers to any interleukin-2 (IL-2) protein or a functional fragment or variant thereof. The term encompasses any naturally occurring IL-2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., rats and mice), unless otherwise specified. The term encompasses unprocessed IL-2 (e.g., the full-length precursor form of IL-2 consisting of amino acid residues 1-153) as well as any form of IL-2 that results from processing in the cell (e.g., the mature form of IL-2 consisting of amino acid residues 21-153). Thus, the term encompasses the protein encoded by the amino acid sequence of SEQ ID NO: 160 as well as sequence variants thereof. The term also includes naturally occurring IL-2 variants. The term also encompasses non-naturally occurring variants of IL-2, such as truncations, deletions, forms in which IL-2 is linked to another molecule, and variants caused by at least one amino acid change in the amino acid sequence (e.g., substitution, addition, or deletion). In some aspects, the variants or homologs have at least 90, 95, 96, 97, 98, 99, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, or 133 contiguous amino acid residues) as compared to a naturally occurring IL-2 polypeptide, such as an IL-2 polypeptide encoded by the amino acid sequence of SEQ ID NO: 159 or 160. Thus, the term IL-2 or IL-2 polypeptide includes an IL-2 protein comprising the amino acid sequence of any of SEQ ID NOs: 1-8, 159, 160, 230, 243-251, 260, 775-792, and 813-822, including variants thereof, such as variants formed by one or more amino acid substitutions in the amino acid sequence of any of SEQ ID NOs: 1-8, 159, 160, 230, 243-251, 260, 775-792 and 813-822.

The term IL-15 or IL-15 polypeptide, as used herein, refers to any interleukin-15 (IL-15) protein or a functional fragment or variant thereof. The term encompasses any naturally occurring IL-15 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., rats and mice), unless otherwise specified. The term encompasses unprocessed IL-15 (e.g., the full-length precursor form of IL-15 consisting of amino acid residues 1-162) as well as any form of IL-15 that results from processing in the cell (e.g., the mature form of IL-15 consisting of amino acid residues 49-162). Thus, the term encompasses the protein encoded by the amino acid sequence of SEQ ID NO: 167, as well as sequence variants thereof. The term also includes naturally occurring IL-15 variants. The term also encompasses non-naturally occurring variants of IL-15, such as truncations, deletions, forms in which IL-15 is linked to another molecule, and variants caused by at least one amino acid change in the amino acid sequence (e.g., substitution, addition, or deletion). In some aspects, variants or homologues have at least 90, 95, 96, 97, 98, 99, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, or 114 contiguous amino acid residues) compared to a naturally occurring IL-15 polypeptide, such as an IL-15 polypeptide encoded by the amino acid sequence of SEQ ID NO: 166 or 167. Thus, the term IL-15 or IL-15 polypeptide includes an IL-15 protein comprising the amino acid sequence of SEQ ID NO: 166 or 167, including variants thereof, such as variants formed by one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 166 or 167.

Functional cytokine fragments contain part of the full-length cytokine protein,

- 117 049880 which retains or has a modified cytokine receptor binding ability (e.g., within at least 50, 80, 90, 95, 96, 97, 98, 99, or 100% of the activity of a full-length cytokine protein). The full-length cytokine protein may be a full-length cytokine protein of a wild-type cytokine protein, or it may be a full-length cytokine protein of a modified version of a cytokine protein, such as a full-length cytokine that has been modified by one or more amino acid substitutions. The ability to bind a cytokine receptor can be demonstrated, for example, by the ability of the cytokine to bind to a cognate cytokine receptor or a component thereof (e.g., one or more chains of a heterotrimeric receptor complex). As used herein, the term "full-length" may refer to the full length of a cytokine in its unprocessed (i.e., precursor) or processed (i.e., mature) form. The term also encompasses a portion of a full-length protein that retains or has a modified cytokine receptor binding capacity (e.g., to the extent of at least 50, 80, 90, 95, 96, 97, 98, 99, or 100% of the activity of a full-length cytokine protein) and has been further modified to include one or more amino acid sequence modifications, such as one or more amino acid deletions, additions, or substitutions. Thus, a functional fragment of a cytokine not only encompasses a portion of a full-length cytokine protein, but also encompasses a variant portion of a full-length cytokine protein. For example, a functional fragment of an IL-2 polypeptide comprises a portion of a full-length IL-2 protein that retains or has a modified IL-2R binding ability (e.g., at least 50, 80, 90, 95, 96, 97, 98, 99, or 100% activity compared to full-length wild-type IL-2). The IL-2R binding ability can be demonstrated, for example, by the ability to bind to the IL-2Ra chain, the IL-2Rβ chain, and/or the IL-2Ry chain of IL-2R, either alone or in combination with each other. A functional fragment of IL-2 comprises, for example, an IL-2 protein that comprises amino acid residues 21-153 of the full-length IL-2 sequence of SEQ ID NO: 159, as well as variants thereof, such as variants that contain one or more substitutions in the amino acid sequence. Thus, a functional fragment of IL-2 comprises, for example, an IL-2 protein comprising the amino acid sequence of any of SEQ ID NOs: 1-8, 160, 230, 243-251, 260, 775792 and 813-822. A functional fragment of IL-15 comprises, for example, an IL-15 protein comprising the amino acid sequence of SEQ ID NO: 167.

The term "activatable," as used herein to describe any cytokine or functional fragment thereof, means that the cytokine or functional fragment thereof has been modified to include a masking moiety and, in some embodiments, other components that allow the cytokine or functional fragment to be activated in a preferred environment. For example, a cytokine or functional fragment thereof that has been modified to include a masking moiety for activation in a preferred environment may be referred to herein as an activatable cytokine, or a masked cytokine, or an activatable masked cytokine. Thus, the term "masked cytokine" includes any cytokine or functional fragment thereof that has been modified to include a masking moiety and, in some embodiments, other components, such as a cleavable peptide, that allow the cytokine or functional fragment to be activated in a preferred environment.

The term "masking moiety" as used herein refers to a peptide that is capable of binding to or otherwise exhibiting affinity for a cytokine or a functional fragment thereof such that the masking moiety blocks, hinders, inhibits (e.g., reduces) or otherwise prevents (e.g., masks) the activity or binding of the cytokine or a functional fragment thereof to its cognate receptor or protein. In some embodiments, the masking moiety is a cytokine receptor or a subunit or a functional fragment thereof.

The term "cytokine receptor" as used herein refers to any receptor in the art that binds to one or more cytokines, including, but not limited to, 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, IL28B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, 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), CD252, CD154, CD178, CD70, CD153, 4-1BB-L, TRAIL, RANKL, APO3L, CD256, CD257, CD258, TL1, AITRL, EDA1, interferon (IFN)-a (alpha), IFN-β (beta), IFN-γ (gamma), growth hormone (GH), erythropoietin (EPO), prolactin (PRL), leukemia inhibitory factor (LIF), oncostatin (OSM) and thrombopoietin (TPO). Some cytokine receptors function in whole or in part as heteromeric complexes of more than one subunit or as homomeric complexes. The term also includes

- 118 049880 cytokine receptor subunits that are capable of binding to one or more cytokines either alone or in complex with one or more other cytokine receptor subunits (e.g. as a heteromeric or homomeric complex). Thus, a reference to a cytokine receptor that is a heteromeric complex of three subunits, for example, includes a reference to a heteromeric complex comprising all three subunits, as well as a reference to each subunit alone or in various combinations with one another. Non-limiting examples of cytokine receptors include 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-2Ro, IL2Re, IL-2Ry, IL-3Ra, IL-4R, IL-5Ra, IL-6R, IL-6ST, IL-7R, IL-9R, IL-10Ro, IL-10Rp, IL-11Ro, IL-12Rp1, IL12Re2, IL-13Ra1, IL-13Ro2, IL-15Ra, IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE, IL-18RAP, IL-18R1, IL-20Ra, IL-20Re, IL-21R, IL-22Ra1, IL-22Ro2, IL-23R, IL-27Ra, IL-28Ra, IL-31RA, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, GMRa (CD116), CD131, GHR, PRLR, EPOR, LIFR (CD118), OSMRe, TPO-R (CD110), CSF-1R, EDAR, TNFRSF1A, TNFRSF1B, LTBR, TNFRSF4, CD40, FAS, TNFRSF6B, CD27, TNFRSF8, TNFRSF9, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11A, TNFRSF11B, TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF14, NGFR, TNFRSF17, TNFRSF18, TNFRSF19, RELT, TNFRSF21, TNFRSF25 and EDA2R.

The term IL-2R, or IL-2 receptor, or IL-2 cytokine receptor, as used herein, refers to a high-affinity IL-2 receptor complex that contains three distinct and non-covalently associated chains: the IL-2Ra chain (also referred to as CD25), the IL-2Re chain (also referred to as CD122), and the IL-2Ry chain (also referred to as CD132). IL-2 is capable of binding to the IL-2Ro chain. IL-2 is also capable of binding to the IL-2Ro chain in combination with the IL2Re chain. IL-2 is also capable of binding with maximal affinity to the IL-2Ra chain in combination with the IL-2Re and IL-2Ry chains. In some cases, the term also encompasses the IL-2Ro chain, the IL-2Re chain, or the IL-2Ry chain, or combinations thereof.

The term IL-15R, or IL-15 receptor, or IL-15 cytokine receptor as used herein refers to a high-affinity IL-15 receptor complex that comprises three distinct and non-covalently associated chains: an IL-15Ra chain, an IL-2Re chain, and an IL-2Ry chain. IL-15 is capable of binding to the IL-15Ro chain, to a heterodimer of the IL-2Re chain and the IL-2Ry chain, and to the IL-15Ra chain in combination with the IL-2Re chain and the IL-2Ry chain. In some cases, the term also encompasses the IL15Ro chain, the IL-2Re chain, or the IL-2Ry chain, or combinations thereof.

The term half-life extending domain refers to a domain that is linked to a target component (e.g., a cytokine or a functional fragment thereof, or a masking fragment) for the purpose of extending the half-life of the target component in serum. The term half-life extending domain includes, for example, antibodies, antibody fragments, bispecific antibodies, albumin, binding proteins (e.g., albumin binding proteins and IgG binding proteins), polyamino acid sequences and antibody derivatives (e.g., scFv, scFc, dual variable domains and antibody derivatives based on the CrossMab approach).

The percentage (%) of amino acid sequence identity with respect to the sequence of a reference polypeptide is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the sequence of the reference polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and without taking into account any conservative substitutions as part of the sequence identity. Alignment for determining the percentage of amino acid sequence identity can be achieved by various methods that are known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. For example, the % amino acid sequence identity of a given amino acid sequence A with respect to, compared to, or against a given amino acid sequence B (which may alternatively be stated as a given amino acid sequence A that has or contains a certain % amino acid sequence identity with respect to, compared to, or against a given amino acid sequence B) is calculated as follows:

100 times the X/Y fraction, where X is the number of amino acid residues scored as identical sequence matches in this program when aligning A and B, and where Y is the total number of amino acid residues in B. It should be understood that if the length of the amino acid sequence of A is not equal to the length of the amino acid sequence of B, the % identity of the amino acid sequence of A and B will not equal the % identity

- 119 049880 amino acid sequence B and A.

Antibody effector functions refer to those biological activities that are inherent in the Fc region (either native sequence Fc regions or amino acid sequence variant Fc regions) of an antibody and vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

Binding affinity as used herein refers to the strength of non-covalent interactions between one binding site of a molecule (e.g., a cytokine) and its binding partner (e.g., a cytokine receptor). In some embodiments, the affinity of a binding protein (e.g., a cytokine) can typically be represented by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein.

An isolated nucleic acid molecule encoding the cytokine polypeptides described herein is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the environment in which it was produced. In some embodiments, the isolated nucleic acid is not associated with all components associated with the production environment. The isolated nucleic acid molecules encoding the cytokine polypeptides and polypeptides herein are in a form different from that in which they occur in nature. Thus, the isolated nucleic acid molecules are different from the nucleic acid encoding the cytokine polypeptides and polypeptides herein that naturally exist in cells.

The term pharmaceutical formulation refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is to be administered. Such formulations are sterile.

The term carriers as used herein includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal exposed thereto at the dosages and concentrations employed. Often, a physiologically acceptable carrier is an aqueous pH buffer solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; a low molecular weight polypeptide (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

As used herein, the term treatment refers to a clinical intervention designed to alter the natural course of a clinical pathology in an individual or cell being treated. Desirable effects of treatment include a decrease in the rate of disease progression, normalization or amelioration of the pathological condition, and remission or improvement of prognosis. An individual is successfully treated, for example, if one or more symptoms associated with a disorder (e.g., a neoplastic disease) are reduced or eliminated. For example, an individual is successfully treated if the treatment results in an improved quality of life for those suffering from the disease, a reduction in the dose of other drugs needed to treat the disease, a decrease in the recurrence of the disease, a decrease in the severity of the disease, a delay in the development or progression of the disease, and/or an increase in the survival of individuals.

As used herein, the term in conjunction with or in combination with refers to the administration of one therapeutic intervention in addition to another therapeutic intervention. Thus, the term in conjunction with or in combination with refers to the administration of one therapeutic intervention before, during, or after the administration of another therapeutic intervention to an individual.

As used herein, the term prevention includes preventing the occurrence or recurrence of a disease in an individual. The individual may be predisposed to, susceptible to, or at risk for developing a disorder, but has not yet been diagnosed with the disorder. In some embodiments, the masked cytokines described herein are used to delay the development of a disorder.

In the context of this document, an individual at risk of developing a disorder

- 120 049880 may or may not have a detectable disease or symptoms of a disease, and may or may not exhibit a detectable disease or symptoms of a disease prior to the use of the treatment methods described herein. At risk means that the individual has one or more risk factors, which are measurable parameters that correlate with the development of a disease, as known in the art. An individual who has one or more of these risk factors has a higher probability of developing the disorder than an individual without one or more of these risk factors.

An effective amount refers to at least an amount effective at doses and for periods of time necessary to achieve a desired or indicated effect, including a therapeutic or prophylactic result. An effective amount can be administered in one or more administrations. A therapeutically effective amount is at least the minimum concentration necessary to achieve a measurable improvement in a particular disorder. A therapeutically effective amount as used herein can vary depending on factors such as the pathological condition, age, sex, and body weight of the patient, and the ability of the antibody to elicit the desired response in the individual. A therapeutically effective amount can also be one in which any toxic or deleterious effects of the masked cytokine are offset by the therapeutically beneficial effects. A prophylactically effective amount refers to an amount effective at doses and for periods of time necessary to achieve the desired prophylactic result. Typically, but not necessarily, because a prophylactic dose is used in subjects before or at an earlier stage of the disease, a prophylactically effective amount may be less than a therapeutically effective amount.

Chronic administration refers to the administration of a drug(s) in a continuous, rather than acute, manner to maintain an initial therapeutic effect (activity) over an extended period of time. Intermittent administration is treatment that is not given sequentially without interruption, but rather is cyclical in nature.

In the context of the present description, an individual or subject means a mammal. A mammal for the purposes of treatment includes humans, domestic and farm animals, as well as zoo animals, animals for use in sports or pets, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual or subject is a human.

Illustrative embodiments

Among the presented embodiments are the following.

1. Masked cytokine containing:

a) a masking fragment; and

b) a cytokine or a functional fragment thereof, wherein the masking fragment is linked to the cytokine or a functional fragment thereof via a first linker.

2. The masked cytokine of embodiment 1, further comprising a half-life extension domain that is linked to either the masking moiety or the cytokine or a functional fragment thereof.

3. A masked cytokine according to embodiment 2, wherein the half-life extending domain is linked to either the masking moiety or the cytokine or a functional fragment thereof via a second linker.

4. A masked cytokine according to Embodiment 2 or Embodiment 3, wherein:

i) the masked cytokine comprises, in the direction from N- to C-terminus or from C- to N-terminus: a) a masking fragment; b) a first linker; c) a cytokine or a functional fragment thereof; and d) a half-life extension domain;

ii) the masked cytokine comprises, in the direction from N- to C-terminus or from C- to N-terminus: a) a masking fragment; b) a first linker; c) a cytokine or a functional fragment thereof; d) a second linker; and e) a half-life extension domain;

iii) the masked cytokine comprises, in an 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 fragment; and d) a half-life extension domain; or iv) the masked cytokine comprises, in an 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 fragment; d) a second linker; and e) a half-life extension domain.

5. The masked cytokine of any one of embodiments 1-4, wherein the cytokine or functional fragment thereof is an IL-2 polypeptide or its

- 121 049880 functional fragment.

6. The masked cytokine of embodiment 5, wherein the IL-2 polypeptide or 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.

7. A masked cytokine according to embodiment 5, wherein the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence obtained by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or functional fragment thereof that reduces the affinity of the IL-2 polypeptide or functional fragment thereof for CD25 (IL-2Rα).

8. The masked cytokine of embodiment 7, wherein the amino acid sequence is formed by introducing one or more of the following amino acid substitutions into any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: R38A, F42A, F42K, F42E, K43A, Y45A, Y45N, Y45R, E62A, E62R, E62S, and L72G.

9. A masked cytokine according to embodiment 5, wherein the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence obtained by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or functional fragment thereof that increases the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rβ or IL-2Ry.

10. The masked cytokine of embodiment 9, wherein the amino acid sequence is formed by introducing one or more of the following amino acid substitutions into any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: H16I, L18C, D20A, D20L, D20F, N29L, L80F, R81D, L85V, I86V, and I92F.

11. A masked cytokine according to embodiment 7 or embodiment 8, wherein the amino acid sequence is formed by the additional introduction of one or more amino acid substitutions that increase the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rβ or IL-2Ry.

12. The masked cytokine of embodiment 11, wherein the one or more amino acid substitutions that increase the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Re or IL-2Ry are selected from the group consisting of H16I, L18C, D20A, D20L, D20F, N29L, L80F, R81D, L85V, I86V and I92F.

13. A masked cytokine according to embodiment 5, wherein the IL2 polypeptide or functional fragment thereof comprises an amino acid sequence obtained by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or functional fragment thereof, which stabilizes the IL2 polypeptide or functional fragment thereof.

14. The masked cytokine of embodiment 13, wherein the amino acid sequence is formed by introducing one of the following amino acid substitutions into any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: C125S, C125A, and C125G.

15. A masked cytokine according to any one of embodiments 7-12, wherein the amino acid sequence is formed by the additional introduction of one or more amino acid substitutions that stabilize the IL-2 polypeptide or functional fragment thereof.

16. The masked cytokine of embodiment 15, wherein the one or more substitutions that stabilize the IL-2 polypeptide or functional fragment thereof are C125S, C125A, or C125G.

17. A masked cytokine according to any one of embodiments 5-16, wherein 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, 261, 826, and 827.

18. A masked cytokine according to any one of embodiments 1-4, wherein the cytokine or functional fragment thereof is an IL-15 polypeptide or functional fragment thereof.

19. The masked cytokine of embodiment 18, wherein the IL-15 polypeptide or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 167.

20. A masked cytokine according to embodiment 18 or embodiment 19, wherein 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 823-827.

21. A masked cytokine according to embodiment 18 or embodiment 19, wherein the masking moiety comprises an amino acid sequence obtained by introducing one or more of the following amino acid substitutions into the amino acid sequence of any of SEQ ID NOs: 232-234 and 823-825: R24A, R26A, K34A, S40A, L42A, and P67A.

- 122 049880

22. The masked cytokine of any one of embodiments 2-21, wherein the half-life extending domain is an antibody or fragment thereof.

23. The masked cytokine of embodiment 22, wherein the antibody or fragment thereof comprises either a heavy chain polypeptide or a light chain polypeptide.

24. The masked cytokine of embodiment 23, wherein the heavy chain polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 168, and 169.

25. A masked cytokine according to embodiment 23, wherein the heavy chain polypeptide comprises one or more amino acid substitutions that alter effector function.

26. The masked cytokine of embodiment 25, wherein the heavy chain polypeptide:

a) is an IgG1 heavy chain polypeptide and contains the following amino acid substitution(s):

i) N297A, N297G or N297Q;

ii) L234A and L235A;

iii) C220S, C226S, C229S and P238S;

iv) C226S, C229S, E233P, L234V and L235A;

v) L234F, L235E and P331S;

vi) S267E and L328F;

vii) D265A;

viii) L234A, L235A and P329G;

b) is an IgG2 heavy chain polypeptide and contains the following amino acid substitution(s):

i) V234A and G237A;

ii) H268Q, V309L, A330S and A331S; or iii) V234A, G237A, P238S, H268A, V309L, A330S and P331S; or

c) is an IgG4 heavy chain polypeptide and contains the following amino acid substitution(s):

i) L235A, G237A and E318A;

ii) S228P, L234A and L235A;

iii) H268Q, V309L, A330S and P331S; or iv) S228P and L235A, numbered in accordance with the Kabat EU numbering system.

27. A masked cytokine according to embodiment 25, wherein the heavy chain polypeptide comprises one or more amino acid substitutions that enhance effector function.

28. The masked cytokine of embodiment 27, wherein the heavy chain polypeptide is an IgG1 heavy chain polypeptide and comprises amino acid substitution(s):

a) S298A, E333A and K334A;

b) S239D and I332E;

c) S239D, A330L and I332E;

d) P247I and A339D or A339Q;

e) D280H and K290S;

f) D280H, K290S and either S298D or S298V;

g) F243L, R292P and Y300L;

h) F243L, R292P, Y300L and P396L;

i) F243L, R292P, Y300L, V305I and P396L;

j) G236A, S239D and I332E;

k) K326A and E333A;

l) K326W and E333S;

m) K290E, S298G and T299A;

n) K290E, S298G, T299A and K326E;

o) K290N, S298G and T299A;

p) K290N, S298G, T299A and K326E;

q) K334V;

r) L235S, S239D and K334V;

s) K334V and Q331M, S239D, F243V, E294L or S298T;

t) E233L, Q311M and K334V;

u) L234I, Q311M and K334V;

v) K334V and S298T, A330M or A330F;

w) K334V, Q311M and either A330M or A330F;

- 123 049880

x) K334V, S298T and either A330M or A330F; y) K334V, S239D and either A330M or S298T;

z) L234Y, Y296W and K290Y, F243V or E294L;

aa) Y296W and either L234Y or K290Y;

ab) S239D, A330S and I332E, ac) V264I;

ad) F243L and V264I;

ae) L328M;

af) I332E;

ag) L328M and I332E;

ah) V264I and I332E;

ai) S239E and I332E;

aj) S239Q and I332E;

ak) S239E;

al) A330Y;

am) I332D;

an) L328I and I332E;

ao) L328Q and I332E;

ap) V264T;

aq) V240I;

ar) V266I;

as) S239D;

at) S239D and I332D;

au) S239D and I332N;

av) S239D and I332Q;

aw) S239E and I332D;

ax) S239E and I332N;

ay) S239E and I332Q;

az) S239N and I332D;

ba) S239N and I332E;

bb) S239Q and I332D;

bc) A330Y and I332E;

bd) V264I, A330Y and I332E;

b) A330L and I332E;

bf) V264I, A330L and I332E;

bg) L234E, L234Y or L234I;

bh) L235D, L235S, L235Y or L235I;

bi) S239T;

bj) V240M;

bk) V264Y;

bl) A330I;

bm) N325T;

bn) I332E and L328D, L328V, L328T or L328I;

bo) V264I, I332E and either S239E or S239Q;

bp) S239E, V264I, A330Y and I332E;

bq) A330Y, I332E and either S239D or S239N;

br) A330L, I332E and either S239D or S239N;

bs) V264I, S298A and I332E;

b) S298A, I332E and either S239D or S239N;

bu) S239D, V264I and I332E;

bv) S239D, V264I, S298A and I332E;

bw) S239D, V264I, A330L and I332E;

bx) S239D, I332E and A330I;

by) P230A;

bz) P230A, E233D and I332E;

ca) E272Y;

cb) K274T, K274E, K274R, K274L or K274Y;

cd) F275W;

ce) N276L;

cf) Y278T;

cg) V302I;

ch) E318R;

- 124 049880 ci) S324D, S324I or S324V;

cj) K326I or K326T;

ck) T335D, T335R or T335Y;

cl) V240I and V266I;

cm) S239D, A330Y, I332E and L234I;

cn) S239D, A330Y, I332E and L235D;

co) S239D, A330Y, I332E and V240I;

cp) S239D, A330Y, I332E and V264T; or cq) S239D, A330Y, I332E and either K326E or K326T, numbered in accordance with the Kabat EU numbering system.

29. The masked cytokine of embodiment 23, wherein the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 157 or 170.

30. A masked cytokine according to embodiment 22, wherein the antibody or fragment thereof is a crystallizable fragment domain (Fc domain) or fragment thereof.

31. A masked cytokine according to embodiment 30, wherein the Fc domain or fragment thereof comprises one or more amino acid substitutions that alter effector function.

32. A masked cytokine according to embodiment 31, wherein the Fc domain or functional fragment thereof:

a) represents the Fc domain of IgG1 and its functional fragment and contains amino acid(s) substitution(s):

i) N297A, N297G or N297Q;

ii) L234A and L235A;

iii) C220S, C226S, C229S and P238S;

iv) C226S, C229S, E233P, L234V and L235A;

v) L234F, L235E and P331S;

vi) S267E and L328F;

vii) D265A;

viii) L234A, L235A and P329G;

b) is the Fc domain of IgG2 or a functional fragment thereof and contains the amino acid(s) substitution(s):

i) V234A and G237A;

ii) H268Q, V309L, A330S and A331S; or iii) V234A, G237A, P238S, H268A, V309L, A330S and P331S; or

c) is the Fc domain of IgG4 or a functional fragment thereof and contains the amino acid(s) substitution(s):

i) L235A, G237A and E318A;

ii) S228P, L234A and L235A;

iii) H268Q, V309L, A330S and P331S; or iv) S228P and L235A, numbered in accordance with the Kabat EU numbering system.

33. A masked cytokine according to embodiment 31, wherein the Fc domain or functional fragment thereof comprises one or more amino acid substitutions that enhance effector function.

34. The masked cytokine of embodiment 33, wherein the Fc domain or functional fragment thereof is an IgG1 Fc domain or functional fragment thereof and comprises amino acid substitution(s):

a) S298A, E333A and K334A;

b) S239D and I332E;

c) S239D, A330L and I332E;

d) P247I and A339D or A339Q;

e) D280H and K290S;

f) D280H, K290S and either S298D or S298V;

g) F243L, R292P and Y300L;

h) F243L, R292P, Y300L and P396L;

i) F243L, R292P, Y300L, V305I and P396L;

j) G236A, S239D and I332E;

k) K326A and E333A;

l) K326W and E333S;

m) K290E, S298G and T299A;

n) K290E, S298G, T299A and K326E;

o) K290N, S298G and T299A;

p) K290N, S298G, T299A and K326E;

- 125 049880

q) K334V;

r) L235S, S239D and K334V;

s) K334V and Q331M, S239D, F243V, E294L or S298T;

t) E233L, Q311M and K334V;

u) L234I, Q311M and K334V;

v) K334V and S298T, A330M or A330F;

w) K334V, Q311M and either A330M or A330F;

x) K334V, S298T and either A330M or A330F;

y) K334V, S239D and either A330M or S298T;

z) L234Y, Y296W and K290Y, F243V or E294L;

aa) Y296W and either L234Y or K290Y;

ab) S239D, A330S and I332E, ac) V264I;

ad) F243L and V264I;

ae) L328M;

af) I332E;

ag) L328M and I332E;

ah) V264I and I332E;

ai) S239E and I332E;

aj) S239Q and I332E;

ak) S239E;

al) A330Y;

am) I332D;

an) L328I and I332E;

ao) L328Q and I332E;

ap) V264T;

aq) V240I;

ar) V266I;

as) S239D;

at) S239D and I332D;

au) S239D and I332N;

av) S239D and I332Q;

aw) S239E and I332D;

ax) S239E and I332N;

ay) S239E and I332Q;

az) S239N and I332D;

ba) S239N and I332E;

bb) S239Q and I332D;

bc) A330Y and I332E;

bd) V264I, A330Y and I332E;

b) A330L and I332E;

bf) V264I, A330L and I332E;

bg) L234E, L234Y or L234I;

bh) L235D, L235S, L235Y or L235I;

bi) S239T;

bj) V240M;

bk) V264Y;

bl) A330I;

bm) N325T;

bn) I332E and L328D, L328V, L328T or L328I;

bo) V264I, I332E and either S239E or S239Q;

bp) S239E, V264I, A330Y and I332E;

bq) A330Y, I332E and either S239D or S239N;

br) A330L, I332E and either S239D or S239N;

bs) V264I, S298A and I332E;

b) S298A, I332E and either S239D or S239N;

bu) S239D, V264I and I332E;

bv) S239D, V264I, S298A and I332E;

bw) S239D, V264I, A330L and I332E;

bx) S239D, I332E and A330I;

by) P230A;

bz) P230A, E233D and I332E;

- 126 049880 ca) E272Y;

cb) K274T, K274E, K274R, K274L or K274Y;

cd) F275W;

ce) N276L;

cf) Y278T;

cg) V302I;

ch) E318R;

ci) S324D, S324I or S324V;

cj) K326I or K326T;

ck) T335D, T335R or T335Y;

cl) V240I and V266I;

cm) S239D, A330Y, I332E and L234I;

cn) S239D, A330Y, I332E and L235D;

co) S239D, A330Y, I332E and V240I;

cp) S239D, A330Y, I332E and V264T; or cq) S239D, A330Y, I332E and either K326E or K326T, numbered in accordance with the Kabat EU numbering system.

35. A masked cytokine according to any one of embodiments 30-32, wherein the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 154.

36. The masked cytokine of any one of embodiments 2-21, wherein the half-life extension domain is an albumin polypeptide or fragment thereof.

37. The masked cytokine of embodiment 36, wherein the albumin polypeptide or fragment thereof comprises the amino acid sequence of SEQ ID NO: 171.

38. The masked cytokine of any one of embodiments 2-21, wherein the half-life extension domain is an albumin binding protein or a fragment thereof.

39. The masked cytokine of embodiment 38, wherein the albumin binding protein or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 172-174 and 252-259.

40. A masked cytokine according to any one of embodiments 2-21, wherein the half-life extension domain is an IgG binding protein or a fragment thereof.

41. A masked cytokine according to embodiment 40, wherein the IgG binding protein or functional fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 175-186.

42. The masked cytokine of any one of embodiments 2-21, wherein the half-life extension domain is a polyamino acid sequence.

43. The masked cytokine of embodiment 42, wherein the polyamino acid sequence is a PAS polypeptide or an XTEN polypeptide.

44. The masked cytokine of embodiment 43, wherein the PAS polypeptide comprises at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1500, at least 2000, at least 2500, or at least 3000 amino acid residues, wherein each amino acid residue is either a proline or an alanine residue.

45. The masked cytokine of embodiment 43, wherein the PAS polypeptide comprises at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1500, at least 2000, at least 2500, or at least 3000 amino acid residues, wherein each amino acid residue is selected from the group consisting of a proline, alanine, and serine residue.

46. The masked cytokine of embodiment 43, wherein the XTEN polypeptide comprises an amino acid sequence of from about 25 to about 500, from about 200 to about 1000, from about 500 to about 1500, from about 1000 to about 2000, or from about 1500 to about 3000 amino acid residues, wherein at least about 70%, 75%, 80%, or 85% of the amino acid sequence consists of non-overlapping sequence motifs in which each of the motifs has from 5 to 100 amino acid residues, from 5 to 50 amino acid residues, or from 9 to 36 amino acid residues, and wherein at least 90%, at least

- 127 049880

91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of each motif consists of four, five, or six types of amino acid residues selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), and wherein the content of any one type of amino acid in the full-length XTEN polypeptide does not exceed about 40%, about 35%, about 30%, about 25%, about 15%, about 10%, or about 8%.

47. The masked cytokine of any one of embodiments 1-46, wherein the masked cytokine further comprises one or more PEG polymer chains attached to the masked cytokine.

48. A masked cytokine according to embodiment 47, wherein one or more PEG polymer chains are attached to the cytokine or a functional fragment thereof and/or a half-life extending domain.

49. A masked cytokine according to any one of embodiments 1-48, wherein the masked cytokine is modified or further modified by altering the amino acid sequence of the masked cytokine such that one or more additional N-linked and/or O-linked glycosylation sites are formed.

50. A masked cytokine according to embodiment 49, wherein the masked cytokine is modified or further modified by altering the amino acid sequence of the masked cytokine such that one or more additional tripeptide sequences asparagine-X-serine (N-X-S) and/or asparagine-X-threonine (N-X-T) are introduced into the amino acid sequence of the masked cytokine, wherein X is any amino acid except proline.

51. A masked cytokine according to embodiment 49 or embodiment 50, wherein the masked cytokine is modified or further modified by altering the amino acid sequence of the masked cytokine such that one or more additional serine or threonine residues are introduced into the amino acid sequence of the masked cytokine.

52. The masked cytokine of any one of embodiments 1-51, wherein the first linker comprises a first cleavable peptide.

53. The masked cytokine of any one of embodiments 1-52, wherein the first linker comprises a first N-terminal spacer domain and/or a first C-terminal spacer domain.

54. A masked cytokine according to embodiment 53, wherein the first linker comprises:

a) a first N-terminal spacer domain, a first cleavable peptide and a first C-terminal spacer domain;

b) the first N-terminal spacer domain and the first cleavable peptide;

c) the first N-terminal spacer domain and the first C-terminal spacer domain;

d) the first cleavable peptide and the first C-terminal spacer domain;

e) the first N-terminal spacer domain; or

f) the first C-terminal spacer domain.

55. The masked cytokine of embodiment 53 or embodiment 54, wherein the first 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.

56. The masked cytokine of any one of embodiments 53-55, wherein the first 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.

57. The masked cytokine of any one of embodiments 53-55, wherein the first 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.

58. The masked cytokine of embodiment 53 or embodiment 54, wherein the first 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.

59. The masked cytokine of embodiment 58, wherein the amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555 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 the C-terminus of the amino acid sequence selected from the group consisting of SEQ

- 128 049880

ID NO: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535 and 538-555.

60. The masked cytokine of embodiments 1-59, wherein the first 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 797812.

61. The masked cytokine of any one of embodiments 3-60, wherein the second linker comprises a second cleavable peptide.

62. The masked cytokine of any one of embodiments 3-61, wherein the second linker comprises a second N-terminal spacer domain and/or a second C-terminal spacer domain.

63. A masked cytokine according to embodiment 62, wherein the second linker comprises:

a) a second N-terminal spacer domain, a second cleavable peptide and a second C-terminal spacer domain;

b) a second N-terminal spacer domain and a second cleavable peptide;

c) a second N-terminal spacer domain and a second C-terminal spacer domain;

d) a second cleavable peptide and a second C-terminal spacer domain;

e) a second N-terminal spacer domain; or

f) the second C-terminal spacer domain.

64. The masked cytokine of embodiments 61-63, wherein the second 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.

65. The masked cytokine of embodiments 62-64, wherein the second 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.

66. The masked cytokine of embodiments 62-65, wherein the second 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.

67. The masked cytokine of embodiment 62 or embodiment 63, wherein the second 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.

68. The masked cytokine of embodiment 67, wherein the amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555 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 the C-terminus of 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.

69. The masked cytokine of embodiments 3-68, wherein the second 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 797812.

70. Masked cytokine containing:

a) the first masking fragment;

b) a cytokine or a functional fragment thereof, wherein the first masking fragment is linked to the cytokine or functional fragment thereof via a first linker; and

c) a second masking fragment, wherein the second masking fragment is linked to the cytokine or a functional fragment thereof via a second linker.

71. The masked cytokine of embodiment 70, further comprising a half-life extension domain that is linked to either the first masking moiety or the second masking moiety.

72. A masked cytokine according to embodiment 71, wherein the half-life extension domain is linked to either the first masking moiety or the second masking moiety via a third linker.

73. A masked cytokine according to embodiment 71 or embodiment 72, wherein:

i) the masked cytokine comprises, in the direction from N- to C-terminus or from C- to N-terminus: a) a first masking fragment; b) a first linker; c) a cytokine or a functional fragment thereof; d) a second

- 129 049880 linker and e) a second masking fragment; and f) a half-life extension domain;

ii) the masked cytokine comprises, in the direction from N- to C-terminus or from C- to N-terminus: a) a first masking fragment; b) a first linker; c) a cytokine or a functional fragment thereof; d) a second linker; e) a second masking fragment; f) a third linker; and g) a half-life extension domain;

iii) the masked cytokine comprises, in an 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 extension domain; or iv) the masked cytokine comprises, in an 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 and e) a first masking moiety; f) a third linker; and g) a half-life extension domain.

74. The masked cytokine of any one of embodiments 70-73, wherein the cytokine or functional fragment thereof is an IL-2 polypeptide or functional fragment thereof.

75. The masked cytokine of embodiment 74, wherein the IL-2 polypeptide or functional fragment thereof comprises:

a) 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; or

b) an amino acid sequence obtained by introducing one or more of the following amino acid substitutions into any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792 and 813-822: H16I, L18C, D20A, D20L, D20F, N29L, R38A, F42A, F42K, F42E, F43A, Y45A, Y45N, Y45R, E62A, E62R, E62S, L72G, L80F, R81D, L85V, I86V, I92F and C125S.

76. The masked cytokine of embodiment 74 or embodiment 75, wherein the first 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, and the second 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, and wherein the amino acid sequence of the first masking moiety and the amino acid sequence of the second masking moiety are different.

77. A masked cytokine according to any one of embodiments 74-76, wherein:

a) the first masking fragment comprises an amino acid sequence of SEQ ID NO: 9 or 231, and the second masking fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 161-165, 187-218, 221-226 and 261; or

b) the first masking fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 161-165, 187-218, 221-226 and 261, and the second masking fragment comprises the amino acid sequence of SEQ ID NO: 9 or 231.

78. A masked cytokine according to any one of embodiments 70-73, wherein the cytokine or functional fragment thereof is an IL-15 polypeptide or functional fragment thereof.

79. The masked cytokine of embodiment 78, wherein the IL-15 polypeptide or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 167.

80. A masked cytokine according to embodiment 78 or embodiment 79, wherein:

a) the first mask fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232-234 and 823-825, and the second mask fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 219-229 and 261;

b) the first mask fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 219-229 and 261, and the second mask fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232-234 and 823-825;

c) the first masking fragment comprises an amino acid sequence obtained by introducing one or more of the following amino acid substitutions into the amino acid sequence of any of SEQ ID NOs: 232-234 and 823-825: R24A, R26A, K34A, S40A, L42A and P67A, and the second masking fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 219-229 and 261; or

d) the first masking fragment 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 fragment comprises an amino acid sequence obtained by introducing one or more of the following amino acid substitutions into the amino acid sequence of any of SEQ ID NOs: 232-234 and 823-825: R24A, R26A, K34A, S40A, L42A and P67A.

81. The masked cytokine of any one of embodiments 71-80, wherein

- 130 049880 half-life extension domain is an antibody or fragment thereof.

82. The masked cytokine of embodiment 81, wherein the antibody or fragment thereof comprises either a heavy chain polypeptide or a light chain polypeptide.

83. The masked cytokine of embodiment 82, wherein the heavy chain polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 168, and 169.

84. The masked cytokine of embodiment 82, wherein the heavy chain polypeptide comprises one or more amino acid substitutions that alter effector function.

85. The masked cytokine of embodiment 84, wherein the heavy chain polypeptide:

a) is an IgG1 heavy chain polypeptide and contains the following amino acid substitution(s):

i) N297A, N297G or N297Q;

ii) L234A and L235A;

iii) C220S, C226S, C229S and P238S;

iv) C226S, C229S, E233P, L234V and L235A;

v) L234F, L235E and P331S;

vi) S267E and L328F;

vii) D265A;

viii) L234A, L235A and P329G;

b) is an IgG2 heavy chain polypeptide and contains the following amino acid substitution(s):

i) V234A and G237A;

ii) H268Q, V309L, A330S and A331S; or iii) V234A, G237A, P238S, H268A, V309L, A330S and P331S; or

c) is an IgG4 heavy chain polypeptide and contains the following amino acid substitution(s):

i) L235A, G237A and E318A;

ii) S228P, L234A and L235A;

iii) H268Q, V309L, A330S and P331S; or iv) S228P and L235A, numbered in accordance with the Kabat EU numbering system.

86. The masked cytokine of embodiment 84, wherein the heavy chain polypeptide comprises one or more amino acid substitutions that enhance effector function.

87. The masked cytokine of embodiment 86, wherein the heavy chain polypeptide is an IgG1 heavy chain polypeptide and comprises amino acid substitution(s):

a) S298A, E333A and K334A;

b) S239D and I332E;

c) S239D, A330L and I332E;

d) P247I and A339D or A339Q;

e) D280H and K290S;

f) D280H, K290S and either S298D or S298V;

g) F243L, R292P and Y300L;

h) F243L, R292P, Y300L and P396L;

i) F243L, R292P, Y300L, V305I and P396L;

j) G236A, S239D and I332E;

k) K326A and E333A;

l) K326W and E333S;

m) K290E, S298G and T299A;

n) K290E, S298G, T299A and K326E;

o) K290N, S298G and T299A;

p) K290N, S298G, T299A and K326E;

q) K334V;

r) L235S, S239D and K334V;

s) K334V and Q331M, S239D, F243V, E294L or S298T;

t) E233L, Q311M and K334V;

u) L234I, Q311M and K334V;

v) K334V and S298T, A330M or A330F;

w) K334V, Q311M and either A330M or A330F;

x) K334V, S298T and either A330M or A330F;

- 131 049880

y) K334V, S239D and either A330M or S298T; z) L234Y, Y296W and K290Y, F243V or E294L;

aa) Y296W and either L234Y or K290Y;

ab) S239D, A330S and I332E, ac) V264I;

ad) F243L and V264I;

ae) L328M;

af) I332E;

ag) L328M and I332E;

ah) V264I and I332E;

ai) S239E and I332E;

aj) S239Q and I332E;

ak) S239E;

al) A330Y;

am) I332D;

an) L328I and I332E;

ao) L328Q and I332E;

ap) V264T;

aq) V240I;

ar) V266I;

as) S239D;

at) S239D and I332D;

au) S239D and I332N;

av) S239D and I332Q;

aw) S239E and I332D;

ax) S239E and I332N;

ay) S239E and I332Q;

az) S239N and I332D;

ba) S239N and I332E;

bb) S239Q and I332D;

bc) A330Y and I332E;

bd) V264I, A330Y and I332E;

b) A330L and I332E;

bf) V264I, A330L and I332E;

bg) L234E, L234Y or L234I;

bh) L235D, L235S, L235Y or L235I;

bi) S239T;

bj) V240M;

bk) V264Y;

bl) A330I;

bm) N325T;

bn) I332E and L328D, L328V, L328T or L328I;

bo) V264I, I332E and either S239E or S239Q;

bp) S239E, V264I, A330Y and I332E;

bq) A330Y, I332E and either S239D or S239N;

br) A330L, I332E and either S239D or S239N;

bs) V264I, S298A and I332E;

b) S298A, I332E and either S239D or S239N;

bu) S239D, V264I and I332E;

bv) S239D, V264I, S298A and I332E;

bw) S239D, V264I, A330L and I332E;

bx) S239D, I332E and A330I;

by) P230A;

bz) P230A, E233D and I332E;

ca) E272Y;

cb) K274T, K274E, K274R, K274L or K274Y;

cd) F275W;

ce) N276L;

cf) Y278T;

cg) V302I;

ch) E318R;

ci) S324D, S324I or S324V;

- 132 049880 cj) K326I or K326T;

ck) T335D, T335R or T335Y;

cl) V240I and V266I;

cm) S239D, A330Y, I332E and L234I;

cn) S239D, A330Y, I332E and L235D;

co) S239D, A330Y, I332E and V240I;

cp) S239D, A330Y, I332E and V264T; or cq) S239D, A330Y, I332E and either K326E or K326T, numbered in accordance with the Kabat EU numbering system.

88. The masked cytokine of embodiment 82, wherein the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 157 or 170.

89. A masked cytokine according to embodiment 81, wherein the antibody or fragment thereof is a crystallizable fragment domain (Fc domain) or fragment thereof.

90. A masked cytokine according to embodiment 89, wherein the Fc domain or fragment thereof comprises one or more amino acid substitutions that alter effector function.

91. A masked cytokine according to embodiment 90, wherein the Fc domain or fragment thereof:

a) is the Fc domain of IgG1 or a fragment thereof and contains amino acid substitution(s):

i) N297A, N297G or N297Q;

ii) L234A and L235A;

iii) C220S, C226S, C229S and P238S;

iv) C226S, C229S, E233P, L234V and L235A;

v) L234F, L235E and P331S;

vi) S267E and L328F;

vii) D265A;

viii) L234A, L235A and P329G;

b) is the Fc domain of IgG2 or a fragment thereof and contains the amino acid(s) substitution(s):

i) V234A and G237A;

ii) H268Q, V309L, A330S and A331S; or iii) V234A, G237A, P238S, H268A, V309L, A330S and P331S; or

c) is the Fc domain of IgG4 or a fragment thereof and contains the amino acid(s) substitution(s):

i) L235A, G237A and E318A;

ii) S228P, L234A and L235A;

iii) H268Q, V309L, A330S and P331S; or iv) S228P and L235A, numbered in accordance with the Kabat EU numbering system.

92. A masked cytokine according to embodiment 90, wherein the Fc domain or fragment thereof comprises one or more amino acid substitutions that enhance effector function.

93. The masked cytokine of embodiment 92, wherein the Fc domain or fragment thereof is an IgG1 Fc domain or fragment thereof and comprises amino acid substitution(s):

a) S298A, E333A and K334A;

b) S239D and I332E;

c) S239D, A330L and I332E;

d) P247I and A339D or A339Q;

e) D280H and K290S;

f) D280H, K290S and either S298D or S298V;

g) F243L, R292P and Y300L;

h) F243L, R292P, Y300L and P396L;

i) F243L, R292P, Y300L, V305I and P396L;

j) G236A, S239D and I332E;

k) K326A and E333A;

l) K326W and E333S;

m) K290E, S298G and T299A;

n) K290E, S298G, T299A and K326E;

o) K290N, S298G and T299A;

p) K290N, S298G, T299A and K326E;

q) K334V;

- 133 049880

r) L235S, S239D and K334V;

s) K334V and Q331M, S239D, F243V, E294L or S298T;

t) E233L, Q311M and K334V;

u) L234I, Q311M and K334V;

v) K334V and S298T, A330M or A330F;

w) K334V, Q311M and either A330M or A330F;

x) K334V, S298T and either A330M or A330F;

y) K334V, S239D and either A330M or S298T;

z) L234Y, Y296W and K290Y, F243V or E294L;

aa) Y296W and either L234Y or K290Y;

ab) S239D, A330S and I332E, ac) V264I;

ad) F243L and V264I;

ae) L328M;

af) I332E;

ag) L328M and I332E;

ah) V264I and I332E;

ai) S239E and I332E;

aj) S239Q and I332E;

ak) S239E;

al) A330Y;

am) I332D;

an) L328I and I332E;

ao) L328Q and I332E;

ap) V264T;

aq) V240I;

ar) V266I;

as) S239D;

at) S239D and I332D;

au) S239D and I332N;

av) S239D and I332Q;

aw) S239E and I332D;

ax) S239E and I332N;

ay) S239E and I332Q;

az) S239N and I332D;

ba) S239N and I332E;

bb) S239Q and I332D;

bc) A330Y and I332E;

bd) V264I, A330Y and I332E;

b) A330L and I332E;

bf) V264I, A330L and I332E;

bg) L234E, L234Y or L234I;

bh) L235D, L235S, L235Y or L235I;

bi) S239T;

bj) V240M;

bk) V264Y;

bl) A330I;

bm) N325T;

bn) I332E and L328D, L328V, L328T or L328I;

bo) V264I, I332E and either S239E or S239Q;

bp) S239E, V264I, A330Y and I332E;

bq) A330Y, I332E and either S239D or S239N;

br) A330L, I332E and either S239D or S239N;

bs) V264I, S298A and I332E;

b) S298A, I332E and either S239D or S239N;

bu) S239D, V264I and I332E;

bv) S239D, V264I, S298A and I332E;

bw) S239D, V264I, A330L and I332E;

bx) S239D, I332E and A330I;

by) P230A;

bz) P230A, E233D and I332E;

ca) E272Y;

- 134 049880 cb) K274T, K274E, K274R, K274L or K274Y;

cd) F275W;

ce) N276L;

cf) Y278T;

cg) V302I;

ch) E318R;

ci) S324D, S324I or S324V;

cj) K326I or K326T;

ck) T335D, T335R or T335Y;

cl) V240I and V266I;

cm) S239D, A330Y, I332E and L234I;

cn) S239D, A330Y, I332E and L235D;

co) S239D, A330Y, I332E and V240I;

cp) S239D, A330Y, I332E and V264T; or cq) S239D, A330Y, I332E and either K326E or K326T, numbered in accordance with the Kabat EU numbering system.

94. A masked cytokine according to embodiment 89, wherein the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 154.

95. The masked cytokine of any one of embodiments 71-77, wherein the half-life extension domain is an albumin polypeptide or a functional fragment thereof.

96. The masked cytokine of embodiment 95, wherein the albumin polypeptide or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 171.

97. The masked cytokine of any one of embodiments 71-77, wherein the half-life extension domain is an albumin binding protein or a functional fragment thereof.

98. The masked cytokine of embodiment 97, wherein the albumin binding protein or functional fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 172-174 and 252-259.

99. The masked cytokine of any one of embodiments 71-77, wherein the half-life extension domain is an IgG binding protein or a functional fragment thereof.

100. A masked cytokine according to embodiment 99, wherein the IgG binding protein or functional fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 175-186.

101. The masked cytokine of any one of embodiments 71-77, wherein the half-life extension domain is a polyamino acid sequence.

102. The masked cytokine of embodiment 101, wherein the polyamino acid sequence is a PAS polypeptide or an XTEN polypeptide.

103. The masked cytokine of embodiment 102, wherein the PAS polypeptide comprises at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1500, at least 2000, at least 2500, or at least 3000 amino acid residues, wherein each amino acid residue is either a proline or an alanine residue.

104. The masked cytokine of embodiment 102, wherein the PAS polypeptide comprises at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1500, at least 2000, at least 2500, or at least 3000 amino acid residues, wherein each amino acid residue is selected from the group consisting of a proline, alanine, and serine residue.

105. The masked cytokine of embodiment 102, wherein the XTEN polypeptide comprises an amino acid sequence of from about 25 to about 500, from about 200 to about 1000, from about 500 to about 1500, from about 1000 to about 2000, or from about 1500 to about 3000 amino acid residues, wherein at least about 70%, 75%, 80%, or 85% of the amino acid sequence consists of non-overlapping sequence motifs,

- 135 049880 of which each of the motifs has from 5 to 100 amino acid residues, from 5 to 50 amino acid residues, or from 9 to 36 amino acid residues, and wherein at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of each of the motifs consists of four, five or six types of amino acid residues selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one type of amino acid in a full-length XTEN polypeptide does not exceed about 40%, about 35%, about 30%, about 25%, approximately 15%, approximately 10%, or approximately 8%.

106. The masked cytokine of any one of embodiments 70-105, wherein the masked cytokine further comprises one or more PEG polymer chains attached to the masked cytokine.

107. A masked cytokine according to embodiment 106, wherein one or more PEG polymer chains are attached to the cytokine or a functional fragment thereof and/or a half-life extension domain.

108. A masked cytokine according to any one of embodiments 70-107, wherein the masked cytokine is modified or further modified by altering the amino acid sequence of the masked cytokine such that one or more additional N-linked and/or O-linked glycosylation sites are formed.

109. A masked cytokine according to embodiment 108, wherein the masked cytokine is modified or further modified by altering the amino acid sequence of the masked cytokine such that one or more additional tripeptide sequences asparagine-X-serine (N-X-S) and/or asparagine-X-threonine (N-X-T) are introduced into the amino acid sequence of the masked cytokine, wherein X is any amino acid except proline.

110. A masked cytokine according to embodiment 108 or embodiment 109, wherein the masked cytokine is modified or further modified by altering the amino acid sequence of the masked cytokine such that one or more additional serine or threonine residues are introduced into the amino acid sequence of the masked cytokine.

111. A masked cytokine according to any one of embodiments 70-110, wherein the first linker comprises a first cleavable peptide.

112. The masked cytokine of any one of embodiments 70-111, wherein the first linker comprises a first N-terminal spacer domain and/or a first C-terminal spacer domain.

113. A masked cytokine according to embodiment 112, wherein the first linker comprises:

a) a first N-terminal spacer domain, a first cleavable peptide and a first C-terminal spacer domain;

b) the first N-terminal spacer domain and the first cleavable peptide;

c) the first N-terminal spacer domain and the first C-terminal spacer domain;

d) the first cleavable peptide and the first C-terminal spacer domain;

e) the first N-terminal spacer domain; or

f) the first C-terminal spacer domain.

114. The masked cytokine of embodiments 111-113, wherein the first 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.

115. The masked cytokine of embodiments 112-114, wherein the first 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.

116. The masked cytokine of embodiments 112-115, wherein the first 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.

117. The masked cytokine of embodiments 111-113, wherein the first 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 NO: 236-242.

118. The masked cytokine of embodiment 117, wherein the amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555 comprises an N-terminus and a C-terminus, and

- 136 049880 an amino acid sequence selected from the group consisting of SEQ ID NO: 236-242 is linked to the N-terminus or C-terminus of an amino acid sequence selected from the group consisting of SEQ ID NO: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535 and 538-555.

119. The masked cytokine of embodiments 70-118, wherein the first 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 797812.

120. The masked cytokine of any one of embodiments 70-119, wherein the second linker comprises a second cleavable peptide.

121. The masked cytokine of any one of embodiments 70-120, wherein the second linker comprises a second N-terminal spacer domain and/or a second C-terminal spacer domain.

122. A masked cytokine according to embodiment 121, wherein the second linker comprises:

a) a second N-terminal spacer domain, a second cleavable peptide and a second C-terminal spacer domain;

b) a second N-terminal spacer domain and a second cleavable peptide;

c) a second N-terminal spacer domain and a second C-terminal spacer domain;

d) a second cleavable peptide and a second C-terminal spacer domain;

e) a second N-terminal spacer domain; or

f) the second C-terminal spacer domain.

123. The masked cytokine of embodiments 120-122, wherein the second 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.

124. The masked cytokine of embodiments 121-123, wherein the second 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.

125. The masked cytokine of embodiments 121-124, wherein the second 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.

126. The masked cytokine of embodiments 120-122, wherein the second 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 NO: 236-242.

127. The masked cytokine of embodiment 126, wherein the amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555 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 the C-terminus of 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.

128. The masked cytokine of embodiments 70-127, wherein the second 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 797812.

129. The masked cytokine of any one of embodiments 72-128, wherein the third linker comprises a third cleavable peptide.

130. The masked cytokine of any one of embodiments 72-129, wherein the third linker comprises a third N-terminal spacer domain and/or a third C-terminal spacer domain.

131. A masked cytokine according to embodiment 130, wherein the third linker comprises:

a) a third N-terminal spacer domain, a third cleavable peptide and a third C-terminal spacer domain;

b) the third N-terminal spacer domain and the third cleavable peptide;

c) a third N-terminal spacer domain and a third C-terminal spacer domain;

d) the third cleavable peptide and the third C-terminal spacer domain;

e) a third N-terminal spacer domain; or

f) the third C-terminal spacer domain.

132. The masked cytokine of embodiments 129-131, wherein the third cleavable peptide comprises an amino acid sequence selected from the group,

- 137 049880 consisting of SEQ ID NO: 96-153, 236-242, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535 and 538-555.

133. The masked cytokine of embodiments 130-132, wherein the third 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.

134. The masked cytokine of embodiments 130-133, wherein the third 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.

135. The masked cytokine of embodiments 129-131, wherein the third 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 NO: 236-242.

136. The masked cytokine of embodiment 135, wherein the amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555 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 the C-terminus of 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.

137. The masked cytokine of embodiments 72-136, wherein the third 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 797812.

138. A masked cytokine according to any one of embodiments 52-137, wherein the first cleavable peptide, the second cleavable peptide and/or the third cleavable peptide is a substrate for a protease that is colocalized in a region or tissue expressing the cytokine receptor.

139. A masked cytokine receptor according to embodiment 138, wherein the cytokine receptor is an IL-2 cytokine receptor or an IL-15 cytokine receptor.

140. The masked cytokine of any one of embodiments 1-139, wherein one or more of the first cleavable peptide, the second cleavable peptide, and the third cleavable peptide are cleaved by one or more enzymes selected from the group consisting of: ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, 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, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1 and TPSG1.

141. The masked cytokine of any one of embodiments 2-69 and 71-140, wherein the half-life extending domain is conjugated to the agent.

142. A masked cytokine according to embodiment 141, wherein the agent is a tubulin polymerization inhibitor, a DNA damaging agent, or a DNA synthesis inhibitor.

143. The masked cytokine of embodiment 142, wherein the agent is a maytansinoid, auristatin, pyrrolobenzodiazepine dimer (PBD), calicheamicin, duocarmycin, indolinobenzodiazepine dimer, or an exatecan Dxd derivative.

144. A masked cytokine according to embodiment 141, wherein the agent is an immunostimulant.

145. A masked cytokine according to embodiment 144, wherein the immunostimulant is a stimulating interferon gene agonist (STING) or a toll-like receptor (TLR) agonist.

- 138 049880

146. The masked cytokine of embodiment 145, wherein the STING agonist is a cyclic dinucleotide (CDN).

147. The masked cytokine of embodiment 146, wherein the CDN is selected from the group consisting of cGAMP, c-di-AMP, c-di-GMP, cAIMP, c-di-IMP, 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide.

148. The masked cytokine of embodiment 145, wherein the TLR agonist is a TLR agonist selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10.

149. Masked cytokine containing:

a) a first half-life extension domain and a second half-life extension domain;

b) a masking fragment; and

c) a cytokine or a functional fragment thereof, wherein the masking fragment is linked to a first half-life extension domain, wherein the cytokine or a functional fragment thereof is linked to a second half-life extension domain, and wherein the first half-life extension domain and the second half-life extension domain contain modifications that facilitate the association of the first and second half-life extension domains.

150. A masked cytokine according to embodiment 149, wherein the masking moiety is linked to the first half-life extension domain via a first linker; and/or wherein the cytokine or functional fragment thereof is linked to the second half-life extension domain via a second linker.

151. A masked cytokine according to embodiment 149 or embodiment 150, wherein the cytokine or functional fragment thereof is an IL-2 polypeptide or functional fragment thereof.

152. The masked cytokine of embodiment 151, wherein the IL-2 polypeptide or 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.

153. The masked cytokine of embodiment 151, wherein the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence obtained by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or functional fragment thereof that reduces the affinity of the IL-2 polypeptide or functional fragment thereof for CD25 (IL-2Ra).

154. The masked cytokine of embodiment 153, wherein the amino acid sequence is formed by introducing one or more of the following amino acid substitutions into any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: R38A, F42A, F42K, F42E, K43A, Y45A, Y45N, Y45R, E62A, E62R, E62S, and L72G.

155. The masked cytokine of embodiment 151, wherein the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence obtained by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or functional fragment thereof that increases the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rβ or IL-2Ry.

156. The masked cytokine of embodiment 155, wherein the amino acid sequence is formed by introducing one or more of the following amino acid substitutions into any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: H16I, L18C, D20A, D20L, D20F, N29L, L80F, R81D, L85V, I86V, and I92F.

157. A masked cytokine according to embodiment 153 or embodiment 154, wherein the amino acid sequence is formed by the additional introduction of one or more amino acid substitutions that increase the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rβ or IL-2Ry.

158. The masked cytokine of embodiment 157, wherein the one or more amino acid substitutions that increase the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rβ or IL-2Ry are selected from the group consisting of H16I, L18C, D20A, D20L, D20F, N29L, L80F, R81D, L85V, I86V, and I92F.

159. A masked cytokine according to embodiment 151, wherein the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence obtained by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or functional fragment thereof that stabilizes the IL-2 polypeptide or functional fragment thereof.

160. The masked cytokine of embodiment 159, wherein the amino acid sequence is formed by introducing one of the following amino acid substitutions into any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: C125S, C125A

- 139 049880 and C125G.

161. A masked cytokine according to any one of embodiments 153-158, wherein the amino acid sequence is formed by the additional introduction of one or more amino acid substitutions that stabilize the IL-2 polypeptide or functional fragment thereof.

162. The masked cytokine of embodiment 161, wherein the one or more substitutions that stabilize the IL-2 polypeptide or functional fragment thereof are C125S, C125A, or C125G.

163. The masked cytokine of embodiments 151-162, wherein 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, 261, 826, and 827.

164. A masked cytokine according to embodiment 149 or embodiment 150, wherein the cytokine or functional fragment thereof is an IL-15 polypeptide or functional fragment thereof.

165. The masked cytokine of embodiment 164, wherein the IL-15 polypeptide or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 167.

166. A masked cytokine according to embodiment 164 or embodiment 165, wherein 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 823-827.

167. A masked cytokine according to embodiment 164 or embodiment 165, wherein the masking moiety comprises an amino acid sequence obtained by introducing one or more of the following amino acid substitutions into the amino acid sequence of any of SEQ ID NOs: 232-234 and 823-825: R24A, R26A, K34A, S40A, L42A, and P67A.

168. The masked cytokine of any one of embodiments 149-167, wherein the first half-life extending domain is a first antibody or fragment thereof, and the second half-life extending domain is a second antibody or fragment thereof.

169. A masked cytokine according to embodiment 168, wherein:

a) the first antibody or fragment thereof comprises a first heavy chain polypeptide and the second antibody or fragment thereof comprises a second light chain polypeptide; or

b) the first antibody or fragment thereof comprises a first light chain polypeptide, and the second antibody or fragment thereof comprises a second heavy chain polypeptide.

170. The masked cytokine of embodiment 169, wherein the first heavy chain polypeptide or the second heavy chain polypeptide comprises one or more amino acid substitutions that alter effector function.

171. The masked cytokine of embodiment 170, wherein the first heavy chain polypeptide or the second heavy chain polypeptide:

a) is an IgG1 isotype and contains the following amino acid substitution(s):

i) N297A, N297G or N297Q;

ii) L234A and L235A;

iii) C220S, C226S, C229S and P238S;

iv) C226S, C229S, E233P, L234V and L235A;

v) L234F, L235E and P331S;

vi) S267E and L328F;

vii) D265A;

viii) L234A, L235A and P329G;

b) is an IgG2 isotype and contains the following amino acid substitution(s):

i) V234A and G237A;

ii) H268Q, V309L, A330S and A331S; or iii) V234A, G237A, P238S, H268A, V309L, A330S and P331S; or

c) is an IgG4 isotype and contains the following amino acid substitution(s):

i) L235A, G237A and E318A;

ii) S228P, L234A and L235A;

iii) H268Q, V309L, A330S and P331S; or iv) S228P and L235A, numbered in accordance with the Kabat EU numbering system.

172. The masked cytokine of embodiment 170, wherein the first heavy chain polypeptide or the second heavy chain polypeptide comprises one or more

- 140,049,880 amino acid substitutions that enhance effector function.

173. The masked cytokine of embodiment 172, wherein the first heavy chain polypeptide or the second heavy chain polypeptide is an IgG1 heavy chain polypeptide and comprises amino acid substitution(s):

a) S298A, E333A and K334A;

b) S239D and I332E;

c) S239D, A330L and I332E;

d) P247I and A339D or A339Q;

e) D280H and K290S;

f) D280H, K290S and either S298D or S298V;

g) F243L, R292P and Y300L;

h) F243L, R292P, Y300L and P396L;

i) F243L, R292P, Y300L, V305I and P396L;

j) G236A, S239D and I332E;

k) K326A and E333A;

l) K326W and E333S;

m) K290E, S298G and T299A;

n) K290E, S298G, T299A and K326E;

o) K290N, S298G and T299A;

p) K290N, S298G, T299A and K326E;

q) K334V;

r) L235S, S239D and K334V;

s) K334V and Q331M, S239D, F243V, E294L or S298T;

t) E233L, Q311M and K334V;

u) L234I, Q311M and K334V;

v) K334V and S298T, A330M or A330F;

w) K334V, Q311M and either A330M or A330F;

x) K334V, S298T and either A330M or A330F;

y) K334V, S239D and either A330M or S298T;

z) L234Y, Y296W and K290Y, F243V or E294L;

aa) Y296W and either L234Y or K290Y;

ab) S239D, A330S and I332E, ac) V264I;

ad) F243L and V264I;

ae) L328M;

af) I332E;

ag) L328M and I332E;

ah) V264I and I332E;

ai) S239E and I332E;

aj) S239Q and I332E;

ak) S239E;

al) A330Y;

am) I332D;

an) L328I and I332E;

ao) L328Q and I332E;

ap) V264T;

aq) V240I;

ar) V266I;

as) S239D;

at) S239D and I332D;

au) S239D and I332N;

av) S239D and I332Q;

aw) S239E and I332D;

ax) S239E and I332N;

ay) S239E and I332Q;

az) S239N and I332D;

ba) S239N and I332E;

bb) S239Q and I332D;

bc) A330Y and I332E;

bd) V264I, A330Y and I332E;

b) A330L and I332E;

bf) V264I, A330L and I332E;

- 141 049880 bg) L234E, L234Y or L234I;

bh) L235D, L235S, L235Y or L235I;

bi) S239T;

bj) V240M;

bk) V264Y;

bl) A330I;

bm) N325T;

bn) I332E and L328D, L328V, L328T or L328I;

bo) V264I, I332E and either S239E or S239Q;

bp) S239E, V264I, A330Y and I332E;

bq) A330Y, I332E and either S239D or S239N;

br) A330L, I332E and either S239D or S239N;

bs) V264I, S298A and I332E;

b) S298A, I332E and either S239D or S239N;

bu) S239D, V264I and I332E;

bv) S239D, V264I, S298A and I332E;

bw) S239D, V264I, A330L and I332E;

bx) S239D, I332E and A330I;

by) P230A;

bz) P230A, E233D and I332E;

ca) E272Y;

cb) K274T, K274E, K274R, K274L or K274Y;

cd) F275W;

ce) N276L;

cf) Y278T;

cg) V302I;

ch) E318R;

ci) S324D, S324I or S324V;

cj) K326I or K326T;

ck) T335D, T335R or T335Y;

cl) V240I and V266I;

cm) S239D, A330Y, I332E and L234I;

cn) S239D, A330Y, I332E and L235D;

co) S239D, A330Y, I332E and V240I;

cp) S239D, A330Y, I332E and V264T; or cq) S239D, A330Y, I332E and either K326E or K326T, numbered in accordance with the Kabat EU numbering system.

174. The masked cytokine of embodiment 169, wherein the first heavy chain polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 168, and 169, and the second heavy chain polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 168, and 169.

175. The masked cytokine of embodiment 169, wherein the first light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 157 or 170, and the second light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 157 or 170.

176. The masked cytokine of embodiment 168, wherein the first antibody or fragment thereof is a first crystallizable fragment domain (Fc domain) or fragment thereof, and the second antibody or fragment thereof is a second Fc domain or fragment thereof.

177. A masked cytokine according to embodiment 176, wherein the first Fc domain or fragment thereof and/or the second Fc domain or fragment thereof comprises one or more amino acid substitutions that alter effector function.

178. The masked cytokine of embodiment 177, wherein the first Fc domain or fragment thereof and/or the second Fc domain or its:

a) is the Fc domain of IgG1 or a fragment thereof and contains amino acid substitution(s):

i) N297A, N297G or N297Q;

ii) L234A and L235A;

iii) C220S, C226S, C229S and P238S;

iv) C226S, C229S, E233P, L234V and L235A;

v) L234F, L235E and P331S;

vi) S267E and L328F;

- 142 049880 vii) D265A;

viii) L234A, L235A and P329G;

b) is the Fc domain of IgG2 or a fragment thereof and contains the amino acid(s) substitution(s):

i) V234A and G237A;

ii) H268Q, V309L, A330S and A331S; or iii) V234A, G237A, P238S, H268A, V309L, A330S and P331S; or

c) is the Fc domain of IgG4 or a fragment thereof and contains the amino acid(s) substitution(s):

i) L235A, G237A and E318A;

ii) S228P, L234A and L235A;

iii) H268Q, V309L, A330S and P331S; or iv) S228P and L235A, numbered in accordance with the Kabat EU numbering system.

179. A masked cytokine according to embodiment 177, wherein the first Fc domain or fragment thereof and/or the second Fc domain or fragment thereof comprises one or more amino acid substitutions that enhance effector function.

180. The masked cytokine of embodiment 179, wherein the first Fc domain or fragment thereof and/or the second Fc domain or fragment thereof is an IgG1 Fc domain or fragment thereof and comprises amino acid substitution(s):

a) S298A, E333A and K334A;

b) S239D and I332E;

c) S239D, A330L and I332E;

d) P247I and A339D or A339Q;

e) D280H and K290S;

f) D280H, K290S and either S298D or S298V;

g) F243L, R292P and Y300L;

h) F243L, R292P, Y300L and P396L;

i) F243L, R292P, Y300L, V305I and P396L;

j) G236A, S239D and I332E;

k) K326A and E333A;

l) K326W and E333S;

m) K290E, S298G and T299A;

n) K290E, S298G, T299A and K326E;

o) K290N, S298G and T299A;

p) K290N, S298G, T299A and K326E;

q) K334V;

r) L235S, S239D and K334V;

s) K334V and Q331M, S239D, F243V, E294L or S298T;

t) E233L, Q311M and K334V;

u) L234I, Q311M and K334V;

v) K334V and S298T, A330M or A330F;

w) K334V, Q311M and either A330M or A330F;

x) K334V, S298T and either A330M or A330F;

y) K334V, S239D and either A330M or S298T;

z) L234Y, Y296W and K290Y, F243V or E294L;

aa) Y296W and either L234Y or K290Y;

ab) S239D, A330S and I332E, ac) V264I;

ad) F243L and V264I;

ae) L328M;

af) I332E;

ag) L328M and I332E;

ah) V264I and I332E;

ai) S239E and I332E;

aj) S239Q and I332E;

ak)S239E;

al) A330Y;

am) I332D;

an) L328I and I332E;

ao) L328Q and I332E;

ap) V264T;

aq) V240I;

- 143 049880 ar) V266I;

as) S239D;

at) S239D and I332D;

au) S239D and I332N;

av) S239D and I332Q;

aw) S239E and I332D;

ax) S239E and I332N;

ay) S239E and I332Q;

az) S239N and I332D;

ba) S239N and I332E;

bb) S239Q and I332D;

bc) A330Y and I332E;

bd) V264I, A330Y and I332E;

b) A330L and I332E;

bf) V264I, A330L and I332E;

bg) L234E, L234Y or L234I;

bh) L235D, L235S, L235Y or L235I;

bi) S239T;

bj) V240M;

bk) V264Y;

bl) A330I;

bm) N325T;

bn) I332E and L328D, L328V, L328T or L328I;

bo) V264I, I332E and either S239E or S239Q;

bp) S239E, V264I, A330Y and I332E;

bq) A330Y, I332E and either S239D or S239N;

br) A330L, I332E and either S239D or S239N;

bs) V264I, S298A and I332E;

b) S298A, I332E and either S239D or S239N;

bu) S239D, V264I and I332E;

bv) S239D, V264I, S298A and I332E;

bw) S239D, V264I, A330L and I332E;

bx) S239D, I332E and A330I;

by) P230A;

bz) P230A, E233D and I332E;

ca) E272Y;

cb) K274T, K274E, K274R, K274L or K274Y;

cd) F275W;

ce) N276L;

cf) Y278T;

cg) V302I;

ch) E318R;

ci) S324D, S324I or S324V;

cj) K326I or K326T;

ck) T335D, T335R or T335Y;

cl) V240I and V266I;

cm) S239D, A330Y, I332E and L234I;

cn) S239D, A330Y, I332E and L235D;

co) S239D, A330Y, I332E and V240I;

cp) S239D, A330Y, I332E and V264T; or cq) S239D, A330Y, I332E and either K326E or K326T, numbered in accordance with the Kabat EU numbering system.

181. A masked cytokine according to embodiment 176, wherein:

a) 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;

b) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155;

c) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 154, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 154;

- 144 049880

d) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 265, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156;

e) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 265;

f) 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: 616;

g) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 616, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155;

h) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 157, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 158;

i) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 158, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 157;

j) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 774;

k) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 774, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796;

l) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 619;

m) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 619, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721;

n) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 772;

o) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 772, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721;

p) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 622;

q) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 622, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793;

r) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 773;

s) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 773, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793;

t) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625;

u) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796;

v) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156;

w) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625; or

x) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO:

- 145 049880

796.

182. The masked cytokine of embodiments 149-167, wherein the first half-life extending domain is a first scFv or fragment thereof, and the second half-life extending domain is a second scFv or fragment thereof.

183. The masked cytokine of embodiments 149-167, wherein the first half-life extending domain is a first Fc domain or fragment thereof, and the second half-life extending domain is a second Fc domain or fragment thereof, and wherein the first Fc domain or fragment thereof is linked to the second Fc domain or fragment thereof.

184. The masked cytokine of embodiment 183, wherein the first Fc domain or fragment thereof is linked to the second Fc domain or fragment thereof via a third linker.

185. A masked cytokine according to any one of embodiments 149-167, wherein:

(a) the first half-life extending domain is an scFv or a fragment thereof, and the second half-life extending domain is an antibody or a fragment thereof; or

b) the first half-life extending domain is an antibody or a fragment thereof, and the second half-life extending domain is an scFv or a fragment thereof.

186. A masked cytokine according to any one of paragraphs 168-181, wherein the modifications facilitating the association of the first and second half-life extending domains comprise:

a) introducing the mutations S354C and T366W into a first antibody or fragment thereof and introducing the mutations Y349C, T366S, L368A and Y407V into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

b) introducing the mutations S354C and T366W into the second antibody or fragment thereof and introducing the mutations Y349C, T366S, L368A and Y407V into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

c) introducing the mutations K392D and K409D into the first antibody or fragment thereof and introducing the mutations D399K and E356K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

d) introducing the mutations K392D and K409D into the second antibody or fragment thereof and introducing the mutations D399K and E356K into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

e) the introduction of the mutations S364H and F405A into the first antibody or fragment thereof and the introduction of the mutations Y349T and T394F into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system; or

f) the introduction of mutations S364H and F405A into the second antibody or fragment thereof and the introduction of mutations Y349T and T394F into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system.

187. The masked cytokine of any one of embodiments 168-181, wherein the amino acid sequence of the first antibody or fragment thereof and the amino acid sequence of the second antibody or fragment thereof are formed by:

a) introducing the mutations S354C and T366W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations Y349C, T366S, L368A and Y407V into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

b) introducing the mutations S354C and T366W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations Y349C, T366S, L368A and Y407V into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

c) introducing the mutations K392D and K409D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

d) introducing the mutations K392D and K409D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

e) introducing the mutations S364H and F405A into the amino acid sequence of SEQ ID NO: 154 or

169, and the introduction of mutations Y349T and T394F into the amino acid sequence of SEQ ID NO: 154 or

169, respectively, numbered in accordance with the Kabat EU numbering system; or

f) introducing the mutations S364H and F405A into the amino acid sequence of SEQ ID NO: 154 or

169, and the introduction of mutations Y349T and T394F into the amino acid sequence of SEQ ID NO: 154 or

169, respectively, numbered in accordance with the Kabat EU numbering system.

188. A masked cytokine according to any one of paragraphs 168-181, wherein the modifications facilitating the association of the first and second half-life extending domains comprise:

a) the introduction of a Y407T mutation into a first antibody or fragment thereof and the introduction of a T366Y mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

- 146 049880

b) the introduction of a Y407A mutation into a first antibody or fragment thereof and the introduction of a T366W mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

c) the introduction of the F405A mutation into the first antibody or fragment thereof and the introduction of the T394W mutation into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

d) the introduction of the F405W mutation into the first antibody or fragment thereof and the introduction of the T394S mutation into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

e) introducing a Y407T mutation into a first antibody or fragment thereof and introducing a T366Y mutation into a second antibody or fragment thereof, numbered according to the Kabat EU numbering system;

f) the introduction of the mutations T366Y and F405A into the first antibody or fragment thereof and the introduction of the mutations T394W and Y407T into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

g) introducing the mutations T366W and F405W into the first antibody or fragment thereof and introducing the mutations T394S and Y407A into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

h) introducing the mutations F405W and Y407A into the first antibody or fragment thereof and introducing the mutations T366W and T394S into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

i) introducing the T366W mutation into the first antibody or fragment thereof and introducing the T366S, L368A and Y407V mutations into the second antibody or fragment thereof, numbered according to the Kabat EU numbering system;

j) introducing a Y407T mutation into a second antibody or fragment thereof and introducing a T366Y mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

k) introducing a Y407A mutation into a second antibody or fragment thereof and introducing a T366W mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

l) the introduction of the F405A mutation into the second antibody or fragment thereof and the introduction of the T394W mutation into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

m) introducing the F405W mutation into the second antibody or fragment thereof and introducing the T394S mutation into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

n) introducing a Y407T mutation into a second antibody or fragment thereof and introducing a T366Y mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

o) the introduction of the mutations T366Y and F405A into the second antibody or fragment thereof and the introduction of the mutations T394W and Y407T into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

p) introducing the mutations T366W and F405W into the second antibody or fragment thereof and introducing the mutations T394S and Y407A into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

q) the introduction of the mutations F405W and Y407A into the second antibody or fragment thereof and the introduction of the mutations T366W and T394S into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system; or

r) introducing the mutation T366W into the second antibody or fragment thereof and introducing the mutations T366S, L368A and Y407V into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system.

189. The masked cytokine of any one of embodiments 168-181, wherein the amino acid sequence of the first antibody or fragment thereof and the amino acid sequence of the second antibody or fragment thereof are formed by:

a) introducing a Y407T mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366Y mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

b) introducing a Y407A mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366W mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

c) introducing the mutation F405A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation T394W into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

d) introducing the mutation F405W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation T394S into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

e) introducing a Y407T mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366Y mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

f) introducing the mutations T366Y and F405A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T394W and Y407T into the amino acid sequence of SEQ ID NO: 154 or

- 147 049880

169, respectively, numbered in accordance with the Kabat EU numbering system;

g) introducing the mutations T366W and F405W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T394S and Y407A into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

h) introducing the mutations F405W and Y407A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T366W and T394S into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

i) introducing the mutation T366W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T366S, L368A and Y407V into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

j) introducing a Y407T mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366Y mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

k) introducing a Y407A mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366W mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

l) introducing the mutation F405A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation T394W into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

m) introducing the mutation F405W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation T394S into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

n) introducing a Y407T mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366Y mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

o) introducing the mutations T366Y and F405A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T394W and Y407T into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

p) introducing the mutations T366W and F405W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T394S and Y407A into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

q) introducing the mutations F405W and Y407A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T366W and T394S into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system; or

r) introducing the mutation T366W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T366S, L368A and Y407V into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system.

190. A masked cytokine according to any one of paragraphs 168-181, wherein the modifications facilitating the association of the first and second half-life extending domains comprise:

a) the introduction of a K409E mutation into a first antibody or fragment thereof and the introduction of a D399K mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

b) the introduction of a K409E mutation into a first antibody or fragment thereof and the introduction of a D399R mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

c) the introduction of a K409D mutation into a first antibody or fragment thereof and the introduction of a D399K mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

d) introducing a K409D mutation into a first antibody or fragment thereof and introducing a D399R mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

e) introducing a K392E mutation into a first antibody or fragment thereof and introducing a D399R mutation into a second antibody or fragment thereof, numbered according to the Kabat EU numbering system;

f) the introduction of a K392E mutation into a first antibody or fragment thereof and the introduction of a D399K mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

g) the introduction of a K392D mutation into a first antibody or fragment thereof and the introduction of a D399R mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

h) the introduction of a K392D mutation into a first antibody or fragment thereof and the introduction of a D399K mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

i) introducing the mutations K409D and K360D into the first antibody or fragment thereof and introducing the mutations D399K and E356K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

j) introducing the mutations K409D and K370D into the first antibody or fragment thereof and introducing the mutations D399K and E357K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

k) introducing the mutations K409D and K392D into the first antibody or fragment thereof and introducing the mutations

- 148 049880

D399K, E356K and E357K in the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

l) the introduction of the mutations K409D and K392D into the first antibody or fragment thereof and the introduction of the mutation D399K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

m) introducing the mutations K409D and K392D into the first antibody or fragment thereof and introducing the mutations D399K and E356K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

n) introducing the mutations K409D and K392D into the first antibody or fragment thereof and introducing the mutations D399K and E357K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

o) the introduction of the mutations K409D and K370D into the first antibody or fragment thereof and the introduction of the mutations D399K and E357K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

p) introducing the mutation D399K into the first antibody or fragment thereof and introducing the mutations K409D and K360D into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

q) introducing the mutations K409D and K439D into the first antibody or fragment thereof and introducing the mutations D399K and E356K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

r) the introduction of a K409E mutation into a second antibody or fragment thereof and the introduction of a D399K mutation into a first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

s) introducing a K409E mutation into a second antibody or fragment thereof and introducing a D399R mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

t) introducing a K409D mutation into a second antibody or fragment thereof and introducing a D399K mutation into a first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

u) introducing a K409D mutation into a second antibody or fragment thereof and introducing a D399R mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

v) the introduction of a K392E mutation into a second antibody or fragment thereof and the introduction of a D399R mutation into a first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

w) introducing a K392E mutation into a second antibody or fragment thereof and introducing a D399K mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

x) the introduction of a K392D mutation into a second antibody or fragment thereof and the introduction of a D399R mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

y) introducing a K392D mutation into a second antibody or fragment thereof and introducing a D399K mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

z) introducing the mutations K409D and K360D into the second antibody or fragment thereof and introducing the mutations D399K and E356K into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

aa) introducing the mutations K409D and K370D into the second antibody or fragment thereof and introducing the mutations D399K and E357K into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

ab) introducing the mutations K409D and K392D into the second antibody or fragment thereof and introducing the mutations D399K, E356K and E357K into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

ac) introducing the mutations K409D and K392D into the second antibody or fragment thereof and introducing the mutation D399K into the first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

ad) introducing the mutations K409D and K392D into the second antibody or fragment thereof and introducing the mutations D399K and E356K into the first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

ae) introducing the mutations K409D and K392D into the second antibody or fragment thereof and introducing the mutations D399K and E357K into the first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

af) introducing the mutations K409D and K370D into the second antibody or fragment thereof and introducing the mutations D399K and E357K into the first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

ag) introducing the mutation D399K into the second antibody or fragment thereof and introducing the mutations K409D and K360D into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system; or ah) introducing the mutations K409D and K439D into the second antibody or fragment thereof and introducing the mutations D399K and E356K into the first antibody or fragment thereof, numbered in accordance with the

- 149 049880 Kabat EU numbering.

191. The masked cytokine of any one of embodiments 168-181, wherein the amino acid sequence of the first antibody or fragment thereof and the amino acid sequence of the second antibody or fragment thereof are formed by:

a) introducing a K409E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

b) introducing a K409E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

c) introducing a K409D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

d) introducing a K409D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

e) introducing a K392E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

f) introducing a K392E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

g) introducing a K392D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

h) introducing a K392D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

i) introducing the mutations K409D and K360D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

j) introducing the mutations K409D and K370D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

k) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K, E356K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

l) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation D399K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

m) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

n) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

o) introducing the mutations K409D and K370D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

p) introducing the mutation D399K into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations K409D and K360D into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

q) introducing the mutations K409D and K439D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

r) introducing a K409E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

s) introducing a K409E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

t) introducing a K409D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and

- 150 049880 introducing the D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered in accordance with the Kabat EU numbering system;

u) introducing a K409D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

v) introducing a K392E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

w) introducing a K392E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

x) introducing a K392D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

y) introducing a K392D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

z) introducing the mutations K409D and K360D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

aa) introducing the mutations K409D and K370D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

ab) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K, E356K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

ac) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation D399K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

ad) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, numbered according to the Kabat EU numbering system;

ae) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

af) introducing the mutations K409D and K370D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

ag) introducing the mutation D399K into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations K409D and K360D into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system; or ah) introducing the mutations K409D and K439D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system.

192. A masked cytokine according to any one of embodiments 150-191, wherein the first linker comprises a first cleavable peptide; and/or wherein the second linker comprises a second cleavable peptide.

193. The masked cytokine of any one of embodiments 150-192, wherein the first linker comprises a first N-terminal spacer domain and/or a first C-terminal spacer domain.

194. A masked cytokine according to embodiment 193, wherein the first linker comprises:

a) a first N-terminal spacer domain, a first cleavable peptide and a first C-terminal spacer domain;

b) the first N-terminal spacer domain and the first cleavable peptide;

c) the first N-terminal spacer domain and the first C-terminal spacer domain;

d) the first cleavable peptide and the first C-terminal spacer domain;

e) the first N-terminal spacer domain; or

f) the first C-terminal spacer domain.

195. The masked cytokine of any one of embodiments 150-194, wherein the second linker comprises a second N-terminal spacer domain and/or a second C-terminal spacer domain.

- 151 049880

196. A masked cytokine according to embodiment 195, wherein the second linker comprises:

a) a second N-terminal spacer domain, a second cleavable peptide and a second C-terminal spacer domain;

b) a second N-terminal spacer domain and a second cleavable peptide;

c) a second N-terminal spacer domain and a second C-terminal spacer domain;

d) a second cleavable peptide and a second C-terminal spacer domain;

e) a second N-terminal spacer domain; or

f) the second C-terminal spacer domain.

197. The masked cytokine of any one of embodiments 192-196, wherein the first 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/or wherein the second cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 96-153, 236-242, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535 and 538-555.

198. The masked cytokine of any one of embodiments 193-197, wherein the first N-terminal spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-95, 235, 263, 268, 269, 727, 794, 799, and 857-878, and/or the first 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.

199. The masked cytokine of embodiments 192-196, wherein the first 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 NO: 236-242.

200. The masked cytokine of embodiment 199, wherein the amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555 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 the C-terminus of 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.

201. The masked cytokine of embodiments 195-200, wherein the second 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/or the second 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.

202. The masked cytokine of embodiments 192-201, wherein the second 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 NO: 236-242.

203. The masked cytokine of embodiment 202, wherein the amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555 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 the C-terminus of 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.

204. The masked cytokine of any one of embodiments 150-203, wherein the first 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; and/or the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 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.

205. The masked cytokine of any one of embodiments 184-204, wherein the third linker comprises a third N-terminal spacer domain and/or a third C-terminal spacer domain.

206. The masked cytokine of embodiment 205, wherein the third 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/or the third 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.

207. The masked cytokine of any one of embodiments 192-206, wherein

- 152 049880 wherein the first cleavable peptide and/or the second cleavable peptide are a substrate for a protease that is colocalized in the region or tissue expressing the cytokine receptor.

208. A masked cytokine receptor according to embodiment 207, wherein the cytokine receptor is an IL-2 cytokine receptor or an IL-15 cytokine receptor.

209. The masked cytokine of any one of embodiments 184-208, wherein the first cleavable peptide and/or the second cleavable peptide is cleaved by one or more enzymes selected from the group consisting of: ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, 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, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1 and TPSG1.

210. The masked cytokine of any one of embodiments 149-209, wherein the first half-life extending domain and/or the second half-life extending domain is conjugated to the agent.

211. A masked cytokine according to embodiment 210, wherein the agent is a tubulin polymerization inhibitor, a DNA damaging agent, or a DNA synthesis inhibitor.

212. The masked cytokine of embodiment 211, wherein the agent is a maytansinoid, auristatin, pyrrolobenzodiazepine dimer (PBD), calicheamicin, duocarmycin, indolinobenzodiazepine dimer, or an exatecan Dxd derivative.

213. A masked cytokine according to embodiment 210, wherein the agent is an immunostimulant.

214. The masked cytokine of embodiment 213, wherein the immunostimulant is a stimulating interferon gene agonist (STING) or a toll-like receptor (TLR) agonist.

215. The masked cytokine of embodiment 214, wherein the STING agonist is a cyclic dinucleotide (CDN).

216. The masked cytokine of embodiment 215, wherein the CDN is selected from the group consisting of cGAMP, c-di-AMP, c-di-GMP, cAIMP, c-di-IMP, 4-(2-chloro-6-fluorobenzyl)-H-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide.

217. The masked cytokine of embodiment 214, wherein the TLR agonist is a TLR agonist selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10.

218. Masked cytokine containing:

a) a first half-life extension domain and a second half-life extension domain;

b) a first masking fragment and a second masking fragment; and

c) a cytokine or a functional fragment thereof, wherein the first masking fragment is linked to a first half-life extension domain, wherein the second masking fragment is linked to the cytokine or a functional fragment thereof, wherein either the second masking fragment or the cytokine or a functional fragment thereof is linked to a second half-life extension domain, and wherein the first half-life extension domain and the second half-life extension domain contain modifications that facilitate the association of the first and second half-life extension domains.

219. A masked cytokine according to embodiment 218, wherein:

a) the first masking moiety is linked to the first half-life extension domain via a first linker; and/or

b) either the second masking fragment or the cytokine or its functional fragment is associated with

- 153 049880 the second half-life extension domain via the second linker.

220. A masked cytokine according to embodiment 218 or embodiment 219, wherein the second masking moiety is linked to the cytokine or functional fragment thereof via a third linker.

221. A masked cytokine according to any one of embodiments 218-220, wherein the cytokine or functional fragment thereof is an IL-2 polypeptide or functional fragment thereof.

222. The masked cytokine of embodiment 221, wherein the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 160, 243-251, 230, 243-251, 260, 775-792, and 813-822.

223. The masked cytokine of embodiment 221, wherein the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence obtained by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or functional fragment thereof that reduces the affinity of the IL-2 polypeptide or functional fragment thereof for CD25 (IL-2Ra).

224. The masked cytokine of embodiment 223, wherein the amino acid sequence is formed by introducing one or more of the following amino acid substitutions into any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: R38A, F42A, F42K, F42E, K43A, Y45A, Y45N, Y45R, E62A, E62R, E62S, and L72G.

225. The masked cytokine of embodiment 221, wherein the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence obtained by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or functional fragment thereof that increases the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rβ or IL-2Ry.

226. The masked cytokine of embodiment 225, wherein the amino acid sequence is formed by introducing one or more of the following amino acid substitutions into any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: H16I, L18C, D20A, D20L, D20F, N29L, L80F, R81D, L85V, I86V, and I92F.

227. A masked cytokine according to embodiment 223 or embodiment 224, wherein the amino acid sequence is formed by the additional introduction of one or more amino acid substitutions that increase the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rβ or IL-2Ry.

228. The masked cytokine of embodiment 227, wherein the one or more amino acid substitutions that increase the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rβ or IL-2Ry are selected from the group consisting of H16I, L18C, D20A, D20L, D20F, N29L, L80F, R81D, L85V, I86V, and I92F.

229. A masked cytokine according to embodiment 221, wherein the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence obtained by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or functional fragment thereof that stabilizes the IL-2 polypeptide or functional fragment thereof.

230. The masked cytokine of embodiment 229, wherein the amino acid sequence is formed by introducing one of the following amino acid substitutions into any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: C125S, C125A, and C125G.

231. A masked cytokine according to any one of embodiments 223-228, wherein the amino acid sequence is formed by the additional introduction of one or more amino acid substitutions that stabilize the IL-2 polypeptide or functional fragment thereof.

232. The masked cytokine of embodiment 231, wherein the one or more substitutions that stabilize the IL-2 polypeptide or functional fragment thereof are C125S, C125A, or C125G.

233. The masked cytokine of embodiments 221-232, wherein the first 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, 261, 826, and 827, and the second 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, 261, 826, and 827, and wherein the amino acid sequence of the first masking moiety and the amino acid sequence of the second masking moiety are different.

234. A masked cytokine according to any one of embodiments 221-233, wherein:

a) the first masking fragment comprises the amino acid sequence of SEQ ID NO: 9 or 231, and the second masking fragment comprises the amino acid sequence selected

- 154 049880 from the group consisting of SEQ ID NO: 10, 161-165, 187-218, 221-226, 261, 826 and 827; or

b) the first masking fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 161-165, 187-218, 221-226, 261, 826 and 827, and the second masking fragment comprises the amino acid sequence of SEQ ID NO: 9 or 231.

235. The masked cytokine of any one of embodiments 218-220, wherein the cytokine or functional fragment thereof is an IL-15 polypeptide or functional fragment thereof.

236. The masked cytokine of embodiment 235, wherein the IL-15 polypeptide or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 167.

237. A masked cytokine according to embodiment 235 or embodiment 236, wherein:

a) the first mask fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232-234 and 823-825, and the second mask fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 219-229, 261, 826 and 827;

b) the first mask fragment comprises 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 mask fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232-234 and 823-825;

c) the first masking fragment comprises an amino acid sequence obtained by introducing one or more of the following amino acid substitutions into the amino acid sequence of any of SEQ ID NOs: 232-234 and 823-825: R24A, R26A, K34A, S40A, L42A and P67A, and the second masking fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 219-229, 261, 826 and 827; or

d) the first masking fragment comprises 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 fragment comprises an amino acid sequence obtained by introducing one or more of the following amino acid substitutions into the amino acid sequence of any of SEQ ID NOs: 232-234 and 823-825: R24A, R26A, K34A, S40A, L42A and P67A.

238. The masked cytokine of any one of embodiments 218-237, wherein the first half-life extending domain is a first antibody or fragment thereof, and the second half-life extending domain is a second antibody or fragment thereof.

239. A masked cytokine according to embodiment 238, wherein:

a) the first antibody or fragment thereof comprises a first heavy chain polypeptide and the second antibody or fragment thereof comprises a second light chain polypeptide; or

b) the first antibody or fragment thereof comprises a first light chain polypeptide, and the second antibody or fragment thereof comprises a second heavy chain polypeptide.

240. The masked cytokine of embodiment 239, wherein the first heavy chain polypeptide or the second heavy chain polypeptide comprises one or more amino acid substitutions that alter effector function.

241. The masked cytokine of embodiment 240, wherein the first heavy chain polypeptide or the second heavy chain polypeptide:

a) is an IgG1 isotype and contains the following amino acid substitution(s):

i) N297A, N297G or N297Q;

ii) L234A and L235A;

iii) C220S, C226S, C229S and P238S;

iv) C226S, C229S, E233P, L234V and L235A;

v) L234F, L235E and P331S;

vi) S267E and L328F;

vii) D265A;

viii) L234A, L235A and P329G;

b) is an IgG2 isotype and contains the following amino acid substitution(s):

i) V234A and G237A;

ii) H268Q, V309L, A330S and A331S; or iii) V234A, G237A, P238S, H268A, V309L, A330S and P331S; or

c) is an IgG4 isotype and contains the following amino acid substitution(s):

i) L235A, G237A and E318A;

- 155 049880 ii) S228P, L234A and L235A;

iii) H268Q, V309L, A330S and P331S; or iv) S228P and L235A, numbered in accordance with the Kabat EU numbering system.

242. The masked cytokine of embodiment 240, wherein the first heavy chain polypeptide or the second heavy chain polypeptide comprises one or more amino acid substitutions that enhance effector function.

243. The masked cytokine of embodiment 242, wherein the first heavy chain polypeptide or the second heavy chain polypeptide is an IgG1 heavy chain polypeptide and comprises amino acid substitution(s):

a) S298A, E333A and K334A;

b) S239D and I332E;

c) S239D, A330L and I332E;

d) P247I and A339D or A339Q;

e) D280H and K290S;

f) D280H, K290S and either S298D or S298V;

g) F243L, R292P and Y300L;

h) F243L, R292P, Y300L and P396L;

i) F243L, R292P, Y300L, V305I and P396L;

j) G236A, S239D and I332E;

k) K326A and E333A;

l) K326W and E333S;

m) K290E, S298G and T299A;

n) K290E, S298G, T299A and K326E;

o) K290N, S298G and T299A;

p) K290N, S298G, T299A and K326E;

q) K334V;

r) L235S, S239D and K334V;

s) K334V and Q331M, S239D, F243V, E294L or S298T;

t) E233L, Q311M and K334V;

u) L234I, Q311M and K334V;

v) K334V and S298T, A330M or A330F;

w) K334V, Q311M and either A330M or A330F;

x) K334V, S298T and either A330M or A330F;

y) K334V, S239D and either A330M or S298T;

z) L234Y, Y296W and K290Y, F243V or E294L;

aa) Y296W and either L234Y or K290Y;

ab) S239D, A330S and I332E, ac) V264I;

ad) F243L and V264I;

ae) L328M;

af) I332E;

ag) L328M and I332E;

ah) V264I and I332E;

ai) S239E and I332E;

aj) S239Q and I332E;

ak) S239E;

al) A330Y;

am) I332D;

an) L328I and I332E;

ao) L328Q and I332E;

ap) V264T;

aq) V240I;

ar) V266I;

as) S239D;

at) S239D and I332D;

au) S239D and I332N;

av) S239D and I332Q;

aw) S239E and I332D;

ax) S239E and I332N;

ay) S239E and I332Q;

az) S239N and I332D;

ba) S239N and I332E;

- 156 049880 bb) S239Q and I332D;

bc) A330Y and I332E;

bd) V264I, A330Y and I332E;

b) A330L and I332E;

bf) V264I, A330L and I332E;

bg) L234E, L234Y or L234I;

bh) L235D, L235S, L235Y or L235I;

bi) S239T;

bj) V240M;

bk) V264Y;

bl) A330I;

bm) N325T;

bn) I332E and L328D, L328V, L328T or L328I;

bo) V264I, I332E and either S239E or S239Q;

bp) S239E, V264I, A330Y and I332E;

bq) A330Y, I332E and either S239D or S239N;

br) A330L, I332E and either S239D or S239N;

bs) V264I, S298A and I332E;

b) S298A, I332E and either S239D or S239N;

bu) S239D, V264I and I332E;

bv) S239D, V264I, S298A and I332E;

bw) S239D, V264I, A330L and I332E;

bx) S239D, I332E and A330I;

by) P230A;

bz) P230A, E233D and I332E;

ca) E272Y;

cb) K274T, K274E, K274R, K274L or K274Y;

cd) F275W;

ce) N276L;

cf) Y278T;

cg) V302I;

ch) E318R;

ci) S324D, S324I or S324V;

cj) K326I or K326T;

ck) T335D, T335R or T335Y;

cl) V240I and V266I;

cm) S239D, A330Y, I332E and L234I;

cn) S239D, A330Y, I332E and L235D;

co) S239D, A330Y, I332E and V240I;

cp) S239D, A330Y, I332E and V264T; or cq) S239D, A330Y, I332E and either K326E or K326T, numbered in accordance with the Kabat EU numbering system.

244. The masked cytokine of embodiment 239, wherein the first heavy chain polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 168, and 169, and the second heavy chain polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 168, and 169.

245. The masked cytokine of embodiment 239, wherein the first light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 157 or 170, and the second light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 157 or 170.

246. The masked cytokine of embodiment 238, wherein the first antibody or fragment thereof is a first crystallizable fragment domain (Fc domain) or fragment thereof, and the second antibody or fragment thereof is a second Fc domain or fragment thereof.

247. A masked cytokine according to embodiment 246, wherein the first Fc domain or fragment thereof and/or the second Fc domain or fragment thereof comprises one or more amino acid substitutions that alter effector function.

248. The masked cytokine of embodiment 247, wherein the first Fc domain or fragment thereof and/or the second Fc domain or its:

a) is the Fc domain of IgG1 or a fragment thereof and contains amino acid substitution(s):

i) N297A, N297G or N297Q;

- 157 049880 ii) L234A and L235A;

iii) C220S, C226S, C229S and P238S;

iv) C226S, C229S, E233P, L234V and L235A;

v) L234F, L235E and P331S;

vi) S267E and L328F;

vii) D265A;

viii) L234A, L235A and P329G;

b) is the Fc domain of IgG2 or a fragment thereof and contains the amino acid(s) substitution(s):

i) V234A and G237A;

ii) H268Q, V309L, A330S and A331S; or iii) V234A, G237A, P238S, H268A, V309L, A330S and P331S; or

c) is the Fc domain of IgG4 or a fragment thereof and contains the amino acid(s) substitution(s):

i) L235A, G237A and E318A;

ii) S228P, L234A and L235A;

iii) H268Q, V309L, A330S and P331S; or iv) S228P and L235A, numbered in accordance with the Kabat EU numbering system.

249. A masked cytokine according to embodiment 247, wherein the first Fc domain or fragment thereof and/or the second Fc domain or fragment thereof comprises one or more amino acid substitutions that enhance effector function.

250. The masked cytokine of embodiment 249, wherein the first Fc domain or fragment thereof and/or the second Fc domain or fragment thereof is an IgG1 Fc domain or fragment thereof and comprises amino acid substitution(s):

a) S298A, E333A and K334A;

b) S239D and I332E;

c) S239D, A330L and I332E;

d) P247I and A339D or A339Q;

e) D280H and K290S;

f) D280H, K290S and either S298D or S298V;

g) F243L, R292P and Y300L;

h) F243L, R292P, Y300L and P396L;

i) F243L, R292P, Y300L, V305I and P396L;

j) G236A, S239D and I332E;

k) K326A and E333A;

l) K326W and E333S;

m) K290E, S298G and T299A;

n) K290E, S298G, T299A and K326E;

o) K290N, S298G and T299A;

p) K290N, S298G, T299A and K326E;

q) K334V;

r) L235S, S239D and K334V;

s) K334V and Q331M, S239D, F243V, E294L or S298T;

t) E233L, Q311M and K334V;

u) L234I, Q311M and K334V;

v) K334V and S298T, A330M or A330F;

w) K334V, Q311M and either A330M or A330F;

x) K334V, S298T and either A330M or A330F;

y) K334V, S239D and either A330M or S298T;

z) L234Y, Y296W and K290Y, F243V or E294L;

aa) Y296W and either L234Y or K290Y;

ab) S239D, A330S and I332E, ac) V264I;

ad) F243L and V264I;

ae) L328M;

af) I332E;

ag) L328M and I332E;

ah) V264I and I332E;

ai) S239E and I332E;

aj) S239Q and I332E;

ak)S239E;

al) A330Y;

- 158 049880 am) I332D;

an) L328I and I332E;

ao) L328Q and I332E;

ap) V264T;

aq) V240I;

ar) V266I;

as) S239D;

at) S239D and I332D;

au) S239D and I332N;

av) S239D and I332Q;

aw) S239E and I332D;

ax) S239E and I332N;

ay) S239E and I332Q;

az) S239N and I332D;

ba) S239N and I332E;

bb) S239Q and I332D;

bc) A330Y and I332E;

bd) V264I, A330Y and I332E;

b) A330L and I332E;

bf) V264I, A330L and I332E;

bg) L234E, L234Y or L234I;

bh) L235D, L235S, L235Y or L235I;

bi) S239T;

bj) V240M;

bk) V264Y;

bl) A330I;

bm) N325T;

bn) I332E and L328D, L328V, L328T or L328I;

bo) V264I, I332E and either S239E or S239Q;

bp) S239E, V264I, A330Y and I332E;

bq) A330Y, I332E and either S239D or S239N;

br) A330L, I332E and either S239D or S239N;

bs) V264I, S298A and I332E;

b) S298A, I332E and either S239D or S239N;

bu) S239D, V264I and I332E;

bv) S239D, V264I, S298A and I332E;

bw) S239D, V264I, A330L and I332E;

bx) S239D, I332E and A330I;

by) P230A;

bz) P230A, E233D and I332E;

ca) E272Y;

cb) K274T, K274E, K274R, K274L or K274Y;

cd) F275W;

ce) N276L;

cf) Y278T;

cg) V302I;

ch) E318R;

ci) S324D, S324I or S324V;

cj) K326I or K326T;

ck) T335D, T335R or T335Y;

cl) V240I and V266I;

cm) S239D, A330Y, I332E and L234I;

cn) S239D, A330Y, I332E and L235D;

co) S239D, A330Y, I332E and V240I;

cp) S239D, A330Y, I332E and V264T; or cq) S239D, A330Y, I332E and either K326E or K326T, numbered in accordance with the Kabat EU numbering system.

251. A masked cytokine according to embodiment 246, wherein:

a) 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;

b) the first Fc domain or fragment thereof comprises the amino acid sequence SEQ ID NO:

- 159 049880

156, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155;

c) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 154, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 154;

d) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 265, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156;

e) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 265;

f) 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: 616;

g) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 616, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155;

h) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 157, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 158;

i) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 158, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 157;

j) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 774;

k) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 774, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796;

l) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 619;

m) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 619, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721;

n) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 772;

o) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 772, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721;

p) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 622;

q) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 622, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793;

r) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 773;

s) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 773, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793;

t) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625;

u) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796;

v) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156;

- 160 049880

w) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625; or

x) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796.

252. The masked cytokine of embodiments 218-237, wherein the first half-life extending domain is a first scFv or fragment thereof, and the second half-life extending domain is a second scFv or fragment thereof.

253. The masked cytokine of embodiments 218-237, wherein the first half-life extending domain is a first Fc domain or fragment thereof, and the second half-life extending domain is a second Fc domain or fragment thereof, and wherein the first Fc domain or fragment thereof is linked to the second Fc domain or fragment thereof.

254. The masked cytokine of embodiment 253, wherein the first Fc domain or fragment thereof is linked to the second Fc domain or fragment thereof via a fourth linker.

255. A masked cytokine according to any one of embodiments 218-237, wherein:

(a) the first half-life extending domain is an scFv or a fragment thereof, and the second half-life extending domain is an antibody or a fragment thereof; or

a) the first half-life extending domain is an antibody or a fragment thereof, and the second half-life extending domain is an scFv or a fragment thereof.

256. A masked cytokine according to any one of paragraphs 238-251, wherein the modifications facilitating the association of the first and second half-life extending domains comprise:

a) introducing the mutations S354C and T366W into a first antibody or fragment thereof and introducing the mutations Y349C, T366S, L368A and Y407V into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

b) introducing the mutations S354C and T366W into the second antibody or fragment thereof and introducing the mutations Y349C, T366S, L368A and Y407V into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

c) introducing the mutations K392D and K409D into the first antibody or fragment thereof and introducing the mutations D399K and E356K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

d) introducing the mutations K392D and K409D into the second antibody or fragment thereof and introducing the mutations D399K and E356K into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

e) the introduction of the mutations S364H and F405A into the first antibody or fragment thereof and the introduction of the mutations Y349T and T394F into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system; or

f) the introduction of mutations S364H and F405A into the second antibody or fragment thereof and the introduction of mutations Y349T and T394F into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system.

257. The masked cytokine of any one of embodiments 238-251, wherein the amino acid sequence of the first antibody or fragment thereof and the amino acid sequence of the second antibody or fragment thereof are formed by:

a) introducing the mutations S354C and T366W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations Y349C, T366S, L368A and Y407V into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

b) introducing the mutations S354C and T366W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations Y349C, T366S, L368A and Y407V into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

c) introducing the mutations K392D and K409D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

d) introducing the mutations K392D and K409D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

e) introducing the mutations S364H and F405A into the amino acid sequence of SEQ ID NO: 154 or

169, and the introduction of mutations Y349T and T394F into the amino acid sequence of SEQ ID NO: 154 or

169, respectively, numbered in accordance with the Kabat EU numbering system; or

f) introducing the mutations S364H and F405A into the amino acid sequence of SEQ ID NO: 154 or

169, and the introduction of mutations Y349T and T394F into the amino acid sequence of SEQ ID NO: 154 or

- 161 049880

169, respectively, numbered in accordance with the Kabat EU numbering system.

258. A masked cytokine according to any one of paragraphs 238-251, wherein the modifications facilitating the association of the first and second half-life extending domains comprise:

a) the introduction of a Y407T mutation into a first antibody or fragment thereof and the introduction of a T366Y mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

b) the introduction of a Y407A mutation into a first antibody or fragment thereof and the introduction of a T366W mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

c) the introduction of the F405A mutation into the first antibody or fragment thereof and the introduction of the T394W mutation into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

d) the introduction of the F405W mutation into the first antibody or fragment thereof and the introduction of the T394S mutation into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

e) introducing a Y407T mutation into a first antibody or fragment thereof and introducing a T366Y mutation into a second antibody or fragment thereof, numbered according to the Kabat EU numbering system;

f) the introduction of the mutations T366Y and F405A into the first antibody or fragment thereof and the introduction of the mutations T394W and Y407T into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

g) introducing the mutations T366W and F405W into the first antibody or fragment thereof and introducing the mutations T394S and Y407A into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

h) introducing the mutations F405W and Y407A into the first antibody or fragment thereof and introducing the mutations T366W and T394S into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

i) introducing the T366W mutation into the first antibody or fragment thereof and introducing the T366S, L368A and Y407V mutations into the second antibody or fragment thereof, numbered according to the Kabat EU numbering system;

j) introducing a Y407T mutation into a second antibody or fragment thereof and introducing a T366Y mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

k) introducing a Y407A mutation into a second antibody or fragment thereof and introducing a T366W mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

l) the introduction of the F405A mutation into the second antibody or fragment thereof and the introduction of the T394W mutation into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

m) introducing the F405W mutation into the second antibody or fragment thereof and introducing the T394S mutation into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

n) introducing a Y407T mutation into a second antibody or fragment thereof and introducing a T366Y mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

o) the introduction of the mutations T366Y and F405A into the second antibody or fragment thereof and the introduction of the mutations T394W and Y407T into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

p) introducing the mutations T366W and F405W into the second antibody or fragment thereof and introducing the mutations T394S and Y407A into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

q) the introduction of the mutations F405W and Y407A into the second antibody or fragment thereof and the introduction of the mutations T366W and T394S into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system; or

r) introducing the mutation T366W into the second antibody or fragment thereof and introducing the mutations T366S, L368A and Y407V into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system.

259. The masked cytokine of any one of embodiments 238-251, wherein the amino acid sequence of the first antibody or fragment thereof and the amino acid sequence of the second antibody or fragment thereof are formed by:

a) introducing a Y407T mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366Y mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

b) introducing a Y407A mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366W mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

c) introducing the mutation F405A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation T394W into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

d) introducing the mutation F405W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation T394S into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

- 162 049880

e) introducing a Y407T mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366Y mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

f) introducing the mutations T366Y and F405A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T394W and Y407T into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

g) introducing the mutations T366W and F405W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T394S and Y407A into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

h) introducing the mutations F405W and Y407A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T366W and T394S into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

i) introducing the mutation T366W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T366S, L368A and Y407V into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

j) introducing a Y407T mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366Y mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

k) introducing a Y407A mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366W mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

l) introducing the mutation F405A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation T394W into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

m) introducing the mutation F405W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation T394S into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

n) introducing a Y407T mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366Y mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

o) introducing the mutations T366Y and F405A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T394W and Y407T into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

p) introducing the mutations T366W and F405W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T394S and Y407A into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

q) introducing the mutations F405W and Y407A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T366W and T394S into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system; or

r) introducing the mutation T366W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T366S, L368A and Y407V into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system.

260. A masked cytokine according to any one of paragraphs 238-251, wherein the modifications facilitating the association of the first and second half-life extending domains comprise:

a) the introduction of a K409E mutation into a first antibody or fragment thereof and the introduction of a D399K mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

b) the introduction of a K409E mutation into a first antibody or fragment thereof and the introduction of a D399R mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

c) the introduction of a K409D mutation into a first antibody or fragment thereof and the introduction of a D399K mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

d) introducing a K409D mutation into a first antibody or fragment thereof and introducing a D399R mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

e) introducing a K392E mutation into a first antibody or fragment thereof and introducing a D399R mutation into a second antibody or fragment thereof, numbered according to the Kabat EU numbering system;

f) the introduction of a K392E mutation into a first antibody or fragment thereof and the introduction of a D399K mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

g) the introduction of a K392D mutation into a first antibody or fragment thereof and the introduction of a D399R mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

h) the introduction of a K392D mutation into a first antibody or fragment thereof and the introduction of a D399K mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

i) the introduction of mutations K409D and K360D into the first antibody or fragment thereof and the introduction of mutations D399K and E356K into the second antibody or fragment thereof, numbered in accordance with the system

- 163 049880 Kabat EU numbering;

j) introducing the mutations K409D and K370D into the first antibody or fragment thereof and introducing the mutations D399K and E357K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

k) introducing the mutations K409D and K392D into the first antibody or fragment thereof and introducing the mutations D399K, E356K and E357K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

l) the introduction of the mutations K409D and K392D into the first antibody or fragment thereof and the introduction of the mutation D399K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

m) introducing the mutations K409D and K392D into the first antibody or fragment thereof and introducing the mutations D399K and E356K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

n) introducing the mutations K409D and K392D into the first antibody or fragment thereof and introducing the mutations D399K and E357K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

o) the introduction of the mutations K409D and K370D into the first antibody or fragment thereof and the introduction of the mutations D399K and E357K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

p) introducing the mutation D399K into the first antibody or fragment thereof and introducing the mutations K409D and K360D into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

q) introducing the mutations K409D and K439D into the first antibody or fragment thereof and introducing the mutations D399K and E356K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

r) the introduction of a K409E mutation into a second antibody or fragment thereof and the introduction of a D399K mutation into a first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

s) introducing a K409E mutation into a second antibody or fragment thereof and introducing a D399R mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

t) introducing a K409D mutation into a second antibody or fragment thereof and introducing a D399K mutation into a first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

u) introducing a K409D mutation into a second antibody or fragment thereof and introducing a D399R mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

v) the introduction of a K392E mutation into a second antibody or fragment thereof and the introduction of a D399R mutation into a first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

w) introducing a K392E mutation into a second antibody or fragment thereof and introducing a D399K mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

x) the introduction of a K392D mutation into a second antibody or fragment thereof and the introduction of a D399R mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

y) introducing a K392D mutation into a second antibody or fragment thereof and introducing a D399K mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

z) introducing the mutations K409D and K360D into the second antibody or fragment thereof and introducing the mutations D399K and E356K into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

aa) introducing the mutations K409D and K370D into the second antibody or fragment thereof and introducing the mutations D399K and E357K into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

ab) introducing the mutations K409D and K392D into the second antibody or fragment thereof and introducing the mutations D399K, E356K and E357K into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

ac) introducing the mutations K409D and K392D into the second antibody or fragment thereof and introducing the mutation D399K into the first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

ad) introducing the mutations K409D and K392D into the second antibody or fragment thereof and introducing the mutations D399K and E356K into the first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

ae) introducing the mutations K409D and K392D into the second antibody or fragment thereof and introducing the mutations D399K and E357K into the first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

af) introducing the mutations K409D and K370D into the second antibody or fragment thereof and introducing the mutations D399K and E357K into the first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

- 164 049880 ag) introducing the D399K mutation into the second antibody or fragment thereof and introducing the K409D and K360D mutations into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system; or ah) introducing the K409D and K439D mutations into the second antibody or fragment thereof and introducing the D399K and E356K mutations into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system.

261. The masked cytokine of any one of embodiments 238-251, wherein the amino acid sequence of the first antibody or fragment thereof and the amino acid sequence of the second antibody or fragment thereof are formed by:

a) introducing a K409E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

b) introducing a K409E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

c) introducing a K409D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

d) introducing a K409D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

e) introducing a K392E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

f) introducing a K392E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

g) introducing a K392D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

h) introducing a K392D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

i) introducing the mutations K409D and K360D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

j) introducing the mutations K409D and K370D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

k) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K, E356K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

l) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation D399K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

m) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

n) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

o) introducing the mutations K409D and K370D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

p) introducing the mutation D399K into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations K409D and K360D into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

q) introducing the mutations K409D and K439D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

r) introducing a K409E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169,

- 165 049880 respectively, numbered in accordance with the Kabat EU numbering system;

s) introducing a K409E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

t) introducing a K409D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

u) introducing a K409D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

v) introducing a K392E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

w) introducing a K392E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

x) introducing a K392D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

y) introducing a K392D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

z) introducing the mutations K409D and K360D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

aa) introducing the mutations K409D and K370D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

ab) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K, E356K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

ac) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation D399K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

ad) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, numbered according to the Kabat EU numbering system;

ae) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

af) introducing the mutations K409D and K370D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

ag) introducing the mutation D399K into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations K409D and K360D into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system; or ah) introducing the mutations K409D and K439D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system.

262. The masked cytokine of any one of embodiments 219-261, wherein the first linker comprises a first cleavable peptide; and/or wherein the second linker comprises a second cleavable peptide.

263. The masked cytokine of any one of embodiments 219-262, wherein the first linker comprises a first N-terminal spacer domain and/or a first C-terminal spacer domain.

264. A masked cytokine according to embodiment 263, wherein the first linker comprises:

a) a first N-terminal spacer domain, a first cleavable peptide and a first C-terminal spacer domain;

b) the first N-terminal spacer domain and the first cleavable peptide;

c) the first N-terminal spacer domain and the first C-terminal spacer domain;

d) the first cleavable peptide and the first C-terminal spacer domain;

- 166 049880

e) the first N-terminal spacer domain; or

f) the first C-terminal spacer domain.

265. The masked cytokine of any one of embodiments 219-264, wherein the second linker comprises a second N-terminal spacer domain and/or a second C-terminal spacer domain.

266. The masked cytokine of embodiment 265, wherein the second linker comprises:

a) a second N-terminal spacer domain, a second cleavable peptide and a second C-terminal spacer domain;

b) a second N-terminal spacer domain and a second cleavable peptide;

c) a second N-terminal spacer domain and a second C-terminal spacer domain;

d) a second cleavable peptide and a second C-terminal spacer domain;

e) a second N-terminal spacer domain; or

f) the second C-terminal spacer domain.

267. The masked cytokine of embodiments 262-266, wherein the first 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/or wherein the second cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 96-153, 236-242, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535 and 538-555.

268. The masked cytokine of embodiments 263-267, wherein the first 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/or wherein the first 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.

269. The masked cytokine of embodiments 262-266, wherein the first 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 NO: 236-242.

270. The masked cytokine of embodiment 269, wherein the amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555 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 the C-terminus of 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.

271. The masked cytokine of embodiments 265-270, wherein the second 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/or the second 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.

272. The masked cytokine of embodiments 262-271, wherein the second 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 NO: 236-242.

273. The masked cytokine of embodiment 272, wherein the amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555 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 the C-terminus of 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.

274. The masked cytokine of embodiments 218-273, wherein the first 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 and/or the second 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.

275. The masked cytokine of any one of embodiments 219-274, wherein the third linker comprises a cleavable peptide.

276. The masked cytokine of any one of embodiments 219-275, wherein

- 167 049880 wherein the third linker comprises a third N-terminal spacer domain and/or a third C-terminal spacer domain.

277. The masked cytokine of embodiment 276, wherein the third linker comprises:

a) a third N-terminal spacer domain, a third cleavable peptide and a third C-terminal spacer domain;

b) the third N-terminal spacer domain and the third cleavable peptide;

c) a third N-terminal spacer domain and a third C-terminal spacer domain;

d) the third cleavable peptide and the third C-terminal spacer domain;

e) a third N-terminal spacer domain; or

f) the third C-terminal spacer domain.

278. The masked cytokine of embodiments 275-277, wherein the third 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/or wherein the third cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 96-153, 236-242, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535 and 538-555.

279. The masked cytokine of embodiments 276-278, wherein the third 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/or the third 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.

280. The masked cytokine of embodiments 275-279, wherein the third 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 NO: 236-242.

281. The masked cytokine of embodiment 280, wherein the amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555 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 the C-terminus of 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.

282. The masked cytokine of embodiments 219-281, wherein the third 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, and/or the third 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.

283. The masked cytokine of any one of embodiments 254-282, wherein the fourth linker comprises a fourth N-terminal spacer domain and/or a fourth C-terminal spacer domain.

284. The masked cytokine of embodiment 283, wherein the fourth 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/or the fourth 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.

285. A masked cytokine according to any one of embodiments 262-284, wherein the first cleavable peptide, the second cleavable peptide and/or the third cleavable peptide is a substrate for a protease that is colocalized in a region or tissue expressing a cytokine receptor.

286. A masked cytokine receptor according to embodiment 285, wherein the cytokine receptor is an IL-2 cytokine receptor or an IL-15 cytokine receptor.

287. The masked cytokine of any one of embodiments 262-286, wherein the first cleavable peptide, the second cleavable peptide, and/or the third cleavable peptide are cleaved by one or more enzymes selected from the group consisting of: ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE

- 168 049880

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, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1 and TPSG1.

288. The masked cytokine of any one of embodiments 218-287, wherein the first half-life extending domain and/or the second half-life extending domain is conjugated to the agent.

289. A masked cytokine according to embodiment 288, wherein the agent is a tubulin polymerization inhibitor, a DNA damaging agent, or a DNA synthesis inhibitor.

290. The masked cytokine of embodiment 289, wherein the agent is a maytansinoid, an auristatin, a pyrrolobenzodiazepine dimer (PBD), a calicheamicin, a duocarmycin, an indolinobenzodiazepine dimer, or an exatecan Dxd derivative.

291. A masked cytokine according to embodiment 288, wherein the agent is an immunostimulant.

292. The masked cytokine of embodiment 291, wherein the immunostimulant is a stimulating interferon gene agonist (STING) or a toll-like receptor (TLR) agonist.

293. The masked cytokine of embodiment 292, wherein the STING agonist is a cyclic dinucleotide (CDN).

294. The masked cytokine of embodiment 293, wherein the CDN is selected from the group consisting of cGAMP, c-di-AMP, c-di-GMP, cAIMP, c-di-IMP, 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide.

295. The masked cytokine of embodiment 292, wherein the TLR agonist is a TLR agonist selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10.

296. A nucleic acid encoding a masked cytokine according to any one of embodiments 1-295 and 320-409.

297. A vector comprising a nucleic acid according to embodiment 296.

298. The vector according to embodiment 297, which is an expression vector.

299. A host cell comprising a nucleic acid according to embodiment 296.

300. A method for producing a masked cytokine, comprising culturing a host cell according to embodiment 299 under conditions that result in the formation of the masked cytokine.

301. The method of embodiment 300, further comprising recovering the masked cytokine produced by the host cell.

302. A masked cytokine generated by the method of embodiment 300 or embodiment 301.

303. A composition comprising a masked cytokine according to any one of embodiments 1-295 and 320-409.

304. A composition comprising a masked cytokine according to embodiment 302.

305. The composition according to embodiment 303 or embodiment 304, further comprising an anti-inflammatory agent or an anti-cancer agent.

306. The composition of embodiment 305, wherein the anti-cancer agent is selected from the group consisting of a PD-1 inhibitor, an EGFR inhibitor, a HER2 inhibitor, a VEGFR inhibitor, a CTLA-4 inhibitor, a BTLA inhibitor, a B7H4 inhibitor, a B7H3 inhibitor, a CSFIR inhibitor, an HVEM inhibitor, a CD27 inhibitor, a KIR inhibitor, an NKG2A inhibitor, an NKG2D agonist, a TWEAK inhibitor, an ALK inhibitor, an antibody targeting CD52, an antibody targeting CCR4, a PD-L1 inhibitor, a KIT inhibitor, a PDGFR inhibitor, a BAFF inhibitor, an HDAC inhibitor, a VEGF ligand inhibitor, a molecule targeting CD19, a molecule targeting FOLR1, DLL3-targeting molecule, DKK1-targeting molecule, MUC1-targeting molecule, MUC16-targeting molecule,

- 169 049880 targeting PSMA, targeting MSLN, targeting NY-ES0-1, targeting B7H3, targeting B7H4, targeting BCMA, targeting CD29, targeting CD151, targeting CD123, targeting CD33, targeting CD37, targeting CDH19, targeting CEA, targeting claudin 18.2, targeting CLEC12A, targeting EGFRVIII, targeting EPCAM, targeting EPHA2, FCRH5 targeting molecule, FLT3 targeting molecule, GD2 targeting molecule, glypican 3 targeting molecule, gpA33 targeting molecule, GPRC5D targeting molecule, IL-23R targeting molecule, IL1RAP targeting molecule, MCSP targeting molecule, RON targeting molecule, ROR1 targeting molecule, STEAP2 targeting molecule, TfR targeting molecule, CD166 targeting molecule, TPBG targeting molecule, TROP2 targeting molecule, proteasome inhibitor, ABL inhibitor, CD30 inhibitor, FLT3 inhibitor, MET inhibitor, RET, ^-1β inhibitor. MEK inhibitor, ROS1 inhibitor, BRAF inhibitor, CD38 inhibitor, RANKL inhibitor, B4GALNT1 inhibitor, SLAMF7 inhibitor, IDH2 inhibitor, mTOR inhibitor, CD20 targeting molecule, BTK inhibitor, PI3K inhibitor, FLT3 inhibitor, PARP inhibitor, CDK4 inhibitor, CDK6 inhibitor, FGFR inhibitor, RAF inhibitor, JAK1 inhibitor, JAK2 inhibitor, JAK3 inhibitor, IL-6 inhibitor, IL-17 inhibitor, Smoothened inhibitor, IL-6R inhibitor, BCL2 inhibitor, PTCH inhibitor, PIGF inhibitor, TGFB inhibitor, CD28 agonist, CD3 agonist, CD40 agonist, GITR agonist, OX40, VISTA agonist, CD137 agonist, LAG3 inhibitor, TIM3 inhibitor, TIGIT inhibitor and IL-2R inhibitor.

307. The composition according to embodiment 305, wherein the anti-inflammatory agent is a cyclooxygenase (COX) inhibitor or an NF-kB inhibitor.

308. The composition according to embodiment 307, wherein the COX inhibitor is a COX-1 and/or COX-2 inhibitor.

309. The composition of embodiment 307 or embodiment 308, wherein the COX inhibitor is selected from the group consisting of SC-560, FR122047, P6, mofezolac, TFAP, flurbiprofen, ketoprofen, celecoxib, rofecoxib, meloxicam, piroxicam, deracoxib, parecoxib, valdecoxib, etoricoxib, chromene derivative, chromane derivative, 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, flosulide, NS-398, L745337, RWJ-63556, L-784512, darbufelone, CS-502, LAS-34475, LAS-34555, S-33516, diclofenac, mefenamic acid, SD-8381, ibuprofen, naproxen, ketorolac, indomethacin, aspirin, naproxen, tolmetin, piroxicam, and meclofenamate.

310. The composition according to embodiment 307, wherein the NF-kB inhibitor is selected from the group consisting of an IKK complex inhibitor, an IkB cleavage inhibitor, an NF-kB nuclear translocation inhibitor, a p65 acetylation inhibitor, an NF-kB DNA binding inhibitor, an NF-kB transactivation inhibitor, and an p53 induction inhibitor.

311. The composition of embodiment 307 or embodiment 308, wherein the NF-kB inhibitor is selected from the group consisting of TPCA-1, NFkB Activation Inhibitor VI (BOT-64), BMS-345541, amlexanox, SC-514 (GK-01140), IMD-0354, IKK-16, BAY-11-7082, MG115, MG-132, lactacystin, epoxomicin, parthenolide, carfilzomib, MLN-4924 (pevonedistate), JSH-23, rolipram, gallic acid, anacardic acid, GYY-4137, p-XSC, CV-3988, prostaglandin E2 (PGE2), LY-294002, wortmannin, mesalamine, quinacrine and flavopiridol.

312. A pharmaceutical composition comprising a masked cytokine according to any one of embodiments 1-295 and 320-409 and a pharmaceutically acceptable carrier.

313. A pharmaceutical composition comprising a masked cytokine according to embodiment 302 and a pharmaceutically acceptable carrier.

314. A kit comprising a masked cytokine according to any one of embodiments 1-295 and 320-409, or a composition according to any one of embodiments 303-311, or a pharmaceutical composition according to embodiment 312 or 313.

315. A method of treating or preventing a neoplastic disease in a subject, comprising administering to the subject an effective amount of a masked cytokine according to any one of embodiments 1-295 and 320-409, or a composition according to any one of embodiments 303-311.

316. The method according to embodiment 315, wherein the neoplastic disease is cancer.

317. The method according to embodiment 316, wherein the cancer is leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer,

- 170 049880 osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer or testicular cancer.

318. A method of treating or preventing an inflammatory or autoimmune disease in a subject, comprising administering to the subject an effective amount of a masked cytokine according to any one of embodiments 1-295 and 320-409, or a composition according to any one of embodiments 303-311.

319. The method of embodiment 318, 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, Sjogren's syndrome, polymyalgia rheumatica, multiple sclerosis (MS), ankylosing spondylitis, type 1 diabetes mellitus, alopecia areata, vasculitis and temporal arteritis, graft versus host disease (GVHD), asthma, COPD, paraneoplastic autoimmune disease, inflammatory cartilage, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, juvenile rheumatoid arthritis of systemic onset, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative reaction, enthesopathy, articular syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, rheumatoid arthritis of systemic onset, enteropathic arthritis, reactive arthritis, Reiter's syndrome, dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myolitis, polymyolitis, dermatomyolitis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, Crohn's disease, ulcerative colitis, celiac disease, rhinosinusitis, rhinosinusitis with polyps, eosinophilic esophogitis, eosinophilic bronchitis, Guillain-Barré disease, thyroiditis (eg, Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, transplant rejection, renal injury, hepatitis C-induced vasculitis, and spontaneous pregnancy loss.

320. Masked cytokine containing:

a) a first half-life extension domain comprising the amino acid sequence of SEQ ID NO: 155 and a second half-life extension domain comprising the amino acid sequence of SEQ ID NO: 156;

b) a masking fragment 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 fragment is linked to a first half-life extension domain, wherein the cytokine or a functional fragment thereof is linked to a second half-life extension domain, and wherein the first half-life extension domain and the second half-life extension domain comprise modifications that facilitate the association of the first and second half-life extension domains.

321. A masked cytokine according to embodiment 320, wherein the masking moiety is linked to the first half-life extension domain via a first linker, and wherein the cytokine or functional fragment thereof is linked to the second half-life extension domain via a second linker.

322. A masked cytokine according to embodiment 321, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 28.

323. A masked cytokine according to embodiment 321 or embodiment 322, wherein the second linker comprises a cleavable peptide comprising the amino acid sequence of SEQ ID NO: 264.

324. The masked cytokine of any one of embodiments 321-323, wherein the second linker comprises the amino acid sequence of SEQ ID NO: 811.

325. A masked cytokine according to any one of embodiments 320-324, wherein the masked cytokine comprises the amino acid sequence of SEQ ID NO: 266.

326. The masked cytokine of any one of embodiments 320-325, wherein the masked cytokine comprises the amino acid sequence of SEQ ID NO: 267.

327. A masked cytokine according to any one of embodiments 320-326, wherein the masked cytokine comprises the amino acid sequences of SEQ ID NOs: 266 and 267.

328. A masked cytokine according to embodiment 1, wherein the masked cytokine comprises an amino acid sequence selected from the group consisting of

- 171 049880 of SEQ ID NO: 585-597, 602, 610-614, 627-636, 642 and 643.

329. A masked cytokine according to embodiment 70, wherein the masked cytokine comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 567 and 598-601.

330. The masked cytokine of embodiment 149, wherein the masked cytokine comprises the amino acid sequences of: SEQ ID NOs: 562 and 563; or SEQ ID NOs: 608 and 603; or SEQ ID NOs: 604 and 603; or SEQ ID NOs: 605 and 603; or SEQ ID NOs: 606 and 603; or SEQ ID NOs: 615 and 617; or SEQ ID NOs: 266 and 267; or SEQ ID NOs: 618 and 620; or SEQ ID NOs: 621 and 623; or SEQ ID NOs: 624 and 626; or SEQ ID NOs: 608 and 267; or SEQ ID NOs: 663 and 664; or SEQ ID NOs: 665 and 666; or SEQ ID NOs: 667 and 267; or SEQ ID NOs: 669 and 267; or SEQ ID NOs: 670 and 671; or SEQ ID NOs: 670 and 671; or SEQ ID NOs: 672 and 267; or SEQ ID NOs: 673 and 267; or SEQ ID NOs: 674 and 267; or SEQ ID NOs: 675 and 267; or SEQ ID NOs: 676 and 267; or SEQ ID NOs: 677 and 267; or SEQ ID NOs: 678 and 267; or SEQ ID NOs: 679 and 267; or SEQ ID NOs: 680 and 267; or SEQ ID NOs: 681 and 267; or SEQ ID NOs: 682 and 267; or SEQ ID NOs: 683 and 267; or SEQ ID NOs: 684 and 267; or SEQ ID NOs: 685 and 267; or SEQ ID NOs: 686 and 267; SEQ ID NOs: 687 and 267; or SEQ ID NOs: 688 and 267; or SEQ ID NOs: 689 and 267; or SEQ ID NOs: 690 and 267; or SEQ ID NOs: 692 and 267; or SEQ ID NOs: 693 and 267; or SEQ ID NOs: 694 and 267; or SEQ ID NOs: 695 and 267; or SEQ ID NOs: 696 and 267; or SEQ ID NOs: 697 and 267; or SEQ ID NOs: 698 and 267; or SEQ ID NOs: 699 and 267; or SEQ ID NOs: 700 and 267; or SEQ ID NOs: 701 and 267; or SEQ ID NOs: 702 and 267; or SEQ ID NOs: 703 and 267; or SEQ ID NOs: 704 and 267; or SEQ ID NOs: 705 and 267; or SEQ ID NOs: 706 and 267; or SEQ ID NOs: 707 and 267; or SEQ ID NOs: 708 and 267; or SEQ ID NOs: 709 and 267; or SEQ ID NOs: 710 and 267; or SEQ ID NOs: 711 and 267; or SEQ ID NOs: 712 and 667; or SEQ ID NOs: 713 and 267; or SEQ ID NOs: 714 and 267; or SEQ ID NOs: 716 and 699; or SEQ ID NOs: 717 and 267; or SEQ ID NOs: 718 and 267; or SEQ ID NOs: 719 and 267; or SEQ ID NOs: 720 and 267; or SEQ ID NOs: 722 and 267; or SEQ ID NOs: 723 and 267; or SEQ ID NOs: 720 and 267; or SEQ ID NOs: 728 and 267; or SEQ ID NOs: 729 and 267; or SEQ ID NOs: 730 and 267; or SEQ ID NOs: 731 and 267; or SEQ ID NOs: 732 and 267; or SEQ ID NOs: 733 and 267; or SEQ ID NOs: 734 and 267; or SEQ ID NOs: 735 and 267; or SEQ ID NOs: 736 and 267; or SEQ ID NOs: 737 and 267; or SEQ ID NOs: 738 and 267; or SEQ ID NOs: 739 and 267; or SEQ ID NOs: 740 and 267; or SEQ ID NOs: 741 and 267; or SEQ ID NOs: 742 and 267; or SEQ ID NOs: 743 and 267; or SEQ ID NOs: 744 and 267; or SEQ ID NOs: 745 and 267; or SEQ ID NOs: 746 and 267; or SEQ ID NOs: 674 and 828; or SEQ ID NOs: 674 and 829; or SEQ ID NOs: 726 and 830; or SEQ ID NOs: 726 and 829; or SEQ ID NOs: 747 and 671; or SEQ ID NOs: 715 and 267; or SEQ ID NOs: 715 and 671; or SEQ ID NOs: 748 and 671; or SEQ ID NOs: 749 and 671; or SEQ ID NOs: 750 and 671; or SEQ ID NOs: 751 and 671; or SEQ ID NOs: 752 and 671; or SEQ ID NOs: 753 and 671; or SEQ ID NOs: 754 and 671; or SEQ ID NOs: 758 and 671; or SEQ ID NOs: 759 and 671; or SEQ ID NOs: 760 and 671; or SEQ ID NO: 761 and 671.

331. A masked cytokine according to embodiment 218, wherein the masked cytokine comprises the amino acid sequences of: SEQ ID NOs: 755 and 616; or SEQ ID NOs: 756 and 616; or SEQ ID NOs: 757 and 616.

332. Masked cytokine containing:

a) a first half-life extension domain and a second half-life extension domain;

b) a first masking fragment and a second masking fragment; and

c) a cytokine or a functional fragment thereof, wherein the first masking fragment is linked to a first half-life extension domain, wherein the second masking fragment is linked to the first masking fragment, wherein the cytokine or a functional fragment thereof is linked to a second half-life extension domain, and wherein the first half-life extension domain and the second half-life extension domain contain modifications that facilitate the association of the first and second half-life extension domains.

333. A masked cytokine according to embodiment 332, wherein:

a) the first masking moiety is linked to the first half-life extension domain via a first linker; and/or

b) the second masking fragment is linked to the first masking fragment via a second linker.

334. A masked cytokine according to embodiment 332 or 333, wherein the cytokine or functional fragment thereof is linked to the second half-life extending domain via a third linker.

335. The masked cytokine of any one of embodiments 332-334, wherein the cytokine or functional fragment thereof is an IL-2 polypeptide or functional fragment thereof.

336. The masked cytokine of embodiment 335, wherein the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 160, 243-251, 230, 243-251, 260, 775-792, and 813-822.

337. The masked cytokine of embodiment 335, wherein the polypeptide

- 172 049880

IL-2 or a functional fragment thereof comprises an amino acid sequence obtained by introducing one or more amino acid substitutions into the amino acid sequence of an IL-2 polypeptide or a functional fragment thereof that reduces the affinity of the IL-2 polypeptide or a functional fragment thereof for CD25 (IL-2Rα).

338. The masked cytokine of embodiment 337, wherein the amino acid sequence is formed by introducing one or more of the following amino acid substitutions into any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: R38A, F42A, F42K, F42E, K43A, Y45A, Y45N, Y45R, E62A, E62R, E62S, and L72G.

339. The masked cytokine of embodiment 335, wherein the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence obtained by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or functional fragment thereof that increases the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rβ or IL-2Ry.

340. The masked cytokine of embodiment 339, wherein the amino acid sequence is formed by introducing one or more of the following amino acid substitutions into any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: H16I, L18C, D20A, D20L, D20F, N29L, L80F, R81D, L85V, I86V, and I92F.

341. A masked cytokine according to embodiment 337 or embodiment 338, wherein the amino acid sequence is formed by the additional introduction of one or more amino acid substitutions that increase the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rβ or IL-2Ry.

342. The masked cytokine of embodiment 341, wherein the one or more amino acid substitutions that increase the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rβ or IL-2Ry are selected from the group consisting of H16I, L18C, D20A, D20L, D20F, N29L, L80F, R81D, L85V, I86V, and I92F.

343. The masked cytokine of embodiment 335, wherein the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence obtained by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or functional fragment thereof that stabilizes the IL2 polypeptide or functional fragment thereof.

344. The masked cytokine of embodiment 343, wherein the amino acid sequence is formed by introducing one of the following amino acid substitutions into any of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: C125S, C125A, and C125G.

345. A masked cytokine according to any one of embodiments 337-342, wherein the amino acid sequence is formed by the further introduction of one or more amino acid substitutions that stabilize the IL-2 polypeptide or functional fragment thereof.

346. The masked cytokine of embodiment 345, wherein the one or more substitutions that stabilize the IL-2 polypeptide or functional fragment thereof are C125S, C125A, or C125G.

347. The masked cytokine of embodiments 335-346, wherein the first 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, 261, 826, and 827, and the second 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, 261, 826, and 827, and wherein the amino acid sequence of the first masking moiety and the amino acid sequence of the second masking moiety are different.

348. A masked cytokine according to any one of embodiments 335-347, wherein:

a) the first masking fragment comprises an amino acid sequence of SEQ ID NO: 9 or 231, and the second masking fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 161-165, 187-218, 221-226, 261, 826 and 827; or

b) the first masking fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 161-165, 187-218, 221-226, 261, 826 and 827, and the second masking fragment comprises the amino acid sequence of SEQ ID NO: 9 or 231.

349. The masked cytokine of any one of embodiments 332-334, wherein the cytokine or functional fragment thereof is an IL-15 polypeptide or functional fragment thereof.

350. The masked cytokine of embodiment 349, wherein the IL-15 polypeptide or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 167.

- 173 049880

351. A masked cytokine according to embodiment 349 or embodiment 350, wherein:

a) the first mask fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232-234 and 823-825, and the second mask fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 219-229, 261, 826 and 827;

b) the first mask fragment comprises 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 mask fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232-234 and 823-825;

c) the first masking fragment comprises an amino acid sequence obtained by introducing one or more of the following amino acid substitutions into the amino acid sequence of any of SEQ ID NOs: 232-234 and 823-825: R24A, R26A, K34A, S40A, L42A and P67A, and the second masking fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 219-229, 261, 826 and 827; or

d) the first masking fragment comprises 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 fragment comprises an amino acid sequence obtained by introducing one or more of the following amino acid substitutions into the amino acid sequence of any of SEQ ID NOs: 232-234 and 823-825: R24A, R26A, K34A, S40A, L42A and P67A.

352. The masked cytokine of any one of embodiments 332-351, wherein the first half-life extending domain is a first antibody or fragment thereof, and the second half-life extending domain is a second antibody or fragment thereof.

353. A masked cytokine according to embodiment 352, wherein:

a) the first antibody or fragment thereof comprises a first heavy chain polypeptide and the second antibody or fragment thereof comprises a second light chain polypeptide; or

b) the first antibody or fragment thereof comprises a first light chain polypeptide, and the second antibody or fragment thereof comprises a second heavy chain polypeptide.

354. The masked cytokine of embodiment 353, wherein the first heavy chain polypeptide or the second heavy chain polypeptide comprises one or more amino acid substitutions that alter effector function.

355. The masked cytokine of embodiment 354, wherein the first heavy chain polypeptide or the second heavy chain polypeptide:

a) is an IgG1 isotype and contains the following amino acid substitution(s):

i) N297A, N297G or N297Q;

ii) L234A and L235A;

iii) C220S, C226S, C229S and P238S;

iv) C226S, C229S, E233P, L234V and L235A;

v) L234F, L235E and P331S;

vi) S267E and L328F;

vii) D265A;

viii) L234A, L235A and P329G;

b) is an IgG2 isotype and contains the following amino acid substitution(s):

i) V234A and G237A;

ii) H268Q, V309L, A330S and A331S; or iii) V234A, G237A, P238S, H268A, V309L, A330S and P331S; or

c) is an IgG4 isotype and contains the following amino acid substitution(s):

i) L235A, G237A and E318A;

ii) S228P, L234A and L235A;

iii) H268Q, V309L, A330S and P331S; or iv) S228P and L235A, numbered in accordance with the Kabat EU numbering system.

356. The masked cytokine of embodiment 353, wherein the first heavy chain polypeptide or the second heavy chain polypeptide comprises one or more amino acid substitutions that enhance effector function.

357. The masked cytokine of embodiment 356, wherein the first heavy chain polypeptide or the second heavy chain polypeptide is an IgG1 heavy chain polypeptide and comprises amino acid substitution(s):

a) S298A, E333A and K334A;

- 174 049880

b) S239D and I332E;

c) S239D, A330L and I332E;

d) P247I and A339D or A339Q;

e) D280H and K290S;

f) D280H, K290S and either S298D or S298V;

g) F243L, R292P and Y300L;

h) F243L, R292P, Y300L and P396L;

i) F243L, R292P, Y300L, V305I and P396L;

j) G236A, S239D and I332E;

k) K326A and E333A;

l) K326W and E333S;

m) K290E, S298G and T299A;

n) K290E, S298G, T299A and K326E;

o) K290N, S298G and T299A;

p) K290N, S298G, T299A and K326E;

q) K334V;

r) L235S, S239D and K334V;

s) K334V and Q331M, S239D, F243V, E294L or S298T;

t) E233L, Q311M and K334V;

u) L234I, Q311M and K334V;

v) K334V and S298T, A330M or A330F;

w) K334V, Q311M and either A330M or A330F;

x) K334V, S298T and either A330M or A330F;

y) K334V, S239D and either A330M or S298T;

z) L234Y, Y296W and K290Y, F243V or E294L;

aa) Y296W and either L234Y or K290Y;

ab) S239D, A330S and I332E, ac) V264I;

ad) F243L and V264I;

ae) L328M;

af) I332E;

ag) L328M and I332E;

ah) V264I and I332E;

ai) S239E and I332E;

aj) S239Q and I332E;

ak) S239E;

al) A330Y;

am) I332D;

an) L328I and I332E;

ao) L328Q and I332E;

ap) V264T;

aq) V240I;

ar) V266I;

as) S239D;

at) S239D and I332D;

au) S239D and I332N;

av) S239D and I332Q;

aw) S239E and I332D;

ax) S239E and I332N;

ay) S239E and I332Q;

az) S239N and I332D;

ba) S239N and I332E;

bb) S239Q and I332D;

bc) A330Y and I332E;

bd) V264I, A330Y and I332E;

b) A330L and I332E;

bf) V264I, A330L and I332E;

bg) L234E, L234Y or L234I;

bh) L235D, L235S, L235Y or L235I;

bi) S239T;

bj) V240M;

bk) V264Y;

- 175 049880 bl) A330I;

bm) N325T;

bn) I332E and L328D, L328V, L328T or L328I;

bo) V264I, I332E and either S239E or S239Q;

bp) S239E, V264I, A330Y and I332E;

bq) A330Y, I332E and either S239D or S239N;

br) A330L, I332E and either S239D or S239N;

bs) V264I, S298A and I332E;

b) S298A, I332E and either S239D or S239N;

bu) S239D, V264I and I332E;

bv) S239D, V264I, S298A and I332E;

bw) S239D, V264I, A330L and I332E;

bx) S239D, I332E and A330I;

by) P230A;

bz) P230A, E233D and I332E;

ca) E272Y;

cb) K274T, K274E, K274R, K274L or K274Y;

cd) F275W;

ce) N276L;

cf) Y278T;

cg) V302I;

ch) E318R;

ci) S324D, S324I or S324V;

cj) K326I or K326T;

ck) T335D, T335R or T335Y;

cl) V240I and V266I;

cm) S239D, A330Y, I332E and L234I;

cn) S239D, A330Y, I332E and L235D;

co) S239D, A330Y, I332E and V240I;

cp) S239D, A330Y, I332E and V264T; or cq) S239D, A330Y, I332E and either K326E or K326T, numbered in accordance with the Kabat EU numbering system.

358. The masked cytokine of embodiment 353, wherein the first heavy chain polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 168, and 169, and the second heavy chain polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 168, and 169.

359. The masked cytokine of embodiment 353, wherein the first light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 157 or 170, and the second light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 157 or 170.

360. The masked cytokine of embodiment 352, wherein the first antibody or fragment thereof is a first crystallizable fragment domain (Fc domain) or fragment thereof, and the second antibody or fragment thereof is a second Fc domain or fragment thereof.

361. A masked cytokine according to embodiment 360, wherein the first Fc domain or fragment thereof and/or the second Fc domain or fragment thereof comprises one or more amino acid substitutions that alter effector function.

362. The masked cytokine of embodiment 361, wherein the first Fc domain or fragment thereof and/or the second Fc domain or its:

a) is the Fc domain of IgG1 or a fragment thereof and contains amino acid substitution(s):

i) N297A, N297G or N297Q;

ii) L234A and L235A;

iii) C220S, C226S, C229S and P238S;

iv) C226S, C229S, E233P, L234V and L235A;

v) L234F, L235E and P331S;

vi) S267E and L328F;

vii) D265A;

viii) L234A, L235A and P329G;

b) is the Fc domain of IgG2 or a fragment thereof and contains the amino acid(s) substitution(s):

i) V234A and G237A;

- 176 049880 ii) H268Q, V309L, A330S and A331S; or iii) V234A, G237A, P238S, H268A, V309L, A330S and P331S; or

c) is the Fc domain of IgG4 or a fragment thereof and contains the amino acid(s) substitution(s):

i) L235A, G237A and E318A;

ii) S228P, L234A and L235A;

iii) H268Q, V309L, A330S and P331S; or iv) S228P and L235A, numbered in accordance with the Kabat EU numbering system.

363. A masked cytokine according to embodiment 361, wherein the first Fc domain or fragment thereof and/or the second Fc domain or fragment thereof comprises one or more amino acid substitutions that enhance effector function.

364. The masked cytokine of embodiment 363, wherein the first Fc domain or fragment thereof and/or the second Fc domain or fragment thereof is an IgG1 Fc domain or fragment thereof and comprises amino acid substitution(s):

a) S298A, E333A and K334A;

b) S239D and I332E;

c) S239D, A330L and I332E;

d) P247I and A339D or A339Q;

e) D280H and K290S;

f) D280H, K290S and either S298D or S298V;

g) F243L, R292P and Y300L;

h) F243L, R292P, Y300L and P396L;

i) F243L, R292P, Y300L, V305I and P396L;

j) G236A, S239D and I332E;

k) K326A and E333A;

l) K326W and E333S;

m) K290E, S298G and T299A;

n) K290E, S298G, T299A and K326E;

o) K290N, S298G and T299A;

p) K290N, S298G, T299A and K326E;

q) K334V;

r) L235S, S239D and K334V;

s) K334V and Q331M, S239D, F243V, E294L or S298T;

t) E233L, Q311M and K334V;

u) L234I, Q311M and K334V;

v) K334V and S298T, A330M or A330F;

w) K334V, Q311M and either A330M or A330F;

x) K334V, S298T and either A330M or A330F;

y) K334V, S239D and either A330M or S298T;

z) L234Y, Y296W and K290Y, F243V or E294L;

aa) Y296W and either L234Y or K290Y;

ab) S239D, A330S and I332E, ac) V264I;

ad) F243L and V264I;

ae) L328M;

af) I332E;

ag) L328M and I332E;

ah) V264I and I332E;

ai) S239E and I332E;

aj) S239Q and I332E;

ak) S239E;

al) A330Y;

am) I332D;

an) L328I and I332E;

ao) L328Q and I332E;

ap) V264T;

aq) V240I;

ar) V266I;

as) S239D;

at) S239D and I332D;

au) S239D and I332N;

av) S239D and I332Q;

- 177 049880 aw) S239E and I332D;

ax) S239E and I332N;

ay) S239E and I332Q;

az) S239N and I332D;

ba) S239N and I332E;

bb) S239Q and I332D;

bc) A330Y and I332E;

bd) V264I, A330Y and I332E;

b) A330L and I332E;

bf) V264I, A330L and I332E;

bg) L234E, L234Y or L234I;

bh) L235D, L235S, L235Y or L235I;

bi) S239T;

bj) V240M;

bk) V264Y;

bl) A330I;

bm) N325T;

bn) I332E and L328D, L328V, L328T or L328I;

bo) V264I, I332E and either S239E or S239Q;

bp) S239E, V264I, A330Y and I332E;

bq) A330Y, I332E and either S239D or S239N;

br) A330L, I332E and either S239D or S239N;

bs) V264I, S298A and I332E;

b) S298A, I332E and either S239D or S239N;

bu) S239D, V264I and I332E;

bv) S239D, V264I, S298A and I332E;

bw) S239D, V264I, A330L and I332E;

bx) S239D, I332E and A330I;

by) P230A;

bz) P230A, E233D and I332E;

ca) E272Y;

cb) K274T, K274E, K274R, K274L or K274Y;

cd) F275W;

ce) N276L;

cf) Y278T;

cg) V302I;

ch) E318R;

ci) S324D, S324I or S324V;

cj) K326I or K326T;

ck) T335D, T335R or T335Y;

cl) V240I and V266I;

cm) S239D, A330Y, I332E and L234I;

cn) S239D, A330Y, I332E and L235D;

co) S239D, A330Y, I332E and V240I;

cp) S239D, A330Y, I332E and V264T; or cq) S239D, A330Y, I332E and either K326E or K326T, numbered in accordance with the Kabat EU numbering system.

365. A masked cytokine according to embodiment 360, wherein:

a) 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;

b) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155;

c) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 154, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 154;

d) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 265, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156;

e) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO:

- 178 049880

265;

f) 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: 616;

g) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 616, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155;

h) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 157, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 158;

i) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 158, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 157;

j) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 774;

k) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 774, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796;

l) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 619;

m) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 619, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721;

n) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 772;

o) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 772, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721;

p) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 622;

q) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 622, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793;

r) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 773;

s) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 773, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793;

t) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625;

u) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796;

v) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156;

w) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625; or

x) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796.

366. The masked cytokine of embodiments 332-351, wherein the first half-life extending domain is a first scFv or fragment thereof, and the second half-life extending domain is a second scFv or fragment thereof.

367. The masked cytokine of embodiments 332-351, wherein

- 179 049880 the first half-life extending domain is a first Fc domain or fragment thereof, and the second half-life extending domain is a second Fc domain or fragment thereof, and wherein the first Fc domain or fragment thereof is linked to the second Fc domain or fragment thereof.

368. The masked cytokine of embodiment 367, wherein the first Fc domain or fragment thereof is linked to the second Fc domain or fragment thereof via a fourth linker.

369. A masked cytokine according to any one of embodiments 232-351, wherein:

(a) the first half-life extending domain is an scFv or a fragment thereof, and the second half-life extending domain is an antibody or a fragment thereof; or

a) the first half-life extending domain is an antibody or a fragment thereof, and the second half-life extending domain is an scFv or a fragment thereof.

370. A masked cytokine according to any one of paragraphs 352-365, wherein the modifications facilitating the association of the first and second half-life extending domains comprise:

a) introducing the mutations S354C and T366W into a first antibody or fragment thereof and introducing the mutations Y349C, T366S, L368A and Y407V into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

b) introducing the mutations S354C and T366W into the second antibody or fragment thereof and introducing the mutations Y349C, T366S, L368A and Y407V into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

c) introducing the mutations K392D and K409D into the first antibody or fragment thereof and introducing the mutations D399K and E356K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

d) introducing the mutations K392D and K409D into the second antibody or fragment thereof and introducing the mutations D399K and E356K into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

e) the introduction of the mutations S364H and F405A into the first antibody or fragment thereof and the introduction of the mutations Y349T and T394F into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system; or

f) the introduction of mutations S364H and F405A into the second antibody or fragment thereof and the introduction of mutations Y349T and T394F into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system.

371. The masked cytokine of any one of embodiments 352-365, wherein the amino acid sequence of the first antibody or fragment thereof and the amino acid sequence of the second antibody or fragment thereof are formed by:

a) introducing the mutations S354C and T366W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations Y349C, T366S, L368A and Y407V into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

b) introducing the mutations S354C and T366W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations Y349C, T366S, L368A and Y407V into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

c) introducing the mutations K392D and K409D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

d) introducing the mutations K392D and K409D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

e) introducing the mutations S364H and F405A into the amino acid sequence of SEQ ID NO: 154 or

169, and the introduction of mutations Y349T and T394F into the amino acid sequence of SEQ ID NO: 154 or

169, respectively, numbered in accordance with the Kabat EU numbering system; or

f) introducing the mutations S364H and F405A into the amino acid sequence of SEQ ID NO: 154 or

169, and the introduction of mutations Y349T and T394F into the amino acid sequence of SEQ ID NO: 154 or

169, respectively, numbered in accordance with the Kabat EU numbering system.

372. A masked cytokine according to any one of paragraphs 352-365, wherein the modifications facilitating the association of the first and second half-life extending domains comprise:

a) the introduction of a Y407T mutation into a first antibody or fragment thereof and the introduction of a T366Y mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

b) the introduction of a Y407A mutation into a first antibody or fragment thereof and the introduction of a T366W mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

c) the introduction of the F405A mutation into the first antibody or fragment thereof and the introduction of the T394W mutation into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

d) introducing the F405W mutation into the first antibody or fragment thereof and introducing the T394S mutation into

- 180 049880 a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

e) introducing a Y407T mutation into a first antibody or fragment thereof and introducing a T366Y mutation into a second antibody or fragment thereof, numbered according to the Kabat EU numbering system;

f) the introduction of the mutations T366Y and F405A into the first antibody or fragment thereof and the introduction of the mutations T394W and Y407T into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

g) introducing the mutations T366W and F405W into the first antibody or fragment thereof and introducing the mutations T394S and Y407A into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

h) introducing the mutations F405W and Y407A into the first antibody or fragment thereof and introducing the mutations T366W and T394S into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

i) introducing the T366W mutation into the first antibody or fragment thereof and introducing the T366S, L368A and Y407V mutations into the second antibody or fragment thereof, numbered according to the Kabat EU numbering system;

j) introducing a Y407T mutation into a second antibody or fragment thereof and introducing a T366Y mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

k) introducing a Y407A mutation into a second antibody or fragment thereof and introducing a T366W mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

l) the introduction of the F405A mutation into the second antibody or fragment thereof and the introduction of the T394W mutation into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

m) introducing the F405W mutation into the second antibody or fragment thereof and introducing the T394S mutation into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

n) introducing a Y407T mutation into a second antibody or fragment thereof and introducing a T366Y mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

o) the introduction of the mutations T366Y and F405A into the second antibody or fragment thereof and the introduction of the mutations T394W and Y407T into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

p) introducing the mutations T366W and F405W into the second antibody or fragment thereof and introducing the mutations T394S and Y407A into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

q) the introduction of the mutations F405W and Y407A into the second antibody or fragment thereof and the introduction of the mutations T366W and T394S into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system; or

r) introducing the mutation T366W into the second antibody or fragment thereof and introducing the mutations T366S, L368A and Y407V into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system.

373. The masked cytokine of any one of embodiments 352-365, wherein the amino acid sequence of the first antibody or fragment thereof and the amino acid sequence of the second antibody or fragment thereof are formed by:

a) introducing a Y407T mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366Y mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

b) introducing a Y407A mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366W mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

c) introducing the mutation F405A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation T394W into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

d) introducing the mutation F405W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation T394S into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

e) introducing a Y407T mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366Y mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

f) introducing the mutations T366Y and F405A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T394W and Y407T into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

g) introducing the mutations T366W and F405W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T394S and Y407A into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

h) introducing the mutations F405W and Y407A into the amino acid sequence of SEQ ID NO: 154 or

- 181 049880

169, and the introduction of the mutations T366W and T394S into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered in accordance with the Kabat EU numbering system;

i) introducing the mutation T366W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T366S, L368A and Y407V into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

j) introducing a Y407T mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366Y mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

k) introducing a Y407A mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366W mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

l) introducing the mutation F405A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation T394W into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

m) introducing the mutation F405W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation T394S into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

n) introducing a Y407T mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a T366Y mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

o) introducing the mutations T366Y and F405A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T394W and Y407T into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

p) introducing the mutations T366W and F405W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T394S and Y407A into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

q) introducing the mutations F405W and Y407A into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T366W and T394S into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system; or

r) introducing the mutation T366W into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations T366S, L368A and Y407V into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system.

374. A masked cytokine according to any one of paragraphs 352-365, wherein the modifications facilitating the association of the first and second half-life extending domains comprise:

a) the introduction of a K409E mutation into a first antibody or fragment thereof and the introduction of a D399K mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

b) the introduction of a K409E mutation into a first antibody or fragment thereof and the introduction of a D399R mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

c) the introduction of a K409D mutation into a first antibody or fragment thereof and the introduction of a D399K mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

d) introducing a K409D mutation into a first antibody or fragment thereof and introducing a D399R mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

e) introducing a K392E mutation into a first antibody or fragment thereof and introducing a D399R mutation into a second antibody or fragment thereof, numbered according to the Kabat EU numbering system;

f) the introduction of a K392E mutation into a first antibody or fragment thereof and the introduction of a D399K mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

g) the introduction of a K392D mutation into a first antibody or fragment thereof and the introduction of a D399R mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

h) the introduction of a K392D mutation into a first antibody or fragment thereof and the introduction of a D399K mutation into a second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

i) introducing the mutations K409D and K360D into the first antibody or fragment thereof and introducing the mutations D399K and E356K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

j) introducing the mutations K409D and K370D into the first antibody or fragment thereof and introducing the mutations D399K and E357K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

k) introducing the mutations K409D and K392D into the first antibody or fragment thereof and introducing the mutations D399K, E356K and E357K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

l) the introduction of the mutations K409D and K392D into the first antibody or fragment thereof and the introduction of the mutation D399K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

- 182 049880

m) introducing the mutations K409D and K392D into the first antibody or fragment thereof and introducing the mutations D399K and E356K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

n) introducing the mutations K409D and K392D into the first antibody or fragment thereof and introducing the mutations D399K and E357K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

o) the introduction of the mutations K409D and K370D into the first antibody or fragment thereof and the introduction of the mutations D399K and E357K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

p) introducing the mutation D399K into the first antibody or fragment thereof and introducing the mutations K409D and K360D into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

q) introducing the mutations K409D and K439D into the first antibody or fragment thereof and introducing the mutations D399K and E356K into the second antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

r) the introduction of a K409E mutation into a second antibody or fragment thereof and the introduction of a D399K mutation into a first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

s) introducing a K409E mutation into a second antibody or fragment thereof and introducing a D399R mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

t) introducing a K409D mutation into a second antibody or fragment thereof and introducing a D399K mutation into a first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

u) introducing a K409D mutation into a second antibody or fragment thereof and introducing a D399R mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

v) the introduction of a K392E mutation into a second antibody or fragment thereof and the introduction of a D399R mutation into a first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

w) introducing a K392E mutation into a second antibody or fragment thereof and introducing a D399K mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

x) the introduction of a K392D mutation into a second antibody or fragment thereof and the introduction of a D399R mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

y) introducing a K392D mutation into a second antibody or fragment thereof and introducing a D399K mutation into a first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

z) introducing the mutations K409D and K360D into the second antibody or fragment thereof and introducing the mutations D399K and E356K into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

aa) introducing the mutations K409D and K370D into the second antibody or fragment thereof and introducing the mutations D399K and E357K into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

ab) introducing the mutations K409D and K392D into the second antibody or fragment thereof and introducing the mutations D399K, E356K and E357K into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system;

ac) introducing the mutations K409D and K392D into the second antibody or fragment thereof and introducing the mutation D399K into the first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

ad) introducing the mutations K409D and K392D into the second antibody or fragment thereof and introducing the mutations D399K and E356K into the first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

ae) introducing the mutations K409D and K392D into the second antibody or fragment thereof and introducing the mutations D399K and E357K into the first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

af) introducing the mutations K409D and K370D into the second antibody or fragment thereof and introducing the mutations D399K and E357K into the first antibody or fragment thereof, numbered according to the Kabat EU numbering system;

ag) introducing the mutation D399K into the second antibody or fragment thereof and introducing the mutations K409D and K360D into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system; or ah) introducing the mutations K409D and K439D into the second antibody or fragment thereof and introducing the mutations D399K and E356K into the first antibody or fragment thereof, numbered in accordance with the Kabat EU numbering system.

375. The masked cytokine of any one of embodiments 352-365, wherein the amino acid sequence of the first antibody or fragment thereof and the amino acid sequence of the second antibody or fragment thereof are formed by:

a) introducing the K409E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and

- 183 049880 introducing the D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered in accordance with the Kabat EU numbering system;

b) introducing a K409E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

c) introducing a K409D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

d) introducing a K409D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

e) introducing a K392E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

f) introducing a K392E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

g) introducing a K392D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

h) introducing a K392D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

i) introducing the mutations K409D and K360D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

j) introducing the mutations K409D and K370D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

k) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K, E356K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

l) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation D399K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

m) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

n) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

o) introducing the mutations K409D and K370D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

p) introducing the mutation D399K into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations K409D and K360D into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

q) introducing the mutations K409D and K439D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

r) introducing a K409E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

s) introducing a K409E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

t) introducing a K409D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

u) introducing a K409D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

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v) introducing a K392E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

w) introducing a K392E mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

x) introducing a K392D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399R mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

y) introducing a K392D mutation into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing a D399K mutation into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

z) introducing the mutations K409D and K360D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

aa) introducing the mutations K409D and K370D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

ab) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K, E356K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

ac) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutation D399K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

ad) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, numbered according to the Kabat EU numbering system;

ae) introducing the mutations K409D and K392D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

af) introducing the mutations K409D and K370D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E357K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system;

ag) introducing the mutation D399K into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations K409D and K360D into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system; or ah) introducing the mutations K409D and K439D into the amino acid sequence of SEQ ID NO: 154 or 169, and introducing the mutations D399K and E356K into the amino acid sequence of SEQ ID NO: 154 or 169, respectively, numbered according to the Kabat EU numbering system.

376. The masked cytokine of any one of embodiments 333-375, wherein the first linker comprises a first cleavable peptide; and/or wherein the second linker comprises a second cleavable peptide.

377. A masked cytokine according to any one of embodiments 333-376, wherein the first linker comprises a first N-terminal spacer domain and/or a first C-terminal spacer domain.

378. A masked cytokine according to embodiment 377, wherein the first linker comprises:

a) a first N-terminal spacer domain, a first cleavable peptide and a first C-terminal spacer domain;

b) the first N-terminal spacer domain and the first cleavable peptide;

c) the first N-terminal spacer domain and the first C-terminal spacer domain;

d) the first cleavable peptide and the first C-terminal spacer domain;

e) the first N-terminal spacer domain; or

f) the first C-terminal spacer domain.

379. The masked cytokine of any one of embodiments 333-378, wherein the second linker comprises a second N-terminal spacer domain and/or a second C-terminal spacer domain.

380. The masked cytokine of embodiment 379, wherein the second linker comprises:

a) a second N-terminal spacer domain, a second cleavable peptide and a second C-terminal spacer domain;

b) a second N-terminal spacer domain and a second cleavable peptide;

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c) a second N-terminal spacer domain and a second C-terminal spacer domain;

d) a second cleavable peptide and a second C-terminal spacer domain;

e) a second N-terminal spacer domain; or

f) the second C-terminal spacer domain.

381. The masked cytokine of embodiments 376-380, wherein the first 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/or wherein the second cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 96-153, 236-242, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535 and 538-555.

382. The masked cytokine of embodiments 377-381, wherein the first 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/or wherein the first 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.

383. The masked cytokine of embodiments 376-380, wherein the first 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 NO: 236-242.

384. The masked cytokine of embodiment 383, wherein the amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555 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 the C-terminus of 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.

385. The masked cytokine of embodiments 379-384, wherein the second 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/or the second 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.

386. The masked cytokine of embodiments 376-385, wherein the second 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 NO: 236-242.

387. The masked cytokine of embodiment 386, wherein the amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555 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 the C-terminus of 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.

388. The masked cytokine of embodiments 333-387, wherein the first 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 and/or the second 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.

389. The masked cytokine of any one of embodiments 334-388, wherein the third linker comprises a third cleavable peptide.

390. The masked cytokine of any one of embodiments 334-389, wherein the third linker comprises a third N-terminal spacer domain and/or a third C-terminal spacer domain.

391. A masked cytokine according to embodiment 390, wherein the third linker comprises:

a) a third N-terminal spacer domain, a third cleavable peptide and a third C-terminal spacer domain;

b) the third N-terminal spacer domain and the third cleavable peptide;

c) a third N-terminal spacer domain and a third C-terminal spacer domain;

d) the third cleavable peptide and the third C-terminal spacer domain;

e) a third N-terminal spacer domain; or

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f) the third C-terminal spacer domain.

392. The masked cytokine of embodiments 389-391, wherein the third 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/or wherein the third cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 96-153, 236-242, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535 and 538-555.

393. The masked cytokine of embodiments 390-392, wherein the third 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/or the third 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.

394. The masked cytokine of embodiments 389-393, wherein the third 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 NO: 236-242.

395. The masked cytokine of embodiment 394, wherein the amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555 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 the C-terminus of 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.

396. The masked cytokine of embodiments 334-395, wherein the third 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, and/or the third 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.

397. The masked cytokine of embodiments 368-396, wherein the fourth linker comprises a fourth N-terminal spacer domain and/or a fourth C-terminal spacer domain.

398. The masked cytokine of embodiment 397, wherein the fourth 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/or the fourth 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.

399. A masked cytokine according to any one of embodiments 376-398, wherein the first cleavable peptide, the second cleavable peptide and/or the third cleavable peptide is a substrate for a protease that is colocalized in a region or tissue expressing a cytokine receptor.

400. A masked cytokine receptor according to embodiment 399, wherein the cytokine receptor is an IL-2 cytokine receptor or an IL-15 cytokine receptor.

401. The masked cytokine of any one of embodiments 376-400, wherein the first cleavable peptide, the second cleavable peptide, and/or the third cleavable peptide are cleaved by one or more enzymes selected from the group consisting of: ABHD12, ADAM12, ABHD12B, ABHD13, ABHD17A, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL1, ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAM10, ADAM15, ADAM17, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, 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, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7,

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TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1 and TPSG1.

402. The masked cytokine of any one of embodiments 332-401, wherein the first half-life extending domain and/or the second half-life extending domain is conjugated to the agent.

403. The masked cytokine of embodiment 402, wherein the agent is a tubulin polymerization inhibitor, a DNA damaging agent, or a DNA synthesis inhibitor.

404. The masked cytokine of embodiment 403, wherein the agent is a maytansinoid, an auristatin, a pyrrolobenzodiazepine dimer (PBD), a calicheamicin, a duocarmycin, an indolinobenzodiazepine dimer, or an exatecan Dxd derivative.

405. A masked cytokine according to embodiment 402, wherein the agent is an immunostimulant.

406. The masked cytokine of embodiment 405, wherein the immunostimulant is a stimulating interferon gene agonist (STING) or a toll-like receptor (TLR) agonist.

407. The masked cytokine of embodiment 406, wherein the STING agonist is a cyclic dinucleotide (CDN).

408. The masked cytokine of embodiment 407, wherein the CDN is selected from the group consisting of cGAMP, c-di-AMP, c-di-GMP, cAIMP, c-di-IMP, 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide.

409. The masked cytokine of embodiment 406, wherein the TLR agonist is a TLR agonist selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10.

Examples

The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present invention. It should be understood that the examples and embodiments described herein are intended for illustrative purposes only and that various modifications or changes in light thereof will be suggested to those skilled in the art and are intended to be included within the spirit and scope of the present application and within the scope of the appended claims.

Although some examples describe the construction, production, and/or testing of masked versions of the IL-2 polypeptide construct or the IL-15 polypeptide construct, some examples also use the original unmasked versions of the IL-2 polypeptide construct or the IL-15 polypeptide construct, for example, for comparison, or other constructs that include one or more components described herein that are tested as controls for comparison. Accordingly, a description of, for example, a study conducted on masked IL-2 polypeptide constructs does not necessarily mean that unmasked versions of the construct were not also tested.

Example 1. Construction of masked IL-2 polypeptides and masked IL15 polypeptides.

Masked IL-2 polypeptide constructs and masked IL-15 polypeptide constructs are prepared in accordance with the teachings herein. In the following examples, some experiments involve the use of masked IL-2 and IL-15 polypeptide constructs in monomeric form, and some experiments involve the use of masked IL-2 and IL-15 polypeptide constructs in dimeric form, such as a dimer formed by disulfide bonds linking two copies of the same masked polypeptide construct (homodimer), or a heterodimer formed by two different polypeptides (see, for example, Tables 8-11).

Masked IL-2 polypeptide constructs are prepared that comprise an IL-2 polypeptide or a functional fragment thereof, a masking fragment, and a half-life extension domain such as albumin, an antibody or a fragment thereof (e.g., an Fc region, a heavy chain and/or a light chain), an albumin-binding peptide, an IgG-binding peptide, or a polyamino acid sequence. Some IL-2 polypeptide constructs are also prepared that comprise an IL-2 polypeptide or a functional fragment thereof linked to a half-life extension domain without also including a masking fragment. Some of the constructs also comprise a linker that comprises a cleavable peptide and links the masking fragment to the IL-2 polypeptide or a functional fragment thereof, resulting in an activatable masked IL-2 polypeptide construct. Some of the constructs also comprise a linker that links the IL-2 polypeptide or a functional fragment thereof to a half-life extension domain. Some of the constructs also contain a linker that links the IL-2 polypeptide or functional fragment thereof to the masking fragment. Masked IL-2 polypeptide constructs that do not contain a cleavable peptide in the linker that links the IL-2 polypeptide or its

- 188 049880 functional fragment with a masking fragment, also called inactivatable masked IL-2 polypeptide constructs or inactivatable IL-2 polypeptide constructs, since they do not contain a cleavable peptide. The structure and composition of exemplary IL-2 polypeptide constructs are presented in Table 4.

Table 4

No kO][STRUKTS11N Cytokine OR STO FUZINOKMNYSH fragment (C) Linker (Li) Masking fragment (MM) Linker (L2) Domain lrvdknia nepjioia piuzhisnn (Η) Structure [direction from N- to C-koYeshu) ΑΜίΜίλίΚΊίΓΗΜ post-cycle 2001 SEO ID KO: 1 - - - SEQID: 154 n-s 2002 SEQID: 1 SEQ!DNO:2fl SEQ ID NO: 9 SEQ ID NO; 154 H-L1-MM-C 2003 SEQIDKO: 1 SEQ ID NO: 21 SEQ ID NO: 9 SEQ ID NO; 154 C-L1-MM-H 20(4 SEQIDKO; 3 - - - SEQ ID NO; 154 N-S 2005 SEQID: 4 - SEQ ID NO: 154 n-s 200$ SEQIDKO: 5 - SEQIDKO: 154 n-s 2014 SEQIDKO:! SEQ ID NO: 10 SEQ ID NO: 15 SEQIDKO: 154 H-C-L2-MM 2017 SEQIDKO; 3 SEQ ID NO; 10 SEQ ID NO: 15 SEQIDKO; 154 H-C-L2-MM 201S. SEQ ID NO; 4 SEQIDKO: 10 SEQ ID NO: 15 SEQ ID NO; 154 H-C-L2-MM 2010 SEQ ID KO: 5 SEQIDKO: 10 SEQ ID NO: 15 SEQID: 154 H-C-L’-MM 2024 SEQ ID NO; 6 SEQ ID NO; 154 N-S 2025 SEQ ID NO: 7 - SEQ ID NO; 154 N-S 202$ SEQ ID NO; 3 SEQIDKO; 154 N-S 2027 SEQ ID NO; 1 SEQ ID NO: 2« SEQ ID NO; ? SEQ ID NO: 30 SEQ ID NO; 154 H-LI-MM-L2-C 202$ SEQIDKO: 1 SEQ ID NO: 20 SEQ ID NO: 0 SEQ ID NO: 31 SEQID: 154 H-LI-MM-L2-C 2020 SEQIDKO; 1 SEQ ID NO: 20 SEQ ID NO:? SEQ ID NO: 32 SEQ ID NO: 154 H-LI-MM-L2-C 2030 SEQIDKO; 1 SEQ ID NO: 20 SEQ ID NO;? SEQ ID NO: 33 SEQIDKO: 154 H-LI-MM-L2-C 2021 SEQ ID KO: 2 SEQ ID NO; 20 SEQIDKO:? SEQ ID NO: 2? SEQIDKO: 154 NTGMMT2-S 2032 SEQ ID KO: 2 SEQ ID NO: 20 SEQ ID NO:? SEQ ID NO: 30 SEQ ID NO: 154 H-LI-MM-L2-C 2033 SEQ ID NO; 2 SEQ ID NO: 20 SEQ ID NO:? SEQ ID NO: 31 SEQ ID NO: 154 H-LI-MM-L2i 2034 SEQ ID KO: 2 SEQ ID NO: 20 SEQ ID NO:? SEQ ID NO: 32 SEQIDKO: 154 H-LI-MM-L2-C

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2« 5 SEQ ID NO: 2 SEQ ID NO; 2« SEQ ID NO: 9 SEQ ID NO; 33 SEQIDNO: 35-1 H-L1-MM-L2-C 2036 SEQ ID NO: 1 SEQ ID NO: 26 SEQ ID NO: 9 SEQIDNO: 154 C-LI-MM-H 2037 SEQ ID NO: 1 SEQ ID NO: 27 SEQ ID NO: 9 SEQIDNO: 154 C-LI-MM-H 203 S SEQ ID NO: 2 SEQ ID NO: 2C SEQ ID NO: 9 - SEQIDNO: 154 C-LI-MM-H 2M9 SEQ ID NO: 2 SEQ ID NO: 2C SEQ ID NO: 9 - SEQIDNO: !54 C-Ll-MM-H 2940 SEQ ID NO: 2 SEQ ID NO: 20 SEQ ID NO: 9 - SEQIDNO: 154 C-LI-MM-H 2041 SEQ ID NO; I - SEQ ID NO: 10 SEQIDNO; 16 SEQIDNO; 154 H-C-L2-MM 2043 SEQ ID NO: 1 SEQ ID NO; 10 SEQIDNO; 17 SEQIDNO: 354 H-C-L2-MM 2943 SEQ ID NO: 1 SEQ ID NO; 10 SEQIDNO: IS SEQIDNO: 354 H-C-L2-MM 2944 SEQ ID NO; I SEQ ID NO; 10 SEQIDNO; 19 SEQIDNO; 354 H-C-L2-MM 2045 SEQ ID NO: 3 SEQ ID NO:10 SEQIDNO: 16 SEQIDNO: 154 H-C-L2-MM 2946 SEQ ID NO: 3 SEQ ID NO; 10 SEQIDNO: 17 SEQIDNO: 154 H-C-L2-MM 2047 SEQ ID NO: 3 SEQ ID NO; 10 SEQIDNO: IS SEQIDNO: 354 H-C-L2-J.LM 294 S SEQ ID NO; 3 SEQ ID NO; 10 SEQIDNO; 19 SEQIDNO; 154 H-C-L2-MM 2049 SEQ ID NO: 4 SEQ ID NO: 10 SEQIDNO: 16 SEQIDNO: 154 H-C-L2-MM 2050 SEQ ID NO: 4 SEQ ID KO: 10 SEQIDNO: 17 SEQIDNO: 154 H-C-L2-MM 2051 SEQ ID NO: 4 SEQ ID NO: 10 SEQIDNO: IS SEQIDNO: 154 H-C-L2-MM 2052 SEQ ID NO: 4 SEQ ID NO: 10 SEQIDNO: 19 SEQIDNO: 154 H-C-L2-MM 2053 SEQ ID NO: 5 SEQ ID NO: 10 SEQIDNO: 16 SEQIDNO: 154 H-C-L2-MM 2054 SEQ ID NO: 5 SEQ ID NO: 10 SEQIDNO: 17 SEQIDNO: 154 H-C-L2-MM 2055 SEQ ID NO: 5 SEQ ID NO: 10 SEQIDNO; 1$ SEQIDNO:! 54 H-C-L2-MM 2056 SEQ ID NO: 5 - SEQ ID NO; 10 SEQIDNO; 19 SEQIDNO; 154 H-C-L2-MM 205? SEQ ID KO: 6 SEQIDKO: 10 SEQIDKO: IS SEQ ID NO: 154 N-S-1LMM 2058 SEQ ID NO; 6 SEQ ID NO; 10 SEQIDNO; 16 SEQ ID NO; 1:4 H-C-L2-MM 2059 SEQ ID NO: 6 SEQ ID NO: 10 SEQIDNO: 17 SEQ ID NO: 154 H-C-L2-MM 2060 SEQ ID NO: 6 SEQ ID NO: 10 SEQIDNO: 18 SEQ ID NO: 154 H-C-L2-MM 2061 SEQ ID NO: 6 SEQIDKO: 10 SEQIDNO: 19 SEQ ID NO: 154 H-C-L2-MM 2062 SEQ ID NO: 7 SEQ ID KO: 10 SEQIDNO: IS SEQ ID NO: 154 H-C-L2-MM 2063 SEQ ID NO: 7 - SEQ ID NO: [0 SEQIDNO: 16 SEQ ID NO: 154 H-C-L’-MSi 2064 SEQ ID NO: 7 SEQ ID NO: 10 SEQIDNO: 17 SEQ ID NO: 154 H-C-L2-MM 2065 SEQ ID NO: 7 SEQ ID NO: 10 SEQIDNO: 18 SEQ ID NO: 154 H-C-L2-MM 2066 SEQ ID NO: 7 SEQIDNO: 10 SEQIDNO: 19 SEQ ID NO: 154 H-C-L2-MM 2067 SEQIDNO: E SEQIDKO: [0 SEQIDNO: IS SEQ ID NO: 154 H-C-L2-MM 206S SEQ ID KO: S SEQIDKO: 10 SEQIDNO: 16 SEQ ID NO: 154 H-C-L2-MM 2069 SEQ ID NO: 8 SEQIDKO: 10 SEQIDNO: 17 SEQ ID NO: 154 H-C-L2-MM 2070 SEQ ID NO: 8 SEQIDNO; [0 SEQIDNO; IS SEQ ID NO; 1:4 H-C-L2-MM 2071 SEQ ID NO: 8 SEQIDNO: 10 SEQIDNO: 19 SEQ ID NO: 154 H-C-L2-MM 2109 SEQ ID NO: 2 SEQ ID NO: 154 H-C AKD32 SEQ ID NO: 1 - - SEQ ID NO: 154 H-C SEQ ID NO: 556 AKO33 SEQ ID NO: 1 SEQ ID NO: 20 SEQ ID NO: 9 SEQ ID NO: 29 SEQ ID NO: 154 H-LI-MM-L2-C SEQ ID NO: 557 .» SEQ ID NO: 1 SEQ ID NO: 21 SEQ ID NO: 9 - SEQ ID NO: 154 C-LI-MM-H SEQ ID NO; 55$ AKD35 SEQ ID NO: 3 SEQ ID NO: 154 H-C SEQ ID NO; 559 AKD36 SEQ ID NO: 4 - SEQ ID NO: 154 H-C SEQ ID NO: 569 07 SEQ ID NO: 5 - - SEQ ID NO: 154 H-C SEQ ID NO: 561

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AK040 SEQ P) NO; 1 SEQ ID NO; 13 SEQ ID NO; 16 SEQ ID NO; 164 H-C-Ll-MM SEQ TO KO; 566 AKO42 SEQ TO NO; 6 - SEQ ID NO: 164 H-C SEQ ID NO: 563 AKO43 SEQ ID NO: 7 SEQ ID NO: 154 H-C SEQ ID NO: 569 AK044 SEQ DNO; 8 - SEQ ID NO; 134 H-C SEQ TO NO; 576 AKS SEQ TONE:1 SEQ ID NO: 20 SEQ ID NO: 9 SEQ ID NO: 3D SEQ ID KO: 154 H-L1-MM-L2-C SEQ ID NO: 57) AKO46 SEQ TONE: 1 SEQ ID NO: 20 SEQ TO NO; 9 SEQ ID NO: 31 SEQ ID NO; 154 H-L1-MM-L2-C SEQ ID NO; 572 aish SEQ ID NO; 1 SEQ ID NO: 20 SEQ ID NO: J SEQ ID NO: 32 SEQ ID NO: 164 H-L1-MM-L2-C SEQ TO NO: 573 AKS SEQ TO NO: 1 SEQ ID NO: 20 SEQ ID ND: 9 SEQ ID KO: 33 SEQ ID KO: 164 H-L1-MM-L2-C SEQ ID NO: 574 AK049 SEQ ID NO; 3 SEQ ID NO; 20 SEQ ID NO; 9 SEQ ID NO; 29 SEQ ID NO; 154 H-L1-MM-L2-C SEQ ID NO; 575 AK<J:O SEQ TO NO; 2 SEQ ID NO; 20 SEQ TO NO: 9 SEQ ID NO: 30 SEQ ID NO; 164 H-LJ-MM-L2-C SEQ TO NO: 576 LKS51 SEQ ID NO: 2 SEQ ID NO: 20 SEQ ID NO: 9 SEQIDNO:3l SEQ ID NO: 154 H-L1-MM-L2-C SEQ ID NO: 577 AK052 SEQ ID NO: 2 SEQ ID NO: 20 SEQ TO NO: 9 SEQ ID NO: 32 SEQ ID NO: 154 H-L1-MM-L2-C SEQ ID NO: 573 AKS SEQ ID NO: 2 SEQ ID NO: 20 SEQ ID NO: J SEQ ID KO: 33 SEQ ID KO: 164 H-L1-MM-L2-C SEQ ID NO: 579 AK054 SEQ ID NO: 1 SEQ ID NO: 26 SEQ ID NO: 9 SEQ ID KO: 154 C-LI.M.M-H SEQ ID NO: 586 AKS SEQ ID NO; 1 SEQ ID NO; 27 SEQ TO NO; 9 SEQ ID NO; 164 C-LI-MM-H SEQ TO NO; 531 AKS SEQ ID NO: 2 SEQ ID NO: 2! SEQ ID NO: 9 SEQ ID NO: 164 C-LI-MM-H SEQ ID NO: 582 AKS57 SEQ ID NO; 2 SEQ ID NO; 26 SEQ ID NO; 9 SEQ ID NO; 154 C-LI-MM-H SEQ ID NO; 533 AKO58 SEQ ID NO: 2 SEQ ID NO: 27 SEQ TO NO: 9 SEQ ID NO: 154 C-LI-MM-H SEQ TO NO: 534 AKS SEQ ID NO: 1 SEQ ID NO: 16 SEQ ID NO: 16 SEQ ID KO: 154 H-C-Ll-MM SEQ ID NO: 585 LKS60 SEQ ID NO: 1 SEQ ID NO: 17 SEQ ID NO: 16 SEQ ID NO: 154 H-C-Ll-MM SEQ ID NO: 586 AK06L SEQ ID NO; 1 SEQ ID NO; IS SEQ ID NO: 16 SEQ ID NO: 164 H-C-Ll-MM SEQ ID NO: 587 AKS62 SEQ ID NO: 1 SEQ ID NO: 19 SEQ ID NO: 16 SEQ ID NO: 154 H-C-Ll-MM SEQ ID NO: 588 LK063 SEQ ID NO: 3 SEQ ID NO; 16 SEQ ID NO; 16 SEQ TO KO; 154 H-C-Ll-MM SEQ ID NO: 5S9 AK064 SEQ ID KO: 3 SEQ ID NO: 17 SEQ TO NO: 16 - SEQ TO NO: 154 H-C-Ll-MM SEQ ID NO: 590 AK065 SEQ ID NO: 3 SEQ ID NO: 18 SEQ ID NO: 16 - SEQ TO KO; 154 H-C-Ll-MM SEQ ID NO: 591 AK666 SEQ ID KO J SEQ ID NO: 19 SEQ ID NO: 16 - SEQ TO KO: 154 H-C-Ll-MM SEQ ID KO: 592 LK0&7 SEQ ID KO; 6 SEQ ID NO: 15 SEQ ID NO; 16 SEQ TO NO; 154 H-C-Ll-MM SEQ ID NO; 593 AK068 SEQ ID KO: 6 SEQ ID NO: 16 SEQ TO NO: 16 - SEQ TO KO: 154 H-C-Ll-MM SEQ ID NO: 594 DK069 SEQ ID NO: 6 SEQ ID NO: 17 SEQ ID NO: 16 - SEQ TO KO; 154 H-C-Ll-MM SEQ ID KO: 595 AK070 SEQ ID NO: 6 SEQ ID NO: IS SEQ ID NO: 16 - SEQ TO KO: 154 H-C-Ll-MM SEQ ID KO: 596 AK071 SEQ ID NO; 6 SEQ ID NO; 19 SEQ ID NO; 16 SEQ TO NO; 154 H-C-Ll-MM SEQ ID NO; 597 AΑΚ1Ι2 SEQ ID NO: 1 SEQ ID NO: IS SEQ ID NO: 261 - SEQ TO KO: 154 H-C-Ll-MM SEQ ID NO: 602 AK686 SEQ ID NO: 2 - - - SEQ TO NO; 154 H-C SEQ ID KO: ¢07 AK632 - SEQ ID NO: 28 SEQ ID NO: 16 - SEQ TO NO; 155 H-LI-MM SEQ ID KO: ¢09 AK083 SEQ ID NO: 1 SEQ ID NO: 15 SEQ ID NO: 226 SEQ TO NO: 154 H-C-Ll-MM SEQ ID NO: 610 AK084 SEQ ID NO; 1 SEQ ID NO; 16 SEQ ID NO; 226 SEQ TO NO; 154 H-C-Ll-MM SEQIDNO;6U AK035 SEQ ID NO: 1 SEQ ID NO: 17 SEQ ID NO: 226 - SEQ TO NO: 154 H-C-Ll-MM SEQ ID NO: ¢12 AΚΰδί SEQ ID NO: 1 SEQ ID NO: 18 SEQ ID NO: 226 - SEQ TO NO: 154 H-C-Ll-MM SEQ ID NO: ¢13 AK087 SEQ ID NO: 1 SEQ ID NO: 19 SEQ ID NO; 226 SEQ TO NO: 154 H-C-Ll-MM SEQ ID NO: 614 AK096 SEQ ID NO; 1 SEQ ID NO; 762 SEQ TO NO: 154 H-C-LI SEQ ID NO: 627 AK097 SEQ ID NO: 1 SEQ ID NO: 763 - - SEQ TO NO: 154 H-C-L! SEQ ID NO: ¢23 AK098 SEQ ID NO: 1 SEQ ID NO: 764 - - SEQ TO NO: 154 H-C-L 1 SEQ ID NO: ¢29 AK099 SEQ ID NO; 1 SEQ ID NO: 765 SEQ TO NO: 154 H-C-LI SEQ ID NO; 630 AK30Q SEQ ID NO; 1 SEQ ID NO: 766 - - SEQ TO NO: 154 H-C-LI SEQ ID NO: 631

- 191 049880

LK101 SEQ ID NO; 1 SEQ ID NO; 767 - SEQ ID NO; 15-1 H-C-Ll SEQ ID NO; ¢32 AK102 SEQ © NO; 1 SEQ ID NO; 768 SEQ ©NO; 154 H-C-Ll SEQ ©NO; ¢33 AKJQ3 SEQ ID NO: 1 SEQ ID NO: 769 - - SEQ ID NO: 154 H-C-Ll SEQ ID NO: ¢34 AK1&4 SEQiDNO: I SEQ ID NO: 770 - - SEQ ©NQ: 154 H-C-Ll SEQ ID KO: ¢35 AK105 SEQ © NO; 1 SEQ ID NO: 771 - SEQ ©NO; 154 H-C-Ll SEQ ©NO; ¢36 AK106 - SEQ ID NO: 28 SEQ ID NO: 261 SEQ ©NO: 154 H-L1-MM SEQ ©NO: ¢37 AKI 12 SEQiDNO: I SEQ ID KO: 15 SEQ ID NO: 261 - SEQ ID NO: 154 H-C-LI-JBt SEQ ID KO: ¢¢2 AK1 13 SEQ ID NO: 5 SEQ ID KO: 15 SEQ ID NO: 261 - SEQ ID NO: 154 H-C-Ll-NDt SEQ ID KO: ¢42 AKIM SEQ © NO: 6 SEQ ID KU 15 SEQ ID NO: 261 SEQ ©NO: 154 H-C-L1-MM SEQ ©NO: 643 AKI 15 SEQ ID NO: 28 SEQ ©NO: Ifl SEQ ©NO: 155 H-L1-MM SEQ ©NO; ¢44 AK1 16 SEQ ID NO: P5 - - - SEQ ID NO: 154 H-C SEQ ID KO: ¢45 AKI 17 SEQ ID NO: 6 - - - SEQ ©NO: 154 H-C SEQ ID KO: ¢46 AKUS SEQ ID NO: 777 SEQ ©NO: 154 H-C SEQ ©NO; ¢47 AKI 19 SEQ ID NO: 778 SEQ ©NO: 154 H-C SEQ ID NO; ¢48 AK120 SEQ ID KO: 77? - - - SEQ ©NO: 154 H-C SEQ ID KO: ¢49 AK121 SEQ ID NO: 780 - SEQ ©NO: 154 H-C SEQ ID NO: ¢50 AKI 22 SEQ ID NO: 781 SEQ ©NO: 154 H-C SEQ ID NO; ¢51 AKI 23 SEQ ID NO: 782 SEQ ©NO: 154 H-C SEQ ID NO; ¢52 AK124 SEQ ID NO: 783 - - - SEQ ©NO: 154 H-C SEQ ID KO: ¢53 LK125 SEQ ID NO: 784 - SEQ ©NO: 154 H-C SEQ ID NO; ¢54 AKI 26 SEQ ID NO: 785 SEQ ©NO: 154 H-C SEQ ID NO; ¢55 AK127 SEQ ID NO: 786 - - - SEQ ©NO: 154 H-C SEQ ID KO; 656 AK128 SEQ ©KO: 787 SEQ ©NO: 154 H-C SEQ ©KO: 657 AK129 SEQ ©KO: 788 SEQ ©NO: 154 H-C SEQ ©KO: 658 AKBO SEQ ID KO: 789 SEQ ©NO: 154 H-C SEQ ©KO: 659 AKI3I SEQ ID KO: 7I SEQ ©NO: 154 H-C SEQ©KO: 660 AKB2 SEQ ©KO: 791 SEQ ©NO: 154 H-C SEQ ©KO: 661 AKU3 SEQ ID KO: 79’ SEQ ©NO: 154 H-C SEQ ©KO: 662

Masked IL-15 polypeptide constructs are prepared that comprise an IL-15 polypeptide or a functional fragment thereof, a masking fragment, and a half-life extension domain such as albumin, an antibody or a fragment thereof (e.g., an Fc region, a heavy chain, and/or a light chain), an albumin-binding peptide, an IgG-binding peptide, or a polyamino acid sequence. Some IL-15 polypeptide constructs are also prepared that comprise an IL-15 polypeptide or a functional fragment thereof linked to a half-life extension domain without also including a masking fragment. Some of the constructs also comprise a linker that comprises a cleavable peptide and links the masking fragment to the IL-15 polypeptide or a functional fragment thereof, resulting in an activatable masked IL-15 polypeptide construct. Some of the constructs also comprise a linker that links the IL-15 polypeptide or functional fragment thereof to the half-life extension domain. Some of the constructs also comprise a linker that links the IL-15 polypeptide or functional fragment thereof to a masking moiety. Masked IL-15 polypeptide constructs that do not comprise a cleavable peptide in the linker that links the IL-15 polypeptide or functional fragment thereof to the masking moiety are also referred to as inactivatable masked IL-15 polypeptide constructs or inactivatable IL-15 polypeptide constructs because they do not comprise a cleavable peptide. Some IL-15 polypeptide constructs are also prepared that comprise a masking moiety comprising the amino acid sequence of SEQ ID NO: 261. The structure and composition of exemplary IL-15 polypeptide constructs are provided in Table 5.

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Table 5

Хз ХСШСГруЕДШ] Cytokine |stp its iJytlWlOitllLHblil fragment(C) Linker (U) Maeknruyushpj fragzhEgfMM) Linker (L2) Domain renewal napiIDja POLOZHNZNI (Η) Structure (nplrshenne cmNKC-tOHlfJ'} 3009 SEQIDNO: 163 SEQ ID TO; 20 SEQ ID NO: 10 SEQ ID TO: 15 SEQIDNO: 151 H-L1-MM-L2-C 3001 SEQIDNQ: 167 SEQ ID TO: 20 SEQ ID NO: 161 SEQ ID TO: 16 SEQ GO NO: 15-3 H-L1-MM-L2-C ZOI SEQIDNO: 167 SEQ ID TO: 20 SEQ ID TO: 162 SEQ TO: 17 SEQIDNO: 151 H-L1-MM-L2-C 3003 SEQIDNO: 167 SEQ TO TO: 20 SEQIDNO: 163 SEQ TO TO: IS SEQIDNO: 151 R-LI-MM-L2-C zom SEQIDNO: 167 SEQ TO TO: 20 SEQ ID NO: IM SEQ ID TO: 19 SEQIDNO: 151 H-L1-MM-L2-C 3005 SEQIDNO: 167 SEQ TO TO: 20 SEQIDNO: 165 SEQ ID TO: 15 SEQIDNO: 151 R-L1-MM-L2-C 3006 SEQIDNO: 167 SEQ TO TO: 20 SEQ ID NO: 21? SEQ ID TO: 16 SEQIDNO: 151 H-L1-MM-L2-C 3007 SEQ ID TO: 167 SEQ ID TO: 20 SEQIDNO: 220 SEQ ID TO: 17 SEQIDNO: 151 R-L1-MM-L2-C 3003 SEQ ID TO: 167 SEQ ID TO: 20 SEQ ID NO: 221 SEQ ID TO: IS SEQIDNO: 151 H-Ll-MM-LZ-C 3000 SEQ ID NO: [67 SEQ ID TO: 20 SEQIDNO: 222 SEQ ID TO: 19 SEQIDNO: 151 R-L1-MM-L2-C ZOYU SEQ ID TO: 167 SEQ ID TO: 20 SEQIDNO: 223 SEQ ID TO: 15 SEQIDNO: 151 C-L2-MM-LI-H zone SEQIDNO: [67 SEQ ID TO: 20 SEQ ID TO: 221 SEQ ID TO: 16 SEQIDNO: 151 CL2-5LM-L1-H 3012 SEQ ID TO: 167 SEQ ID TO: 20 SEQIDNO: 225 SEQ ID TO: 17 SEQIDNO: 151 C-L2-MM-L1-H 3013 SEQ ID NO: [67 SEQ ID TO: 20 SEQ ID TO: 226 SEQ ID TO; IS SEQIDNO: 151 C-L2-NLM-L1-H ZOYU SEQ ID NO: 167 SEQ ID TO: 20 SEQIDNO: 227 SEQ ID TO: 19 SEQIDNO: 151 C-L2-5IM-L1-H 3015 SEQ ID NO: [67 SEQ TO TO: 20 SEQ ID TO: 223 SEQ ID TO; 15 SEQIDNO: 151 C-L2MM-L1H 3016 SEQ ID NO: [67 SEQ ID TO: 20 SEQIDNO: 229 SEQ ID TO: 16 SEQIDNO: 151 C-L2-MM-LI-H 3017 SEQ ID NO; [67 SEQ ID TO: 20 SEQ ID NO; 232 SEQ ID TO: 17 SEQIDNO; 151 C-L2-5LM-L1-H 301S SEQ ID NO: [67 SEQ ID TO: 20 SEQ ID TO: 233 SEQ ID TO: IS SEQIDNO: 151 C-L2.5LM-LPH ZOYU SEQ ID NO; [67 SEQ ID TO: 20 SEQ ID NO; 23-1 SEQ ID TO: 19 SEQIDNO: 151 C-L2-5EM-L1-H

Also prepared are masked IL-2 polypeptide constructs that comprise an IL-2 polypeptide or a functional fragment thereof, a first masking fragment, a second masking fragment, and a half-life extension domain such as albumin, an antibody or a fragment thereof (e.g., an Fc region, a heavy chain, and/or a light chain), an albumin-binding peptide, an IgG-binding peptide, or a polyamino acid sequence. Some of the constructs also comprise a linker that links the first masking fragment to the IL-2 polypeptide or a functional fragment thereof. Some of the constructs also comprise a linker that links the second masking fragment to the IL-2 polypeptide or a functional fragment thereof. Some of the constructs comprise a cleavable peptide in a linker linking the first masking moiety to the IL-2 polypeptide or functional fragment thereof and/or a linker linking the second masking moiety to the IL-2 polypeptide or functional fragment thereof, resulting in an activatable masked IL-2 polypeptide construct. Some of the constructs also comprise a linker linking the second masking moiety to the half-life extension domain. Masked IL-2 polypeptide constructs that do not comprise a cleavable peptide in any of the linkers that link the IL-2 polypeptide or functional fragment thereof to the first masking moiety or the second masking moiety are also referred to as non-activatable masked IL-2 polypeptide constructs or non-activatable IL-2 polypeptide constructs, since they do not comprise a cleavable peptide. The structure and composition of exemplary IL-2 polypeptide constructs are provided in Table 6.

- 193 049880

Table 6

KCIItClJlVKCU-IIC MpSKMruKCh£L|P f|Sh1MS1TT (MM1) 2[tiHKC]i (Ll) Cytokine Idk his M9UH fragment (C) ,Ίκι ι«μ (,L2) MacKicpvipQiiiit l^pillUCH g (MM2) Linker (L3) Domain njxuneriiK ICC|M1U,17 HU.'iyiTIJIICl] Structure (|-|G]G1|>LP.TSN1SS u g N> to the C' end) AiniliCiKHC.lOTIIM ctc.u.ic.iansTC.TWiif>c tt. 2015 SEQJDNO: 9 SEQ [0X0:20 SEQ [D NO: i SEQ 3D NO: >9 SEQ ID NO: ю SEQTDNO: 1? SEQ3DNO: IS H-Ll-MMl-L2-<-L3-MM2 2&7J SEQ ID NO: 9 SEQ [DNO: 20 SEQ ID NO: 1 SEQ 3D NO: 29 SEQ ID NO: L0 SEQ ID NO: 16 SEiQiDNO: 164 E1-b1^SH1-b2-S-b3-L1M2 2073 SEQ ID NO: 9 SEQ [DNO; 20 SEQ ID NO: 1 SEQ ID NO: 29 SEQ ID NO: ίΰ SEQ ID NO: 1 SEQ ID NO: 15.1 H»LIAtMi.L2.C»l3AfM2 Railway SEQJDNO: 9 SEQ (DNO: 20 SEQ [D NO: 1 SEQ 3D NO: >9 SEQ ID NO: L0 SEQ ID NO: ]$ SEQ 3D NO: 154 H‘LI-MMI-L2<‘L3‘MM2 2075 SEQ ]D NO: 9 SEQ [DNO:JO SEQ ID NO: 1 SEQ 3D NO: 29 SEQ ID NO: L0 SEQ ID NO: 19 SEiQiDNO: l?J E1-Y-MM1-E2-C-i-MM2 2076 SEQ ID NO: 9 SEQ [D NO: 20 SEQ ID NO: 2 SEQ ID NO: 29 SEQ ID NO: ίΰ SEQ ID NO: 16 SEQ ID NO: 154 NtNiAGM1.E2.CrLZA1M2 SEQJDNO; 9 SEQ [DNO: 20 SEQ [D NO: 2 SEQ 3D NO; 29 SEQ ID NO: L0 SEQ ID NO: ]7 SEQ 3D NO: |54 H-LbMStl-L2-<>L3>MM2 2078 SEQ ID NO: 9 SEQ [DXO:JO SEQ ID NO: 2 SEQ ID NO: 29 SEQ ID NO: ίΰ SEQ ID NO: 18 SEQ ID NO: 151 H-LI-MMI-L2-C-L3-MM2 2079 SEQ ID NO; 9 SEQ ID NO: 20 SEQ ID NO: 2 SEQ 3D NO; 29 SEQ ID NO: ίΰ SEQ ID NO: 19 SEQ ID NO; 154 E1-b1-5bM1-E2-S-Y-L1M2 AK041 SEQ ID NO: 9 SEQ [DNO: 20 SEQ ID NO. 1 SEQ ID NO: 29 SEQ ID NO: ίΰ SEQ ID NO: 15 SEQ ID NO: 154 H-L1-MM1-L2-C-L3-MM2 SEQ ID NO: 56 A K.072 SEQJDNO: 9 SEQ ID NO: 20 SEQ ID NO: I SEQ 3D NO: 29 SEQ ID NO: L0 SEQTDNO: 16 SEQ 3D NO: 154 SEQ ID NO: 59S AK07J SEQ ID NO; 9 SEQ ID NO: 20 SEQ ID NO: 1 SEQ 3D NO; 29 SEQ ID NO: L0 SEQ ID NO: 1 SEiQiDNO; 154 E1-b1-MM1-1D-S-i-L1M2 SEQ ID NO: 599 AKO-71 SEQ ID NO: 9 SEQ [DNO; 20 SEQ ID NO. 1 SEQ ID NO: 29 SEQ ID NO: L0 SEQ ID NO: IS SEQ ID NO: 154 Ntb1A1MIE.2-CrbZL1M2 SEQ ID NO: 6OD AK.075 SEQJDNO: S SEQ ID NO: 20 SEQ ID NO: i SEQ 3D NO: 29 SEQ ID NO: L0 SEQTDNO 19 SEQ3DNO: 154 SEQ ID NO: 601

Also prepared are masked IL-15 polypeptide constructs that comprise an IL-15 polypeptide or a functional fragment thereof, a first masking fragment, a second masking fragment, and a half-life extension domain such as albumin, an antibody or a fragment thereof (e.g., an Fc region, a heavy chain, and/or a light chain), an albumin-binding peptide, an IgG-binding peptide, or a polyamino acid sequence. Some of the constructs also comprise a linker that links the first masking fragment to the IL-15 polypeptide or a functional fragment thereof. Some of the constructs also comprise a linker that links the second masking fragment to the IL-15 polypeptide or a functional fragment thereof. Some of the constructs comprise a cleavable peptide in a linker linking a first masking fragment to an IL-15 polypeptide or a functional fragment thereof and/or a linker linking a second masking fragment to an IL-15 polypeptide or a functional fragment thereof, resulting in the formation of an activatable masked IL-15 polypeptide construct. Some of the constructs also comprise a linker linking the second masking fragment to a half-life extension domain. Some of the constructs also comprise a second half-life extension domain that is associated with the first half-life extension domain. Masked IL-15 polypeptide constructs that do not contain a cleavable peptide in any of the linkers that link the IL-15 polypeptide or functional fragment thereof to the first masking fragment or the second masking fragment are also referred to as non-activatable masked IL-15 polypeptide constructs or non-activatable IL-2 polypeptide constructs because they do not contain a cleavable peptide. The structure and composition of exemplary IL-15 polypeptide constructs are provided in Table 7.

Table 7

No. soft-plow Multi-functional fragment (MMI) Linker (Ll) Yugtoyush or its functional phrase Mcaci (C) .Ъpeker (L2) Mgkknrukshnj fragment (MM2) Linker CL 3) Domain extension pernodp po.t/zhnish (Η) Structure (direction orN- to: CKOHtiy) L>[|||gotaes.tol!al sequence 3020 SEQ ID NO:232 SEQ ID NO: 20 SEQ ID NO: ]ti? SEQ ID NO: 29 SEQJDNO. 10 SEQ] DNO: 15 SEQ ID NO: 154 H.LlTMMlrL2.C,L3.MM2 3021 ' 3022 SEQ IDNO:232 SEQ IDNO:J52 SEQ ID NO: 20 SEQ ID NO: 20 SEQ ID NO: 167 SEQ ID NO: J67 SEQ ID NO: 29 SEQ ID NO: 29 SEQ[DNO: L6I SEQ[DNO: L62 SEQ JD NO: ] 6 | SEQ ID NO. 154 SEQ JD NO: 17 j SEQ ID NO: 154 H-L1-.MM1-L2-C-L3-MM2 H-Ll-A£Ml-L2-C-LJ-M>i2 3023 SEQ ID NO;2J2 SEQ ID NO; 20 SEQ ID NO: 167 SEQ ID NO: 29 SEQ [DNO;L63 SEQIDNO; IS | SEQ ID NO: 154 H-L1-SLMI-L2-C-LJ-MM2 3024 SEQ [DNO: 2LL SEQ ID NO; 20 SEQJDNO: 167 SEQ ID NO: 29 SEQ [DNO;L64 SEQ] DNO; 19 1 SEQJDNO: 154 [[-L]*MMl*L2-<'-L.UMM2 3025 SEQ ID NO: 2.1. iJ-QJDNO: 2D SEQ ID NO: ]67 SEQ ID NO: 29 SEQIDNQ: ίι5<. S>;Q]|>NO: 15 I sEQ IDN'O: 154 H.], )AlMl-t _r 7.<-.l,.l.\[Xt2 3026 SEQ [DNO: 2.1.1 SEQ ID NO: 20 SEQ ID NO: 167 SEQ ID NO: 29 SEQ tDNO: 219 SEQ]DNO:J6| SEQ1DNOM54 Η.1.]ΛΐΜΐ.[.2.ί'.Ι..1Λ[Μ2 3027 SEQ ID NO:233 SEQ ID NO: 20 SEQ ID NO: 167 SEQ ID NO: 29 SEQ tDNO: 220 SEQ JD NO: 17 1 SEQ ID NO: 154 Η.ί1ΔΐΜΜ2.Ο.Ι.3ΑίΜ2 3028 SEQ ID NO: 2.14 SEQ ID NO; 20 SEQ ID NO: 167 SEQ [DNO: 29 SEQ [DNO; 22! SEQJDNO; 18 | SEQJDNO; 154 3029 SEQ tDNO: 2.14 SEQ ID NO: 20 SEQ ID NO: 167 SEQ ID NO: 29 SEQ[DNO: 222 SEQIDNO: 19 1 SEQ ID NO; 154 Η.],]ΔΐΜΐ-υ·<·-Ι..Ι·ΜΜ2 3030 SEQ [DNO: 2.14 SEQ ID NO: 20 SEQ ID NO: ]67 SEQ ID NO: 29 SEQ [DNO: 227 SEQJDNO: JSI SEQJDNO: 154 ΜΜ2-[.3·ύ-Ι.2-ΜΜί.].]·Η 303] SEQ ID NO:234 SEQ ID NO: 20 SEQ ID NO: 167 SEQ ID NO; 29 SEQ [DNO: 22S SEQ JD NO: 16 I SEQ ID NO; 1 54 MM2’L3-C-L2’MML-L1-H 3032 ' AK40I SEQ ID NO:23-1 SEQ ID NO:261 SEQ ID NO: 20 SEQ ID NO: 28 SEQ ID NO: 167 SEQ ID NO: 167 SEQ ID NO: 29 SEQ ID NO: .48 SEQ [DNO: 229 SEQ [DNO: 825 SEQ JD NO: 17 j SEQ ID NO. 154 SEQ ID NO: 2? j SEQ ID NO: 155 MM2-L3-C-L2-MML-L1-H H-L1-ALM1-L2-MM2-LJ-C SEQJDNO: 753 - - - - | SEQ ID NO: 616 H SEQJDNO: 616 LK403 SEQ [DNO: 261 SEQ ID NO; 28 SEQJDNO: 167 SEQJDNO; 313 SEQ [DNO; 825 SEQJDNO;J7| SEQJDNO: 155 SEQ ID NO: 756 * - I SEQ ID NO: 616 H SEQlONQriiJ^ AK402 SEQ [DNO: 261 SEQ ID NO: 2S SEQ ID NO: 167 SEQ ID NO: 319 SEQ [DNO: S2< SEQJDNO: 27 1 SEQJDNO; 3 55 H-].]-MML-[.2-MM2-[.j-C SEQJDNO; 737 - j SEQ ID NO: 616 H SEQlDN0;dl6

Masked IL-2 polypeptide constructs are also prepared that comprise an IL-2 polypeptide or a functional fragment thereof, a masking fragment, a first half-life extending domain, a second half-life extending domain such as albumin, an antibody or fragment thereof (e.g., an Fc region, a heavy chain, and/or a light chain), an albumin-binding peptide, an IgG-binding peptide, or a polyamino acid sequence. The masking fragment is linked to

- 194 049880 a first half-life extension domain, the IL-2 polypeptide or a functional fragment thereof is linked to a second half-life extension domain, and the first half-life extension domain and the second half-life extension domain comprise modifications that facilitate the association of the first and second half-life extension domains. In one exemplary embodiment, the masking fragment is linked to the first half-life extension domain and comprises the amino acid sequence of SEQ ID NO: 267, and the IL-2 polypeptide or a functional fragment thereof is linked to the second half-life extension domain and comprises the amino acid sequence of SEQ ID NO: 266, and the first half-life extension domain and the second half-life extension domain comprise modifications that facilitate the association of the first and second half-life extension domains. In one exemplary embodiment of an unmasked IL-2 polypeptide construct, the embodiment comprises an IL-2 polypeptide or a functional fragment thereof linked to a first half-life extension domain and comprises a second half-life extension domain, wherein the IL-2 polypeptide or a functional fragment thereof is linked to the first half-life extension domain and comprises the amino acid sequence of SEQ ID NO: 266, and the second half-life extension domain comprises the amino acid sequence of SEQ ID NO: 265. Some of the constructs also comprise a linker that links the masking moiety to the first half-life extension domain and/or a linker that links the IL-2 polypeptide or a functional fragment thereof to the second half-life extension domain. The first and second half-life extension domains of some constructs are also linked. In some constructs, the first and second extended half-life domains of some constructs are linked via a linker. Some of the constructs contain a cleavable peptide in a linker linking the masking moiety to the first half-life extension domain and/or a linker linking the IL-2 polypeptide or a functional fragment thereof to the second half-life extension domain, resulting in an activatable masked IL-2 polypeptide construct. Masked IL-2 polypeptide constructs that do not contain a cleavable peptide in either a linker linking the IL-2 polypeptide or a functional fragment thereof to the second half-life extension domain or a linker linking the masking moiety to the first half-life extension domain are also referred to as non-activatable masked IL-2 polypeptide constructs or non-activatable IL-2 polypeptide constructs, since they do not contain a cleavable peptide. The structure and composition of exemplary IL-2 polypeptide constructs are provided in Table 8.

Table 8

He designs Cytokine or its functional fragment (C) Linker (LI) Masking fragment (MM) Linker (L2) Half-life extension domain (H) Structure (direction from N- to C-terminus) Amino acid sequence 2007/2008 SEQ ID NO: 1 SEQ ID NO: 22 SEQIDNO: 157 C-L1-H SEQ ID NO: 23 SEQ ID NO: 9 SEQIDNO: 158 MM-L1-H 2009/2010 SEQ ID NO: 24 SEQ ID NO: 9 SEQIDNO: 158 MM-L1-H SEQ ID NO: 1 SEQ ID NO: 25 SEQIDNO: 157 C-L1-H 2080/2081 SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 1 SEQ ID NO: 11 SEQIDNO: 156 H-L1-C 2080/2082 SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 1 SEQ ID NO: 12 SEQIDNO: 156 H-L1-C 2080/2083 SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 1 SEQ ID NO: 13 SEQIDNO: 156 H-L1-C 2080/2084 SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 1 SEQ ID NO: 14 SEQIDNO: 156 H-L1-C 2080/2085 SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 3 SEQ ID NO: 11 SEQIDNO: 156 H-L1-C 2080/2086 SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 3 SEQ ID NO: 12 SEQIDNO: 156 H-L1-C 2080/2087 SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 3 SEQ ID NO: 13 SEQIDNO: 156 H-L1-C 2080/2088 SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 3 SEQ ID NO: 14 SEQIDNO: 156 H-L1-C 2080/2089 SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM

- 195 049880

.N° of construction Cytokine or its functional fragment (C) Linker (LI) M a c κι rpy Yushin fragment (MM) Linker (L2) Half-life extension domain (H) Structure (direction from N- to C-terminus) Amino acid sequence SEQ ID NO: 4 SEQ ID NO: 11 SEQIDNO: 156 H-Ll-C 2080/2090 - SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 4 SEQ ID NO: 12 SEQIDNO: 156 H-Ll-C 2080/2091 - SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 4 SEQ ID NO: 13 SEQIDNO: 156 H-Ll-C 2080/2092 - SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 4 SEQ ID NO: 14 SEQIDNO: 156 H-Ll-C 2080/2093 - SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 5 SEQ ID NO: 11 SEQIDNO: 156 H-Ll-C 2080/2094 - SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 5 SEQ ID NO: 12 SEQIDNO: 156 H-Ll-C 2080/2095 - SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 5 SEQ ID NO: 13 SEQIDNO: 156 H-Ll-C 2080/2096 - SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 5 SEQ ID NO: 14 SEQIDNO: 156 H-Ll-C 2080/2097 - SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 6 SEQ ID NO: 11 SEQIDNO: 156 H-Ll-C 2080/2098 - SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 6 SEQ ID NO: 12 SEQIDNO: 156 H-Ll-C 2080/2099 - SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQ ID NO: 6 SEQ ID NO: 13 SEQIDNO: 156 H-Ll-C 2080/2100 - SEQ ID NO: 28 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM

.V? constructions Cytokine or its functional fragment (C) Linker (LI) Masking fragment (MM) Linker (L2) Half-life extension domain (H) Structure (direction or N- to C-terminus) Amino acid sequence SEQ ID NO: 6 SEQIDNO: 14 - SEQIDNO: 156 H-Ll-C 2080/2101 - SEQ ID NO: 28 SEQIDNO: 10 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 7 SEQIDNO: 11 - SEQIDNO: 156 H-Ll-C 2080/2102 - SEQ ID NO: 28 SEQIDNO: 10 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 7 SEQIDNO: 12 - SEQIDNO: 156 H-Ll-C 2080/2103 - SEQ ID NO: 28 SEQIDNO: 10 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 7 SEQIDNO: 13 - SEQIDNO: 156 H-Ll-C 2080/2104 - SEQ ID NO: 28 SEQIDNO: 10 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 7 SEQIDNO: 14 - SEQIDNO: 156 H-Ll-C 2080/2105 - SEQ ID NO: 28 SEQIDNO: 10 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 8 SEQIDNO: 11 - SEQIDNO: 156 H-Ll-C 2080/2106 - SEQ ID NO: 28 SEQIDNO: 10 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 8 SEQIDNO: 12 - SEQIDNO: 156 H-Ll-C 2080/2107 - SEQ ID NO: 28 SEQIDNO: 10 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 8 SEQIDNO: 13 - SEQIDNO: 156 H-Ll-C 2080.2108 - SEQ ID NO: 28 SEQIDNO: 10 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 8 SEQIDNO: 14 - SEQIDNO: 156 H-Ll-C 5001/5002 SEQ ID NO: 260 SEQ ID NO: 262 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 266 - - SEQ ID NO: 265 H SEQ ID NO: 265 5001/5003 SEQ ID NO: 260 SEQ ID NO: 262 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 266 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AK.038 SEQ ID NO: 1 SEQ ID NO: 22 - SEQIDNO: 157 C-Ll-H SEQ ID NO: 562

- 196 049880

He designs Cytokine or its functional fragment (C) Linker (LI) Masking fragment (MM) Linker (L2) Half-life extension domain (H) Structure (direction from N- to C-terminus) Amino acid nutrition SEQ ID NO: 23 SEQ ID NO: 9 SEQIDNO: 158 MM-L1-H SEQIDNO: 563 AK039 SEQ ID NO: 24 SEQ ID NO: 9 SEQIDNO: 158 MM-L1-H SEQIDNO: 564 SEQ ID NO: 1 SEQ ID NO: 25 SEQIDNO: 157 C-Ll-H SEQIDNO: 565 AK076 SEQ ID NO: 1 SEQ ID NO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 603 SEQ ID NO: 795 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQIDNO: 603 AKO77 SEQ ID NO: 1 SEQ ID NO: 12 SEQIDNO: 156 H-Ll-C SEQIDNO: 604 SEQ ID NO: 795 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQIDNO: 603 AKO78 SEQ ID NO: 1 SEQ ID NO: 13 SEQIDNO: 156 H-Ll-C SEQIDNO: 605 SEQ ID NO: 795 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQIDNO: 603 AK079 SEQ ID NO: 1 SEQ ID NO: 14 SEQIDNO: 156 H-Ll-C SEQIDNO: 606 SEQ ID NO: 795 SEQ ID NO: 10 SEQIDNO: 155 H-L1-MM SEQIDNO: 603 AK081 SEQ ID NO: 1 SEQ ID NO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 608 SEQ ID NO: 265 H SEQIDNO: 265 AKO88 SEQ ID NO: 1 SEQIDNO: 155 H-C SEQIDNO: 615 SEQ ID NO: 616 H SEQIDNO: 616 AK089 SEQ ID NO: 1 SEQIDNO: 155 H-C SEQIDNO: 615 SEQ ID NO: 15 SEQ ID NO: 10 SEQIDNO: 156 H-L1-MM SEQIDNO: 617 AK090 SEQ ID NO: 1 SEQIDNO: 721 H-C SEQIDNO: 618 SEQ ID NO: 619 H SEQIDNO: 619 AK091 SEQ ID NO: 1 SEQIDNO: 721 H-C SEQIDNO: 618 SEQ ID NO: 15 SEQ ID NO: 10 SEQIDNO: 772 H-L1-MM SEQIDNO: 620 AK092 SEQ ID NO: 1 SEQIDNO: 793 H-C SEQIDNO: 621

X? constructions Cytokine or its functional fragment (C) Linker (LI) .M a c κι ipy юптш fragment (MM) Linker (L2) Half-life extension domain (H) Structure (direction οι N- to C-terminus) Amino acid sequence - - SEQ ID NO: 622 N SEQ ID NO: 622 AK093 SEQ ID NO: 1 - SEQIDNO: 793 H-C SEQ ID NO: 621 - SEQIDNO: 15 SEQIDNO: 10 - SEQIDNO: 773 H-L1-MM SEQ ID NO: 623 AK094 SEQ ID NO: 1 - SEQIDNO: 796 H-C SEQ ID NO: 624 - - SEQ ID NO: 625 H SEQ ID NO: 625 AKO95 SEQ ID NO: 1 - SEQIDNO: 796 H-C SEQ ID NO: 624 - SEQIDNO: 15 SEQIDNO: 10 - SEQIDNO: 774 H-L1-MM SEQ ID NO: 626 AK107 - SEQIDNO: 15 SEQ ID NO: 261 - SEQIDNO: 156 H-L1-MM SEQ ID NO: 638 SEQ ID NO: 1 - SEQIDNO: 155 H-C SEQ ID NO: 615 AK108 - SEQIDNO: 15 SEQ ID NO: 261 - SEQIDNO: 772 H-L1-MM SEQ ID NO: 639 SEQ ID NO: 1 - SEQIDNO: 721 H-C SEQ ID NO: 618 AK109 - SEQIDNO: 15 SEQ ID NO: 261 - SEQIDNO: 773 H-L1-MM SEQ ID NO: 640 SEQ ID NO: 1 - SEQIDNO: 793 H-C SEQ ID NO: 621 AKIO - SEQIDNO: 15 SEQ ID NO: 261 - SEQIDNO: 774 H-L1-MM SEQ ID NO: 641 SEQ ID NO: 1 - SEQIDNO: 796 H-C SEQ ID NO: 624 AKIU SEQ ID NO: 1 SEQIDNO: 11 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 608 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AKI 63 SEQ ID NO: 1 SEQIDNO: 11 - SEQIDNO: 793 H-Ll-C SEQ ID NO: 663 - - SEQ ID NO: 622 H SEQ ID NO: 622 AK164 SEQ ID NO: 1 SEQIDNO: 11 - SEQIDNO: 793 H-Ll-C SEQ ID NO: 663 - SEQ ID NO: 795 SEQ ID NO: 261 - SEQIDNO: 773 H-L1-MM SEQ ID NO: 664 AK165 SEQ ID NO: 1 SEQIDNO: 11 - SEQIDNO: 796 H-Ll-C SEQ ID NO: 665

- 197 049880

Design No. Cytokine or its functional fragment (C) Linker (LI) Masking fragment (MM) Linker (L2) Half-life extension domain (H) Structure (direction from N- to C-cone) Amino acid eating SEQ ID NO: 625 N SEQIDNO: 625 AK166 SEQ ID NO: 1 SEQ ID NO: 11 SEQIDNO: 796 H-Ll-C SEQIDNO: 665 SEQ ID NO: 795 SEQ ID NO: 261 SEQIDNO: 774 H-L1-MM SEQIDNO: 666 AK167 SEQ ID NO:3 SEQ ID NO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 667 SEQ ID NO: 265 H SEQIDNO: 265 AK168 SEQ ID NO:3 SEQ ID NO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 667 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK169 SEQ ID NO: 6 SEQ ID NO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 669 SEQ ID NO: 265 H SEQIDNO: 265 AK170 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 SEQ ID NO: 6 SEQ ID NO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 669 AK171 SEQ ID NO: 1 SEQ ID NO: 11 SEQIDNO: 155 H-Ll-C SEQIDNO: 670 SEQ ID NO: 616 H SEQIDNO: 616 AK172 SEQ ID NO: 1 SEQ ID NO: 11 SEQIDNO: 155 H-Ll-C SEQIDNO: 670 SEQ ID NO: 795 SEQ ID NO: 261 SEQIDNO: 156 H-L1-MM SEQIDNO: 671 AK184 SEQ ID NO: 1 SEQ ID NO: 800 SEQIDNO: 156 H-Ll-C SEQIDNO: 672 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK185 SEQ ID NO: 1 SEQ ID NO: 185 SEQIDNO: 156 H-Ll-C SEQIDNO: 673 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK186 SEQ ID NO: 1 SEQ ID NO: 802 SEQIDNO: 156 H-Ll-C SEQIDNO: 674 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK187 SEQ ID NO: 1 SEQ ID NO: 803 SEQIDNO: 156 H-Ll-C SEQIDNO: 675

Design No. Cytokine or its functional fragment (C) Linker (LI) Masking fragment (MM) Linker (L2) Half-life extension domain (H) Structure (direction οι N- to C-terminus) Amino acid sequence SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK188 SEQ ID NO: 3 SEQ ID NO: 804 SEQIDNO: 156 H-Ll-C SEQIDNO: 676 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK189 SEQ ID NO: 1 SEQ ID NO: 805 SEQIDNO: 156 H-Ll-C SEQIDNO: 677 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK190 SEQ ID NO: 1 SEQ ID NO: 806 SEQIDNO: 156 H-Ll-C SEQIDNO: 678 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK191 SEQ ID NO: 3 SEQIDNO: 807 SEQIDNO: 156 H-Ll-C SEQIDNO: 679 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK192 SEQ ID NO: 3 SEQ ID NO: 805 SEQIDNO: 156 H-Ll-C SEQIDNO: 680 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AKI 93 SEQ ID NO: 3 SEQ ID NO: 806 SEQIDNO: 156 H-Ll-C SEQIDNO: 681 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK194 SEQ ID NO: 1 SEQIDNO: 808 SEQIDNO: 156 H-Ll-C SEQIDNO: 682 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK195 SEQ ID NO: 1 SEQ ID NO: 809 SEQIDNO: 156 H-Ll-C SEQIDNO: 683 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK196 SEQ ID NO: 1 SEQIDNO: 810 SEQIDNO: 156 H-Ll-C SEQIDNO: 684 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK197 SEQ ID NO: 3 SEQIDNO: 808 SEQIDNO: 156 H-Ll-C SEQIDNO: 685 SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK198 SEQ ID NO: 3 SEQ ID NO: 809 SEQIDNO: 156 H-Ll-C SEQIDNO: 686

- 198 049880

constructions Cytokine plp its functional fragment (C) Linker (LI) Masking fragment (MM) Linker (L2) Half-life extension domain (H) Structure (direction or N- to C-terminus) Amino acid sequence - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 Ak199 SEQ ID NO: 3 SEQ ID NO: 810 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 687 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AQ200 SEQ ID NO: 1 SEQ ID NO: 811 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 688 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AQ201 SEQ ID NO: 1 SEQ ID NO: 812 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 689 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AQ202 SEQ ID NO: 1 SEQ ID NO: 263 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 690 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AQ203 SEQ ID NO: 3 SEQ ID NO: 811 - SEQIDNO 156 H-Ll-C SEQ ID NO: 266 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 A204 SEQ ID NO: 3 SEQ ID NO: 812 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 692 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AQ205 SEQ ID NO: 3 SEQ ID NO: 263 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 693 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 A206 SEQ ID NO: 3 SEQ ID NO: 800 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 694 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AQ207 SEQ ID NO: 3 SEQ ID NO: 801 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 695 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AQ208 SEQ ID NO: 1 SEQ ID NO: 804 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 696 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO 155 H-L1-MM SEQ ID NO: 267 AQ209 SEQ ID NO: 3 SEQ ID NO: 802 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 697

Ns constructions Cptokpn or its functional fragment (C) Linker (LI) Masking num fragment (MM) Linker (L2) Half-life extension domain (H) Structure (direction from N- to C-terminus) Amino acid sequence - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK210 SEQ ID NO: 1 SEQIDNO: 807 SEQIDNO: 156 H-Ll-C SEQIDNO: 698 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK211 SEQ ID NO: 3 SEQ ID NO: 339 SEQIDNO: 156 H-Ll-C SEQIDNO: 699 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK212 SEQ ID NO: 1 SEQ ID NO: 339 SEQIDNO: 156 H-Ll-C SEQIDNO: 700 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK215 SEQ ID NO: 815 SEQIDNO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 701 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK216 SEQ ID NO: 816 SEQIDNO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 702 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK217 SEQ ID NO: 817 SEQIDNO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 703 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK218 SEQ ID NO: 818 SEQIDNO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 704 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK219 SEQ ID NO: 819 SEQIDNO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 705 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK220 SEQ ID NO: 820 SEQIDNO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 706 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK221 SEQ ID NO: 821 SEQIDNO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 707 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK222 SEQ ID NO: 822 SEQIDNO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 708

- 199 049880

constructions Cytokine plp its fun ctional and fragment (C) Linker (LI) Masking fragment (MM) Linker (L2) Half-life extension domain (H) Structure (direction from N- to C-cone) Amino acid sequence - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK223 SEQ ID NO: 813 SEQ ID NO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 709 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK224 SEQ ID NO: 814 SEQ ID NO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 710 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK225 SEQ ID NO: 3 SEQ ID NO: 803 SEQIDNO: 156 H-Ll-C SEQIDNO: 711 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK228 SEQ ID NO: 1 SEQ ID NO: 339 SEQIDNO: 156 H-Ll-C SEQIDNO: 700 - SEQ ID NO: 265 H SEQIDNO: 265 AK231 - SEQIDNO: 156 H SEQIDNO: 156 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK232 SEQ ID NO: 3 SEQ ID NO: 11 SEQIDNO: 156 H-Ll-C SEQIDNO: 667 - SEQ ID NO: 795 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 712 AK233 SEQ ID NO: 1 SEQ ID NO: 691 SEQIDNO: 156 H-Ll-C SEQIDNO: 713 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK234 SEQ ID NO: 3 SEQ ID NO: 691 SEQIDNO: 156 H-Ll-C SEQIDNO: 714 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK235 SEQ ID NO: 3 SEQ ID NO: 802 SEQIDNO: 156 H-Ll-C SEQIDNO: 697 - SEQ ID NO 265 H SEQIDNO: 265 AK252 - SEQ ID NO: 15 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 716 SEQ ID NO: 3 SEQ ID NO: 339 SEQIDNO: 156 H-Ll-C SEQIDNO: 699 AK253 SEQ ID NO: 3 SEQ ID NO: 339 SEQIDNO: 156 H-Ll-C SEQIDNO: 699

.Vs constructions Cytokine plp its functional fragment (C) Linker (Li) Masking the thorn fragment (MM) Linker (L2) Half-life extension domain (H) Structure (direction from N- to C-cone) Amino acid sequence - SEQ ID NO: 265 N SEQ ID NO: 265 AK.304 SEQ ID NO: 815 SEQ ID NO: 802 - SEQIDNO: 156 H-Ll-C SEQIDNO: 717 - SEQ ID NO: 265 N SEQ ID NO: 265 AK.305 SEQ ID NO: 815 SEQ ID NO: 802 - SEQIDNO: 156 H-Ll-C SEQIDNO: 717 SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK.306 SEQ ID NO: 816 SEQ ID NO: 802 - SEQIDNO: 156 H-Ll-C SEQIDNO: 718 - SEQ ID NO: 265 H SEQ ID NO: 265 AK.307 SEQ ID NO: 816 SEQ ID NO: 802 - SEQIDNO: 156 H-Ll-C SEQIDNO: 718 SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK308 SEQ ID NO: 818 SEQ ID NO: 802 - SEQIDNO: 156 H-Ll-C SEQIDNO: 719 - SEQ ID NO: 265 H SEQ ID NO: 265 AK309 SEQ ID NO: 818 SEQ ID NO: 802 - SEQIDNO: 156 H-Ll-C SEQIDNO: 719 SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AKIO SEQ ID NO: 819 SEQ ID NO: 802 - SEQIDNO: 156 H-Ll-C SEQIDNO: 720 - SEQ ID NO: 265 H SEQ ID NO: 265 AK311 SEQ ID NO: 819 SEQ ID NO: 802 - SEQIDNO: 156 H-Ll-C SEQIDNO: 720 SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK312 SEQ ID NO: 820 SEQ ID NO: 802 - SEQIDNO: 156 H-Ll-C SEQIDNO: 722 - SEQ ID NO: 265 H SEQ ID NO: 265 AK313 SEQ ID NO: 820 SEQ ID NO: 802 - SEQIDNO: 156 H-Ll-C SEQIDNO: 722 SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK314 SEQ ID NO: 813 SEQ ID NO: 802 - SEQIDNO: 156 H-Ll-C SEQIDNO: 723

- 200 049880

Design No. Cytokine or its functional fragment (C) Linker (LI) Masking fragment (MM) Linker (L2) Domain of extension of half-life (N) Structure (direction from N- to C-terminus) Amino acid sequence - - SEQ ID NO: 265 N SEQ ID NO: 265 AK315 SEQ ID NO: 813 SEQ ID NO: 802 - SEQIDNO: 156 H-Ll-C SEQIDNO: 723 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK316 SEQ ID NO: 1 SEQ ID NO: 802 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 674 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK325 SEQ ID NO: 815 SEQ ID NO: 11 - SEQIDNO: 156 H-Ll-C SEQIDNO: 701 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK326 SEQ ID NO: 816 SEQ ID NO: 11 - SEQIDNO: 156 H-Ll-C SEQIDNO: 702 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK327 SEQ ID NO: 818 SEQ ID NO: 11 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 704 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK328 SEQ ID NO: 819 SEQ ID NO: 11 - SEQIDNO: 156 H-Ll-C SEQ ID NO: 705 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK329 SEQ ID NO: 820 SEQ ID NO: 11 - SEQIDNO: 156 H-Ll-C SEQIDNO: 706 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AKZZO SEQ ID NO: 813 SEQ ID NO: 11 - SEQIDNO: 156 H-Ll-C SEQIDNO: 709 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK349 SEQ ID NO: 813 SEQ ID NO: 807 - SEQIDNO: 156 H-Ll-C SEQIDNO: 728 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK350 SEQ ID NO: 813 SEQ ID NO: 797 - SEQIDNO: 156 H-Ll-C SEQIDNO: 729 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK351 SEQ ID NO: 813 SEQ ID NO: 798 - SEQIDNO: 156 H-Ll-C SEQIDNO: 730

.Ns constructions Cytokine or its functional fragment (C) Linker (LI) Masking fragment (MM) Linker (L2) Half-life extension domain (H) Structure (direction or N- to C-terminus) Amino acid sequence - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK352 SEQ ID NO: 813 SEQ ID NO: 724 SEQIDNO: 156 H-Ll-C SEQ ID NO: 731 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK353 SEQ ID NO: 813 SEQ ID NO: 725 SEQIDNO: 156 H-Ll-C SEQIDNO: 732 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK354 SEQ ID NO: 813 SEQ ID NO: 536 SEQIDNO: 156 H-Ll-C SEQIDNO: 733 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK355 SEQ ID NO: 813 SEQ ID NO: 537 SEQIDNO 156 H-Ll-C SEQIDNO: 734 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK357 SEQ ID NO: 813 SEQ ID NO: 502 SEQIDNO 156 H-Ll-C SEQIDNO: 735 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK358 SEQ ID NO: 813 SEQ ID NO: 503 SEQIDNO: 156 H-Ll-C SEQIDNO: 736 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK359 SEQ ID NO: 813 SEQ ID NO: 492 SEQIDNO: 156 H-Ll-C SEQIDNO: 737 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK360 SEQ ID NO: 813 SEQ ID NO: 493 SEQIDNO: 156 H-Ll-C SEQIDNO: 738 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK361 SEQ ID NO: 813 SEQ ID NO: 416 SEQIDNO: 156 H-Ll-C SEQIDNO: 739 - SEQ ID NO: 28 SEQ ID NO: 261 SEQIDNO: 155 H-L1-MM SEQIDNO: 267 AK362 SEQ ID NO: 813 SEQ ID NO: 417 SEQIDNO: 156 H-Ll-C SEQ ID NO: 740 - SEQ ID NO: 28 SEQ ID NO 261 SEQIDNO 155 H-L1-MM SEQIDNO: 267 AK363 SEQ ID NO: 813 SEQ ID NO: 418 SEQIDNO: 156 H-Ll-C SEQIDNO: 741

- 201 049880

He designs Cytokine pip its functional fragment (C) Linker (LI) Masking fragment (MM) Linker (L2) Half-life extension domain (H) Structure (direction from N- to C-terminus) Amino acid sequence - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AK364 SEQ ID NO: 813 SEQ ID NO: 419 - SEQIDNO: 156 H-L1-C SEQ ID NO: 742 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AK365 SEQ ID NO: 813 SEQ ID NO: 350 - SEQIDNO: 156 H-L1-C SEQ ID NO: 743 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AK366 SEQ ID NO: 813 SEQ ID NO: 351 - SEQIDNO: 156 H-L1-C SEQ ID NO: 744 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AK367 SEQ ID NO: 813 SEQ ID NO: 352 - SEQIDNO: 156 H-L1-C SEQ ID NO: 745 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AK368 SEQ ID NO: 813 SEQ ID NO: 353 - SEQIDNO: 156 H-L1-C SEQ ID NO: 746 - SEQ ID NO: 28 SEQ ID NO: 261 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 267 AK.317 SEQ ID NO: 1 SEQ ID NO: 802 - SEQIDNO: 156 H-L1-C SEQ ID NO: 674 - SEQ ID NO: 28 SEQ ID NO: 826 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 828 AK.324 SEQ ID NO: 1 SEQ ID NO: 802 - SEQIDNO: 156 H-L1-C SEQ ID NO: 674 - SEQ ID NO: 28 SEQ ID NO: 827 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 829 AK.341 SEQ ID NO: 3 SEQ ID NO: 802 - SEQIDNO: 156 H-L1-C SEQ ID NO: 726 - SEQ ID NO: 28 SEQ ID NO: 826 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 830 AK.342 SEQ ID NO: 3 SEQ ID NO: 802 - SEQIDNO: 156 H-L1-C SEQ ID NO: 726 - SEQ ID NO: 28 SEQ ID NO: 827 - SEQIDNO: 155 H-L1-MM SEQ ID NO: 829

Also prepared are masked IL-15 polypeptide constructs that comprise an IL-15 polypeptide or a functional fragment thereof, a masking fragment, a first half-life extension domain, a second half-life extension domain such as albumin, an antibody or a fragment thereof (e.g., an Fc region, a heavy chain and/or a light chain), an albumin-binding peptide, an IgG-binding peptide, or a polyamino acid sequence. The masking fragment is linked to the first half-life extension domain, the IL-15 polypeptide or a functional fragment thereof is linked to the second half-life extension domain, and the first half-life extension domain and the second half-life extension domain contain modifications that facilitate the association of the first and second half-life extension domains. Some of the constructs also comprise a linker that links the masking moiety to the first half-life extension domain and/or a linker that links the IL-15 polypeptide or a functional fragment thereof to the second half-life extension domain. The first and second half-life extension domains of some constructs are also linked. In some constructs, the first and second extended half-life domains of some constructs are linked via a linker. Some of the constructs comprise a cleavable peptide in the linker that links the masking moiety to the first half-life extension domain and/or a linker that links the IL-15 polypeptide or a functional fragment thereof to the second half-life extension domain, resulting in the formation of an activatable masked IL-15 polypeptide construct. Masked IL-15 polypeptide constructs that do not contain a cleavable peptide in either the linker that links the IL-15 polypeptide or a functional fragment thereof to the second half-life extension domain or the linker that links the masking fragment to the first half-life extension domain are also referred to as non-activatable masked IL-15 polypeptide constructs or non-activatable IL-15 polypeptide constructs because they do not contain a cleavable peptide. The structure and composition of exemplary IL15 polypeptide constructs are provided in Table 9.

- 202 049880

Table 9

No of the design Cytokine or its fuEscilanalpine fragment (C) Linker (LI) M?SK1]ruYu[Sh]G1 fragment (MM) Linker (L21 Half-life extension domain (Ή) Structure* (direction from N- to S'Kchniu) Amino acid sequence 3033 - SEQ ID NO: 23 SEQ ID NO: 10 SEQ ID NO 155 H-Ll-MM SEQIDNO: 167 SEQ ID NO: 11 SEQ ID NO 156 H-Ll-C 3034 - SEQ ID NO: 28 SEQ ID NO: 10 - SEQ ID NO 155 MM-LI-Π SEQIDNO: 167 SEQIDNO: 11 - - SEQ ID NO 156 C-Ll-H 3035 - SEQ ID NO: 28 SEQ ID NO: 10 - SEQ ID NO 155 ASH-LI-H SEQIDNO: 167 SEQ ID NO: J1 - - SEQ ID NO 156 C-Ll-H 3036 - SEQ ID NO: 2$ SEQ ID NO: 232 SEQ ID NO 155 H-Ll-MM SEQIDNO; 167 SEQ ID NO: 11 - SEQIDNO 156 H-LI-C 3037 - SEQ ID NO: 23 SEQ ID NO: 233 SEQ ID NO 155 IL'ALI-H SEQIDNO; 167 SEQIDNO: 11 SEQ ID NO 156 C-Ll-H 3038 SEQ ID NO: 28 SEQ ID NO: 233 - SEQ ID NO 155 H-Ll-MM SEQIDNO: 107 SEQIDNO: 11 - * SEQ ID NO 156 H-LbC AK243 SEQIDNO: 167 SEQ ID NO: titiS - - SEQ ID NO 156 H-LI-C SEQIDNO: 715 - - - - SEQ ID NO 265 II SEQIDNO: 265 AK247 SEQ YUKO: 167 SEQ ID NO: 345 SEQIDNO 156 H-LI-C SEQIDNO: 747 - SEQIDNO: 799 SEQ ID NO: 261 - SEQIDNO 156 H-Ll-MM SEQ ID NO: 671 AK2-1S SEQIDNO: 167 SEQ ID NO: 663 SEQIDNO 156 H-Ll-C SEQIDNO; 715 SEQ ID NO: 23 SEQ ID NO: 261 SEQ ID NO 155 H-Ll-MM SEQIDNO: 267 AK24SL· SEQIDNO: 107 SEQ ID NO: ΰ<53 - SEQ ID NO 156 H-Ll-C SEQIDNO: 715 - SEQ ID NO: 799 SEQ ID NO: 261 SEQ ID NO 156 H-Ll-MM SEQIDNO: 671 AK249 SEQIDNO: Ιΰ7 SEQ ID NO: 355 SEQ ID NO 156 H-LI-C SEQ ID NO: 748 - SEQ ID NO: 799 SEQ ID NO: 261 - SEQ IE NO 156 H-Ll-MM SEQ ID NO; 671 AK250 SEQ ID NO; 167 SEQIDNO; 354 SEQ ID NO: $23 SEQ ID NO: 27 SEQIDNO 156 H-LIC-L2-MM SEQ ID NO: 749 SEQIDNO: 799 SEQ ID NO: 261 SEQIDNO 156 H-Ll-MM SEQ ID NO: 671 AK25J SEQ ID NO: 167 SEQIDNO: 354 SEQ ID NO: 824 SEQ ID NO: 27 SEQIDNO L56 H-LI-C-L2-MM SEQ ID NO; 75Q - SEQ ID NO: 799 SEQ ID NO: 261 - SEQ ID NO 156 H-Ll-MM SEQ ID NO; 671 AK41S SEQ ID NO; 167 SEQ P> NO; 348 SEQIDNO 156 H-LJ-C SEQ ID NO: 751 SEQIDNO; 799 SEQ ID NO: 261 SEQIDNO 156 H-Ll-MM SEQ ID NO: 671 AK419 SEQ ID NO: 167 SEQIDNO: 668 SEQ ID NO: 823 SEQ ID NO: 27 SEQIDNO 156 H-LI-C-L2-MM SEQ ID NO; 752 - SEQ ID NO: 799 SEQ ID NO: 261 - SEQ ID NO L56 H-Ll-MM SEQ ID NO; 671 AK420 SEQ ID NO: 167 SEQ ID NO: 668 SEQ ID NO: $24 SEQ ID NO: 27 SEQIDNO 156 H-LI-C-L2-MM SEQ ID NO: 753 SEQIDNO: 799 SEQ ID NO: 261 SEQIDNO 156 H-Ll-MM SEQ ID NO: 671 AK42J SEQ ID NO: 167 SEQ ID NO: 349 - - SEQIDNO 156 H-LJ-C SEQ ID NO; 754 - SEQ ID NO: 799 SEQ ID NO: 261 - SEQ ID NO 156 H-Ll-MM SEQ ID NO: 671 AK399 SEQ ID NO; 167 SEQIDNO; 320 SEQ ID NO; $23 SEQ ID NO; 321 SEQIDNO 156 H-LIC-L2-MM SEQ ID NO: 75S SEQIDNO: 799 SEQ ID NO: 261 SEQIDNO 156 H-Ll-MM SEQ ID NO: 671 AK405 SEQ ID NO: 167 SEQIDNO: 320 SEQ ID NO: 823 SEQ ID NO: 322 SEQIDNO L56 H-LI-C-L2-MM SEQ ID NO; 759 - SEQ ID NO: 799 SEQ ID NO: 261 - SEQ ID NO 156 H-Ll-MM SEQ ID NO; 671 AK400 SEQ ID NO; 167 SEQ ID NO: 349 SEQ ID NO: $24 SEQ ID NO: 30 SEQ Y NO 156 H-LI.C-L2-MM SEQ ID NO: 760 SEQIDNO: 799 SEQ ID NO: 261 SEQIDNO 156 H-Ll-MM SEQ ID NO: 671 AK404 SEQ ID NO: 167 SEQ ID NO: 349 SEQ ID NO: 823 SEQ ID NO: 30 SEQIDNO 156 H-LI-C-L2-MM SEQ ID NO: 761 - SEQ ID NO: 799 SEQ ID NO: 261 - SEQ IE NO 156 H-Ll-MM SEQ ID NO: 671

Also prepared are masked IL-2 polypeptide constructs that comprise an IL-2 polypeptide or a functional fragment thereof, a first masking fragment, a second masking fragment, a first half-life extension domain, a second half-life extension domain such as albumin, an antibody or a fragment thereof (e.g., an Fc region, a heavy chain and/or a light chain), an albumin-binding peptide, an IgG-binding peptide, or a polyamino acid sequence. The first masking fragment is linked to the first half-life extension domain, the second masking fragment is linked to the IL-2 polypeptide or a functional fragment thereof, or the second masking fragment, or the IL-2 polypeptide or a functional fragment thereof is linked to the second half-life extension domain, and the first half-life extension domain and the second half-life extension domain contain modifications that facilitate the association of the first and second half-life extension domains. Some of the constructs also contain a linker that links the first masking fragment to the first period extension domain.

- 203 049880 half-life, and/or a linker that links the second masking fragment to the IL-2 polypeptide or a functional fragment thereof. The first and second half-life extension domains of some constructs are also linked. In some constructs, the first and second extended half-life domains of some constructs are linked via a linker. Some of the constructs contain a cleavable peptide in the linker linking the first masking fragment to the first half-life extension domain and/or contain a cleavable peptide in the linker linking the second masking fragment to the IL-2 polypeptide or a functional fragment thereof, resulting in the formation of an activatable masked IL-2 polypeptide construct. Masked IL-2 polypeptide constructs that do not contain a cleavable peptide in either the linker that links the first masking moiety to the first half-life extension domain or the linker that links the second masking moiety to the IL-2 polypeptide or a functional fragment thereof are also referred to as non-activatable masked IL-2 polypeptide constructs or non-activatable IL-2 polypeptide constructs because they do not contain a cleavable peptide. The structure and composition of exemplary IL-2 polypeptide constructs are provided in Table 10.

Table 10

No. of the yuistrushi C|P0KNNIYAT1 one hundred functional fragment (C) Linker {LI) Masknruyushtsp fragment (MM) Linker |L2) Dzdssn extension of the period of gyuluzhtsh· UO Structure (nasflvlsnns from N’ ks-koashu) 4009 SEQ ID NO; 28 SEQ ID NO; 10 SEQIDNO; 155 H-LJ-Ш! SEQIDNO: 1 SEQ ID NO: 11 SEQ ID NO: 9 SEQ ID NO: 15 SEQIDNO: 156 HLI-C-L2-MM 400J - SEQ ID NO: 28 SEQ ID NO; Ю - SEQIDNO; 155 H-LI-MM SEQIDN03 SEQIDNO; 11 SEQ ID NO; 9 SEQIDNO; 16 SEQIDNO; 156 HLI-C-L2-MM 4002 - SEQ ID NO: 2S SEQ ID NO: 10 - SEQIDNO: 155 ΙΙ·ΕΙΑίιΜ SEQ1D NO: 4 SEQIDNO: 11 SEQ ID NO: 9 SEQIDNO; 17 SEQIDNO: I5G II Lt-C-L2-MM 4003 SEQIDNO: 28 SEQ ID NO: 10 SEQIDNO: 155 MM-Lt-H SEQ ID NO: $ SEQIDNO: 11 SEQ ID NO: 9 SEQ ID NO: IS SEQIDNO: 156 MML2-C-LI-H 4004 - SEQ ID NO: 2S SEQ ID NO: 10 - SEQIDNO: 155 MM-Lt-H SEQ ID NO; 6 SEQIDNO; 11 SEQ ID NO; 9 SEQIDNO; 19 SEQIDNO: 156 MML2-C-LI-H 4005 - SEQ ID NO: 2S SEQ ID NO: JO - SEQIDNO: 155 MM-LI-H SEQ ID NO: 7 SEQIDNO; 11 SEQ ID NO: 9 SEQ ID NO: 15 SEQIDNO: 156 MM-L2-C-LI-H 4006 - SEQIDNO: 2S SEQ ID NO: JO SEQ ID NO: 155 H-Ll-MM SEQ ID NO: S SEQIDNO: 11 SEQ ID NO: 9 SEQ ID NO; 16 SEQ ID NO: 156 II-L1-C-L2-M1M

Also prepared are masked IL-15 polypeptide constructs that comprise an IL-15 polypeptide or a functional fragment thereof, a first masking fragment, a second masking fragment, a first half-life extension domain, a second half-life extension domain such as albumin, an antibody or a fragment thereof (e.g., an Fc region, a heavy chain and/or a light chain), an albumin-binding peptide, an IgG-binding peptide, or a polyamino acid sequence. The first masking fragment is linked to the first half-life extension domain, the second masking fragment is linked to the IL-15 polypeptide or a functional fragment thereof, or the second masking fragment, or the IL-15 polypeptide or a functional fragment thereof is linked to the second half-life extension domain, and the first half-life extension domain and the second half-life extension domain contain modifications that facilitate the association of the first and second half-life extension domains. Some of the constructs also comprise a linker that links the first masking moiety to the first half-life extension domain and/or a linker that links the second masking moiety to the IL-15 polypeptide or a functional fragment thereof. The first and second half-life extension domains of some constructs are also linked. In some constructs, the first and second extended half-life domains of some constructs are linked via a linker. Some of the constructs comprise a cleavable peptide in the linker linking the first masking moiety to the first half-life extension domain and/or comprise a cleavable peptide in the linker linking the second masking moiety to the IL-15 polypeptide or a functional fragment thereof, resulting in the formation of an activatable masked IL15 polypeptide construct. Masked IL-15 polypeptide constructs that do not contain a cleavable peptide in either the linker that links the first masking moiety to the first half-life extension domain or the linker that links the second masking moiety to the IL-15 polypeptide or a functional fragment thereof are also referred to as non-activatable masked IL-15 polypeptide constructs or non-activatable IL-15 polypeptide constructs because they do not contain a cleavable peptide. The structure and composition of exemplary IL-2 polypeptide constructs are provided in Table 11.

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Table 11

He designs Cytokine OR ΰΓΰ function]Iu1GZL11LTP fragment (C) Linker (L1) Masking fragment (MM) Linker (L2.) House of extension of the period of loluzhpzsh! (Il) Structure (nllrayleyns ΰτ Nto the C-terminus) 3034 - SEQ1DNO: 23 SEQ ID NO: 10 - SEQ ID NO: 155 H-L1-.MM SEQIDNO: 167 SEQ ID NO: 11 SEQ ID NO: 232 SEQ ID NO: 235 SEQ ID NO: J56 H-L1-C-L2-1MM 3035 - SEQ ID NO: 23 SEQ ID NO: 10 - SEQ ID NO: J55 H-L1-MM SEQIDNO: 167 SEQ ID NO: 11 SEQ ID NO: 233 SEQ ID NO: 235 SEQ ID NO: 156 H-L1-C-L2-MM 3036 - SEQ ID NO: 23 SEQ ID NO: 10 - SEQ ID NO: J55 H-L1-MM SEQIDNO; 16? SEQ ID NO; II SEQ ID NO; 234 SEQ ID NO; 233 SEQ ID NO; 156 H-Ll-C-L2-MM 3038 - SEQ ID NO: 2S SEQ ID NO; 10 - SEQ ID NO: J55 MM-L1-H SEQIDKO: 167 SEQ Π> NO; IJ SEQ ID NO: 232 SEQ ID NO: 235 SEQ ID NO; 156 MM.L2-GLNH zozz - SEQ ID NO: 2£ SEQ ID NO: Ю - SEQ ID NO: 155 M.M-1D-N SEQIDNO: 16? SEQ ID NO: IJ SEQ ID NO: 233 SEQ ID NO: 235 SEQ ID NO: 156 MM-L2-C-L1-H 3040 - SEQ ID NO: 2S SEQ ID NO: 10 - SEQ ID NO: 155 MM-L1-H SEQID NO: 16? SEQ ID NO: IJ SEQ ID NO: 234 SEQ ID NO: 235 SEQ ID NO; 156 MM-L2-C-L1-H

Example 2. Characterization of masked IL-2 and IL-15 polypeptides in vitro.

The masked IL-2 polypeptide constructs and the masked IL-15 polypeptide constructs obtained in Example 1 were characterized using several cellular and functional in vitro assays.

Receiving.

Plasmids encoding constructs (e.g., IL-2 masked polypeptide constructs and IL-15 masked polypeptide constructs) were transfected into either Expi293 cells (Life Technologies A14527) or HEK293-6E cells (National Research Council; NRC). Transfections were performed with 1 mg of total DNA using PEIpro (Polyplus Transfection, 115-100) at a 1:1 ratio with total DNA. Each of the DNA and PEI was added to 50 ml of OptiMem medium (Life Technologies 31985088) and sterile filtered. DNA and PEI were combined for 10 min and added to Expi293 cells at a cell density of 1.8-2.8*106 cells/mL or 0.85-1.20*106 cells/m for expi293 cells or HEK293 cells, respectively, and at least 95% viability. HEK293-6E transfections were performed at a cell density and viability of at least 95% following the same protocol used for Expi293 transfections. After 5-7 days, cells were pelleted by centrifugation at 3000 x g and the supernatant was filtered through a 0.2 μm membrane. Protein A resin (CaptivA, Repligen CA-PRI-0005) was added to the filtered supernatant and incubated for at least 2 h at 4°C with shaking. The resin was packed into a column, washed with 15 column volumes of 20 mM citrate, pH 6.5, and then washed with 15 column volumes of 20 mM citrate, 500 mM sodium chloride, pH 6.5. Bound protein was eluted from the column with 20 mM citrate, 100 mM NaCl, pH 2.9.

The titer (mg/L) of illustrative resulting constructs, including parental (e.g. unmasked) and masked constructs, is presented in Table 12 below.

Table 12

Design ID Titer (mg/l) Design ID Titer (mg/l) Design ID Titer (mg/l) Design ID Titer (mg/l) AK032 5.8 AK063 3.5 AK165 13.5 AK314 60 AK033 9.6 AK064 1,5 AK166 17.1 AK315 59.8 AK034 13.9 AK065 3.2 AK167 56.4 AK316 69.2 AK035 16.7 AK066 3.5 AK168 36.1 AK325 3.2 AK036 25 AK076 1,4 AK184 21.6 AK326 33.1 AK037 11,1 AK077 1,5 AK188 12.8 AK327 68.5 AK039 3.5 AK078 4,4 AK203 83.2 AK328 28.8 AK040 0,7 AK079 4.3 AK209 27.3 AK329 181 AK042 1,9 AK080 2,3 AK211 43.8 AK330 51 AK043 1,1 AK081 23.5 AK212 18.2 AK341 58 AK044 15 AK083 3,3 AK225 44.1 AK342 24 AK045 9.3 AK084 3.2 AK226 20.2 AK349 39.6 AK046 12.5 AK085 2,1 AK227 27.4 AK350 7,8 AK047 18,5 AK086 3.2 AK228 20.1 AK351 9.3 AK048 24 AK087 3.2 AK231 19.3 AK352 9.9 AK049 12.2 AK088 41 AK233 36.4 AK353 11 AK050 12.8 AK090 14.8 AK234 33.3 AK354 9.8 AK051 11.9 AK092 6.3 AK235 35.9 AK355 13.7

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AK052 10 AK094 17.1 AK252 68 AK357 13.1 AK053 15.2 AK106 10,5 AK253 41.4 AK358 12.4 AK054 11.9 AK107 0 AK304 19.9 AK359 10.4 AK055 13.2 AK109 12.6 AK305 53.2 AK360 11,1 AK056 9.2 AK110 23.8 AK306 29.3 AK361 13.7 AK057 10.8 AK111 12.7 AK307 62.9 AK362 9.3 AK058 13.5 AK112 21.6 AK308 74.5 AK363 13.2 AK059 3.7 AK113 25.3 AK309 90.8 AK364 7.9 AK060 1,2 AK114 29 AK310 44 AK365 10.9 AK061 3.5 AK115 15 AK311 64.9 AK366 8.3 AK062 0,6 AK163 7.5 AK312 154 AK367 10.3 AK248 47.7 AK164 4 AK313 81.2 AK368 7.1

SDS-PAGE analysis.

For SDS-PAGE analysis, protein samples were prepared using 4x Laemmli sample buffer (BioRad catalog #1610747). For reduced samples, 0.1 M Bond Breaker TCEP (Thermo Scientific 77720) was added and samples were heated for 5 min at 65°C. Proteins were loaded onto a 12-well 4-12% Bis-Tris NuPage protein gel (Invitrogen NP0322BOX) with 4 μg protein per well. The gel was stained with SimplyBlue SafeStain (Invitrogen LC6065).

As shown in Fig. 8, SDS-PAGE analysis was performed on flow-through (FT) samples (i.e., proteins that did not bind to the Protein A column) and elution (E) samples (i.e., proteins that bound to and were eluted from the Protein A column) after preparation and purification of exemplary constructs (AK304, AK305, AK307, AK308, AK309, AK310, AK311, AK312, AK313, AK314, and AK315). These exemplary data demonstrate that the constructs described herein can be successfully prepared and purified.

Reporter bioassays.

Reporter bioassays are performed on masked IL-2 polypeptide constructs and masked IL-15 polypeptide constructs, along with unmasked parental constructs or other controls, to monitor downstream pathway activation, such as the JAK-STAT pathway.

In some studies, HEK-Blue IL-2 reporter cells (Invivogen) were used to examine JAK-STAT pathway activation according to the following method. HEK-Blue IL-2 passage 6 (p6) cells (97% viable) were washed 2x with assay medium (DMEM+10% heat-inactivated FBS), seeded in 3 plates at 5e4 cells/well in 150 μl assay medium, and incubated in assay medium for approximately 2 h to allow attachment to the plate. Each assay construct was diluted to 300 pM in assay medium, then diluted 1:2 on the plate. 50 μl of each dilution was added for a final stock concentration of 75 pM. HEK-Blue IL-2 cell supernatant was collected after 24 h and incubated with Quantiblue (180 μl + 20 μl supernatant) along with 3 wells/plate of assay medium at 37°C for 1 h. Optical density was read using a Biotek Neo2 at 625 nm. In one study, an exemplary IL-15 AK248 masked polypeptide construct was tested according to this protocol, with or without pretreatment with MMP10 protease, along with recombinant human IL-15 (rhIL-15) as a positive control. As shown in Fig. 22A and 22B, no activation of the JAK-STAT pathway was detected using the IL-15 AK248 masked polypeptide construct (without protease cleavage), but high levels of JAK-STAT pathway activation were observed after cleavage with an activating protease. This demonstrates that the exemplary IL-15 polypeptide can be effectively masked as well as activated by protease cleavage.

In some studies, CTLL2 cells were used to study JAKSTAT pathway activation according to the following method. CTLL2 cells were seeded at 40,000 cells per well in RPMI with 10% FBS. Dilutions of constructs of interest (e.g., the masked IL-15 polypeptide construct AK248, with or without MMP protease pretreatment, or a positive control such as rhIL-15) were added and incubated at 37°C. After 6 h, Bio-Glo reagent was added and luminescence was measured using a BioTek Synergy Neo2 plate reader.

Receptor binding.

The binding of the masked IL-2 polypeptide constructs and the masked IL-15 polypeptide constructs prepared in Example 1 was assessed. For the masked IL-2 polypeptide constructs, in some experiments the ELISA plates were coated with a receptor subunit such as IL-2Ra (also referred to as CD25), IL-2Rβ (also referred to as CD122), or IL-2Ry (also referred to as CD132), or combinations thereof. For the masked IL-15 polypeptide constructs, in some experiments the ELISA plates were coated with a subunit

- 206 049880 receptor such as IL-15Ra (also referred to as CD122), IL-2Re, or IL-2Ry (also referred to as CD132), or combinations thereof. Dilutions of the masked IL-2 polypeptide constructs or the masked IL-15 polypeptide constructs allow binding to the receptor subunit(s) and are detected with an HRP-conjugated anti-huFc detection antibody. Binding of the masked IL-2 polypeptide constructs and the masked IL-15 polypeptide constructs is determined under conditions with and without protease cleavage.

Intracellular receptor binding.

Intracellular receptor binding of the masked IL-2 polypeptide constructs and the masked IL-15 polypeptide constructs prepared in Example 1 is assessed. Dilutions of the masked IL-2 polypeptide constructs or the IL-15 polypeptide constructs are capable of binding to peripheral blood lymphocytes or tissue culture cells such as CTLL2 cells and are detected by fluorescence-activated cell sorting (FACS) using a FITC-conjugated anti-huFc antibody or a FITC-conjugated anti-albumin detection antibody. Binding of the masked IL-2 polypeptide constructs and the IL-15 polypeptide constructs is determined under conditions with and without protease cleavage.

Receptor binding affinity.

The binding affinity of the masked IL-2 polypeptide constructs and the masked IL-15 polypeptide constructs prepared in Example 1 is assessed. For example, surface plasmon resonance (SPR) is performed at room temperature and/or 37°C. IL-2 receptors (e.g., IL-2Ra, IL-2Rβ, or IL-2Ry, or combinations thereof) are coupled to CM5 microarrays (GE Healthcare) via EDC/NHS chemistry. IL-15 receptors (e.g., IL-15Ra, IL-2Rβ, or IL-2Ry, or combinations thereof) are coupled to CM5 microarrays (GE Healthcare) via EDC/NHS chemistry. The masked IL-2 polypeptide constructs or the masked IL-15 polypeptide constructs are passed through the chips over a range of concentrations to obtain dissociation constants for the rates of association and dissociation. The binding affinity of masked IL-2 polypeptide constructs and IL-15 polypeptide constructs was determined under conditions with and without protease cleavage.

For some SPR studies examining the binding of masked and unmasked IL-2 polypeptide constructs, Reichert carboxymethyldextran hydrogel surface sensor chips were coated and immobilized with the construct of interest (e.g., masked IL-2 polypeptide construct or unmasked IL-2 polypeptide construct) at a concentration of 30 μg/mL in 10 mM sodium acetate, pH 5.0, diluted 1:1 in EDC and NHS. Dilutions of CD25-Fc or Fc-CD122 were prepared in PBS (CD25: 16 nM, 8 nM, 4 nM, 2 nM, 1 nM and CD122: 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM). Using a 4Channel SPR Reichert, dilutions of CD25 or CD122 were passed through clamps with the immobilized construct to determine the association rate at 25°C. At equilibrium (approximately 3-4 min), the running buffer was replaced with PBS to determine dissociation rates greater than 1 min. Between each cycle, the chip was regenerated with 10 mM glycine, pH 2.0. A similar approach was used to study the binding of masked and unmasked IL-15 polypeptide constructs to their respective binding partners at appropriate concentrations, in accordance with the teachings and examples herein.

Figures 9A-9D depict the results of an SPR assay that examined the binding of exemplary masked IL-2 polypeptide constructs (AK215 and AK216) to CD25-Fc. Figure 9A depicts the interaction between AK215 and CD25-Fc, Figure 9B depicts the interaction between AK216 and CD25-Fc, and Figure 9C depicts the interaction between a recombinant human IL2 (rhIL2) control and CD25-Fc. Figure 9D is a table summarizing the data obtained for the association constant (ka), dissociation constant (kd), equilibrium dissociation constant (KD), and the chi ² -value and U-value for each interaction. These results demonstrate that these exemplary masked IL-2 polypeptide constructs did not exhibit detectable binding to CD25-Fc, whereas the rhIL2 control exhibited detectable binding.

Figures 10A-10D depict the results of an SPR assay that examined the binding of exemplary masked IL-2 polypeptide constructs (AK216 and AK218) to CD122-Fc. Figure 10A depicts the interaction between AK216 and CD122-Fc, Figure 10B depicts the interaction between AK218 and CD122-Fc, and Figure 10C depicts the interaction between a recombinant human IL2 control (rhIL2) and CD122-Fc. Figure 10D is a table summarizing the data obtained for the association constant (ka), dissociation constant (kd), equilibrium dissociation constant (KD), and the chi ² -value and U-value for each interaction. These results demonstrate that these exemplary masked IL-2 polypeptide constructs did not exhibit detectable binding to CD122-Fc, whereas the rhIL2 control did exhibit detectable binding. Additional exemplary SPR data are provided below in Table 13 for the various constructs tested, including masked and unmasked constructs. For some constructs, KDs were determined where appropriate for

- 207 049880 constructs with or without protease pre-cleavage.

Table 13

Construction KD for CD25 (without protease cleavage) KD for CD122 (without protease cleavage) KD for CD122 (after protease cleavage) rhIL2 1.2 nM 124 nM But. AK032 1.76 nM 260 nM But. AK033 No binding found 368 nM* But. AK034 No binding found Not determined But. AK035 No binding found 110 nM But. AK042 No binding found 4.1 nM But. AK049 No binding found 4.67 nM But. AK056 No binding found Not determined But. AK076 2.28 nM No binding found AK077 4.77 nM No binding found AK078 3.41 nM No binding found AK081 1.66 nM 489 nM* But. AK109 1.67 nM No binding found 118 nM AK110 0.911 nM No binding found 195 nM AK111 0.4 nM No binding found 235 nM AK112 0.724 nM No binding found No binding found AK113 No binding found 191 nM 74.3 nM AK114 No binding found 10.2 nM 13.8 nM AK168 No binding found Not determined 175 nM AK215 No binding found AK216 No binding found AK217 1.9 nM AK218 Weak coupling AK219 Weak coupling AK220 Weak or no binding detected AK221 Weak coupling AK222 Weak or no binding detected AK223 No binding found AK224 No binding found

Split.

The rate of degradation of the masked IL-2 polypeptide constructs is assessed by performing receptor binding assays as described above after incubation of the masked IL-2 peptide constructs in the presence or absence of protease and, if present, inactivated at various time points, such as by adding EDTA. The rate of degradation of the masked IL-15 polypeptide constructs is assessed by performing receptor binding assays as described above after incubation of the masked IL15 peptide constructs in the presence or absence of protease and, if present, inactivated at various time points, such as by adding EDTA. The rate of degradation is also assessed by reducing and non-reducing polyacrylamide gel electrophoresis (PAGE) and whole mass mass spectrometric analysis and peptide map analysis. The rate of cleavage is also assessed using an ex vivo assay in which the masked IL-2 polypeptide constructs or the masked IL-15 polypeptide constructs are exposed to human, mouse, or cynomolgus monkey peripheral blood lymphocytes or normal human tissue or human tumor tissue.

For some protease activation studies, MMP10 was diluted to 50 ng/µl in MMP Cleavage Buffer and activated with 1 mM APMA for 2 h at 37°C. 5 µl of protease (250 ng total) of activated protease was incubated with 1 µM masked cytokine constructs (e.g., masked IL-2 polypeptide constructs) and incubated at 37°C for 2 h. Cleavage was assessed by SDS-PAGE using AnykD™ Criterion™ TGX Stain-Free™ Protein Gels. A similar approach is used to study cleavage by other proteases.

Fig. 11A shows an illustrative structure of the masked IL-2 polypeptide before (left) and

- 208 049880 after (right) digestion with a protease such as a tumor-associated protease. Fig. 11B depicts SDS-PAGE analysis of an exemplary IL-2 masked polypeptide construct that was incubated in the absence (left lane) or presence (right lane) of the MMP10 protease.

Proliferation.

The proliferation of IL-2- and IL-15-responsive tissue culture cell lines such as CTLL2, YT, TF1B, LGL, HH, and CT6 was assessed following treatment with the masked IL-2 polypeptide constructs or the masked IL-15 polypeptide constructs generated in Example 1. For experiments using the masked IL-2 polypeptide constructs, cells were plated in 96-well tissue culture plates in IL-2-free medium for 2-4 hours and then treated with various concentrations of the masked IL-2 polypeptide constructs. For experiments using the masked IL-15 polypeptide constructs, cells were plated in 96-well tissue culture plates in IL-15-free medium for 2-4 hours and then treated with various concentrations of the masked IL-15 polypeptide constructs. After incubation at 37°C for 24-48 h, cell numbers are determined by the addition of MTS, Alamar blue, luciferase or similar metabolic detection reagent and colorimetric, fluorescent or luciferase readings are detected using a spectrophotometric plate reader.

Proliferation of immune cells after treatment with the masked IL-2 polypeptide constructs or the masked IL-15 polypeptide constructs prepared in Example 1 is also assessed. Human, mouse, or cynomolgus monkey peripheral blood mononuclear cells (PBMCs) are treated with the constructs at various concentrations, and proliferation of cell types such as natural killer (NK) cells, CD8+ T cells, CD4+ T cells, and/or Treg cells is determined by cell type-specific staining and fluorescence-activated cell sorting (FACS) analysis. In some experiments, some PBMCs are treated with controls for comparison. In some experiments, some PBMCs are treated with aldesleukin as a control for masked IL-2 polypeptide treatment. In some experiments, IL-2 masked polypeptide constructs and IL-15 masked polypeptide constructs are tested under conditions with and without protease cleavage (e.g., activation). In some experiments, NK cells are stained as CD45+CD3 ^- CD56'. CD8+ T cells are stained as CD45+CD3 ⁺ CD8 ⁺ , CD4+ T cells are stained as CD45+CD3 ⁺ CD4 ⁺ CD25 ^- , and Treg cells are stained as CD45+CD3 ⁺ CD4 ⁺ CD25+. FOXP3+. In some experiments, PBMC are treated for five days. In some experiments, PBMC are also stained with Ki67, a marker of cell proliferation. In some experiments, PBMC are labeled with CFSE (Sigma-Aldrich) prior to treatment and proliferation is measured by determining the dilution rate of CFSE. In some experiments, each construct, as well as aldesleukin and/or other controls, are administered at one or more concentrations, such as one or more concentrations ranging from 0.0001 to 500 nM.

Activation of STAT5.

Also assessed is the activation of signal transducer and activator of transcription 5 (STAT5) following treatment with the masked IL-2 polypeptide constructs or the masked IL-15 polypeptide constructs prepared in Example 1. PBMCs are treated with the constructs for a specified period of time and then immediately fixed to preserve the phosphorylation status of proteins such as STAT5. In some experiments, some PBMCs are treated with controls for comparison. In some experiments, some PBMCs are treated with aldesleukin as a control for treatment with masked IL-2 polypeptide. In some experiments, masked IL-2 polypeptide constructs and masked IL-15 polypeptide constructs are tested under conditions with and without protease cleavage (e.g., activation). In some experiments, PBMCs are treated for 10, 15, 20, or 25 min. In some experiments, each construct, as well as aldesleukin and/or other controls, are administered at one or more concentrations, such as one or more concentrations ranging from 0.0001 to 500 nM. In some experiments, fixed and permeabilized PBMCs are then stained with an antibody specific for phosphorylated STAT5 (phospho-STAT5) and analyzed by flow cytometry. In some experiments, total and phosphorylated STAT5 levels are measured. The phospho-STAT5 status of some cell types, such as NK cells, CD8+ T cells, CD4+ T cells, and/or Treg cells, is determined by cell type-specific staining. In some experiments, NK cells are stained as CD45+CD3 ^- CD56+, CD8+ T cells are stained as CD45+CD3 ⁺ CD8 ⁺ , CD4+ T cells are stained as CD45+CD3 ⁺ CD4 ⁺ CD25 ^- , and Treg cells are stained as CD45+CD3 ⁺ CD4 ⁺ CD25+. FOXP3+.

STAT5 activation in mouse cell lines such as CTLL-2 cells is also assessed following treatment with the masked IL-2 polypeptide constructs or the masked IL-15 polypeptide constructs prepared in Example 1. In some experiments, some CTLL-2 cells are treated with controls for comparison. In some experiments, some CTLL-2 cells are

- 209 049880 are treated with aldesleukin as a control for treatment with masked IL-2 polypeptide. In some experiments, masked IL-2 polypeptide constructs and masked IL-15 polypeptide constructs are tested under conditions with and without protease cleavage (e.g., activation). In some experiments, CTLL-2 cells are treated for 10 min, 15 min, 20 min, or 25 min and then fixed to preserve the phosphorylation status of proteins such as STAT5. In some experiments, each construct, as well as aldesleukin and/or other controls, are administered at one or more concentrations. In some experiments, total and phosphorylated STAT5 levels are measured.

In some studies, levels of IL-2-induced intracellular STAT5 activation (pSTAT5 signal) were determined as follows. Frozen human PBMCs were thawed in a water bath and added to 39 mL of prewarmed medium (RPMI1640 medium plus 10% FBS, 1% P/S, 1% NEA), centrifuged, and recovered in medium at a concentration of 10E6 cells/mL. Cells were seeded at 5E5 cells/well in a 96-well plate. IL-2 (e.g., rhIL-2 or an exemplary IL-2-containing polypeptide construct) diluted in medium was added to each well and incubated at 37°C for 20 min. Cells were then fixed with 200 μL/well of fixation buffer (eBiosciences) at 4°C overnight. After centrifugation, fixed cells were resuspended in 200 μl cold BD Phosflow buffer and incubated at 4°C for 30 min. After washing the cells twice, they were treated with Biolegend Human TruStain FcX (a total of 2.5 μl in 50 μl per sample in staining buffer) for 5 min on ice. Staining antibodies; 5 μl pSTAT5-APC (pY694, BD), 10 μl CD56-BV421 (5.1H11, Biolegend), 10 μl CD4-PerCP/Cy5.5 (A161A1, Biolegend) and 10 μl CD3-FITC (UCHT1, Biolegend) were added and incubated for 30 min on ice, protected from light. Cells were washed twice, resuspended and analyzed by flow cytometry.

Fig. 12A-12D depict the results of STAT5 activation studies as described above using exemplary constructs AK032, AK035, AK041, or rhIL-2 as a control. STAT5 activation levels (%) are shown for NK cells, CD8+ T cells, effector T cells (Teff), and regulatory T cells (Treg). Constructs AK032 and AK035 comprise an IL-2 polypeptide linked to an Fc domain, and construct AK041 comprises an IL-2 polypeptide linked to a CD25 domain and a CD122 domain. As demonstrated, the engineered IL-2 polypeptide constructs in some embodiments can reduce Treg cell activation while maintaining or enhancing CD8+ T cell and NK cell activation.

Fig. 13A-13C depict the results of STAT5 activation studies as described above using exemplary constructs AK081 and AK032. Construct AK081 was tested with and without pre-exposure to MMP10. An isotype control as well as a negative control other than IL-2 were also tested. STAT5 activation levels (%) are shown for NK cells, CD8+ T cells, and CD4+ T cells. Constructs AK032 and AK081 contain an IL-2 polypeptide linked to an Fc domain, and construct AK081 contains a cleavable peptide in the linker connecting the IL-2 polypeptide to the Fc domain. As demonstrated, the unmasked monomeric AK081 IL-2 polypeptide construct stimulates STAT5 activation of PBMCs with or without protease activation, similar to the unmasked dimeric AK032 IL-2 polypeptide construct.

Figs. 14A-14D depict the results of STAT5 activation studies as described above using exemplary constructs AK081 and AK111, as well as controls that included rhIL-2 and an anti-RSV antibody. An untreated control was also tested. Construct AK111 is an exemplary masked IL-2 polypeptide construct that includes the wild-type form of IL-2 polypeptide (excluding the C125A mutation). As shown in Figs. 14A-14C, the AK111 masked IL-2 polypeptide construct demonstrated reduced STAT5 activation compared to the AK081 unmasked IL-2 polypeptide construct. Fig. 14D shows the _EC50 (pM) and fold change data for constructs AK081, AK111, and the rhIL-2 control.

Figs. 15A-15D depict the results of STAT5 activation studies as described above using exemplary constructs AK167 and AK168, as well as controls that included rhIL-2 and an RSV antibody. An untreated control was also tested. Construct AK168 is an exemplary masked IL-2 polypeptide construct that includes a mutant form of IL-2 polypeptide that eliminates or attenuates CD25 binding. Construct AK167 is the original unmasked form of construct AK168 that contains the same mutant IL-2 polypeptide. As depicted in Figs. 15A-15C, the unmasked AK167 construct exhibited reduced STAT5 activation compared to the rhIL-2 control, and the masked IL-2 polypeptide construct AK168 did not induce detectable STAT5 activation. In Fig. 15D shows the EC50 (pM) and fold change data for constructs AK167, AK168, and the rhIL-2 control. The EC50 of construct AK168 was not detectable (no data).

Fig. 16A-16D depict the results of STAT5 activation studies as described above using exemplary constructs AK165 and AK166, as well as isotype controls and

- 210 049880 IL-2-Fc controls that were previously exposed (+ MMP10) or not exposed to the MMP10 protease. Construct AK166 is an exemplary masked IL-2 polypeptide construct that includes the wild-type form of the IL-2 polypeptide (except for the C125A mutation). Construct AK165 is the original unmasked form of construct AK166 that contains the same IL-2 polypeptide. The legend depicted in Fig. 16A also applies to Fig. 16B, and the legend depicted in Fig. 16C also applies to Fig. 16D. As depicted in Fig. 16A-16D, STAT5 activation was significantly reduced for the masked AK166 construct (without protease cleavage), but was restored to levels similar to the IL2-Fc control after exposure to the activating protease MMP10.

17A-17C depict the results of STAT5 activation studies as described above using exemplary AK109 and AK110 constructs, as well as isotype control and IL-2-Fc control, that were previously exposed (+ MMP10) or not exposed to the MMP10 protease. The AK109 and AK110 constructs are exemplary masked IL-2 polypeptide constructs that contain half-life extension domains having various heterodimerization mutations. The legend depicted in Fig. 17B also refers to Fig. 17A. As depicted in Figs. 17A-17C, STAT5 activation was significantly reduced for the masked AK109 and AK110 construct (without protease cleavage), but significantly increased to levels approaching those of the IL2-Fc control following exposure to the activating MMP10 protease.

Figures 18A-18D depict the results of STAT5 activation studies as described above using constructs AK211, AK235, AK253, AK306, AK310, AK314, and AK316, as well as the rhIL-2 control. This includes constructs that are the original unmasked constructs (AK235, AK253, AK306, AK310, AK314) that contain various mutations that modulate CD25 binding. Figure 18D depicts the _EC50 data for each of the constructs studied, as well as the rhIL-2 control.

Figures 19A-19D depict the results of STAT5 activation studies as described above using constructs AK081, AK167, AK216, AK218, AK219, AK220, and AK223 that were activated with protease, as well as using an rhIL-2 control. An untreated control was also tested. This included masked IL-2 polypeptide constructs (AK216, AK218, AK219, AK220, and AK223) that contained various mutations that modulate CD25 binding. The constructs were pretreated with an activating protease prior to testing their ability to activate STAT5. Figure 19D depicts the _EC50 data for each of the constructs tested, as well as for the rhIL-2 control.

Figures 20A-20C depict the results of STAT5 activation studies as described above using constructs AK081, AK189, AK190, and AK210, as well as an anti-RSV antibody control. This includes masked IL-2 polypeptide constructs (AK189, AK190, AK210) that contain an IL-2 polypeptide having the C125A mutation and contain the same cleavable peptide sequence (RAAAVKSP; SEQ ID NO: 121), but have different linker sequences due to differences in the amino acid residues at the N-terminus of the protease cleavage sequence. The legend depicted in Figure 20A also applies to Figures 20B and 20C.

Figures 21A-21C depict the results of STAT5 activation studies as described above using constructs AK167, AK191, AK192, and AK193, as well as an anti-RSV antibody control. This includes masked IL-2 polypeptide constructs (AK189, AK190, AK210) that contain an IL-2 polypeptide having the R38A, F42A, Y45A, E62A, and C125A mutations and contain the same cleavable peptide sequence (RAAAVKSP; SEQ ID NO: 121), but have different linker sequences due to differences in the amino acid residues at the N-terminus of the protease cleavage sequence. The legend depicted in Figure 21A also applies to Figures 20B and 20C.

Example 3. Characterization of masked IL-2 and IL-15 polypeptides in vivo.

Pharmacokinetics.

The pharmacokinetics of the masked IL-2 polypeptide constructs and the masked IL-15 polypeptide constructs obtained in Example 1 are assessed in vivo using mouse models.

Mice are treated with constructs intravenously or subcutaneously, and the plasma concentration of the construct is measured over time. In some experiments, some mice are treated with controls for comparison. In some experiments, some mice are treated with aldesleukin as a control for treatment with the masked IL-2 polypeptide. In some experiments, treated mice have tumors. In some experiments, treated mice do not have tumors. In some experiments, mice are treated with constructs and blood is collected at different times during the course of treatment, which may include blood collection prior to treatment and processing to obtain blood plasma. In some experiments, blood is collected at different time points over two, three, four, or more weeks of treatment. In some experiments, the average plasma concentration of the administered constructs is measured, and

- 211 049880 as well as aldesleukin and/or other controls. Masked IL-2 polypeptide constructs are detected in plasma samples after dilution in PBS Tween by IL-2 and human Fc-specific ELISA and quantified using a standard curve generated for each construct. Masked IL-15 polypeptide constructs are detected in plasma samples after dilution in PBS Tween by IL-15 and human Fc-specific ELISA and quantified using a standard curve generated for each construct. The percentage of full-length and cleaved constructs is determined by Western blotting using HRP-conjugated anti-huFc and HRP-conjugated anti-huIL-2 and by whole mass and peptide mass spectrometry.

The pharmacokinetics of the masked IL-2 polypeptide constructs and the masked IL-15 polypeptide constructs in tumors are also assessed in vivo using mouse models. Tumor-bearing mice are treated with the constructs intravenously or subcutaneously, and the concentration of the construct in the mouse tumors is assessed. In some experiments, some mice are treated with controls for comparison. In some experiments, some mice are treated with aldesleukin as a control for treatment with the masked IL-2 polypeptide. Tumors are analyzed for the presence of the constructs, as well as the presence of certain proteases. In some experiments, tumors are analyzed for the presence and percentage of full-length and cleaved constructs.

Some pharmacokinetic studies were performed according to the following method. Female C57BL/6 mice were purchased from Charles River Laboratories and were 8-10 weeks old at the start of the study. MC38 tumor cells (5/105 per mouse) were injected subcutaneously into the right flank of each mouse. When the tumor size reached ~100 ^mm3 (day 0), mice received a single intravenous dose of 2 mg/kg of the construct of interest (e.g., unmasked IL-2 parental polypeptide construct, masked IL-2 polypeptide construct, or uncleavable masked IL-2 polypeptide construct) in PBS. Constructs studied include, for example, AK032, AK081, AK111, AK167, AK168, AK191, AK197, AK203, AK209, and AK211. Plasma was collected 5 min, 1, 2, and 5 days after drug administration. Drug levels were determined by ELISA using anti-human IgG (clone M1310G05, Biolegend) as capture antibody and various detection antibodies. HRP- or biotin-conjugated detection antibodies to human IgG (ab97225, Abcam) or CD122 (clone 9A2, Ancell) and IL-2 (Poly5176, Biolegend) were used to determine total and uncleaved drug levels, respectively.

Figures 23A-23D depict the results of pharmacokinetic studies as described above in tumor-bearing mice using constructs AK032, AK081, AK111, AK167, and AK168, as well as an anti-RSV antibody control. Figure 23A is a simplified representation of the structure of each of the constructs studied. As indicated, AK111 and AK168 are exemplary masked IL-2 polypeptide constructs. Constructs AK167 and AK168 contain mutations (R38A, F42A, Y45A, and E62A) that abolish or reduce binding to CD25. Figure 23B depicts plasma Fc levels (μg/mL) upon detection of human IgG, and Figure 23C depicts plasma Fc-CD122 levels (μg/mL) when human CD122 is detected, and Fig. 23D depicts plasma Fc-IL2 levels (μg/mL) when human IL-2 is detected.

Figures 24A-24D depict the results of pharmacokinetic studies as described above in tumor-bearing mice using constructs AK167, AK191, AK197, AK203, AK209, and AK211, as well as an anti-RSV antibody control. Figure 24A is a simplified representation of the structure of each of the constructs studied. As indicated, AK168, AK191, AK197, AK203, and AK209 are exemplary masked IL-2 polypeptide constructs, each of which contains a different cleavable peptide sequence in the linker connecting the IL-2 polypeptide to the half-life extender domain. Figure 24B depicts plasma Fc levels (μg/mL) upon detection of human IgG, Figure 24C depicts plasma Fc-IL2 levels (μg/mL) when human IL-2 is detected, and Fig. 24D depicts plasma Fc-CD122 levels (μg/mL) when human CD122 is detected. As shown in Figs. 24B, 24C, and 24D, Fc levels, Fc-IL2 levels, and Fc-CD122 plasma levels are similar among the masked IL-2 polypeptide constructs tested.

Bioactivity in mice.

The in vivo bioactivity of the masked IL-2 polypeptide constructs and the masked IL-15 polypeptide constructs prepared in Example 1 was assessed in vivo using mouse models such as C57BL/6 mice. Mice were treated with the constructs and assessed for in vivo bioactivity. In some experiments, some mice were treated with controls for comparison. In some experiments, some mice were treated with aldesleukin as a control for treatment with the masked IL-2 polypeptide. In some experiments, mice treated with aldesleukin were

- 212 049880 were treated and had tumors. In some experiments, mice that were treated did not have tumors. In some experiments, mice were assessed for dose-dependent expansion of immune cells. In some experiments, mice were treated with different doses of the construct, aldesleukin, or another control. In some experiments, mice were treated for two weeks. Blood was collected from the mice at various time points and then stained with antibodies to immune cell markers of interest. Some experiments also assessed the longitudinal kinetics of proliferation and expansion of specific circulating cell types, such as CD8+ T cells, NK cells, and Treg cells, as well as the ratio of CD8+ T cells and NK cells to CD4+ CD25' FoxP3 ⁺ Treg cells. In some experiments, mice are assessed for vascular fluid leakage, for example by assessing edema and lymphocyte infiltration in specific organs such as the lungs and liver, as determined by organ wet weight and histological analysis.

Some studies assessed vascular leakage to evaluate potential toxicity-related effects mediated by IL-2-based therapy using the following method. Repeat dose toxicity studies were performed using female C57BL/6 mice purchased from Charles River Laboratories and 8-10 weeks old and weighing 18-22 g at study entry. Groups of 5 mice received daily intraperitoneal injections of masked and unmasked IL-2 constructs in PBS daily for 4 or 5 days. The constructs studied included AK081, AK111, AK167, and AK168. A control antibody was also administered as a control. Two hours after the last dose, all mice were injected intravenously with 0.1 ml of 1% Evans Blue (Sigma, Cat. #E2129) in PBS. Two hours after Evans blue administration, mice were anesthetized and perfused with 10 U/ml heparin in PBS. Spleens, lungs, and livers were collected and fixed in 3 ml 4% PFA for 2 days at 4°C before measuring the optical density of the supernatant at 650 nm with NanoDrop OneC (Thermo Fisher Scientific, Waltham, MA) as an indicator of vascular Evans blue leakage. Fixed organs were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Histopathology and quantification were performed by NovoVita Histopathology Laboratory, LLC. (Allston, MA) according to standard procedures. Fig. 30A-30D depict the results of an in vivo study, as described above, to assess fluid leakage through vessels using exemplary masked IL-2 polypeptide constructs AK111 and AK168, as well as unmasked IL-2 polypeptide constructs AK081 and AK167, and an anti-RSV antibody control. Fig. 30A depicts the percentage (%) of body weight loss, and Figs. 30B, 30C, and 30D depict the weight in grams of the liver, lungs, and spleen, respectively, for each.

Fluid leakage through the vessels, as indicated by measuring the degree of dye leakage into the tissue, was also assessed for the AK081, AK111, AK167 and AK168 constructs along with the RSV antibody control, the results are shown in Fig. 31A and 31B for the liver and lung, respectively. The degree of dye leakage was measured based on the absorbance at 650 nm.

Fluid leakage through the vessels, as indicated by measuring the extent of perivascular invasion of mononuclear cells into liver and lung tissue, was also assessed for constructs AK081, AK111, AK167, and AK168 along with the RSV antibody control, and the results are depicted in Figs. 32A and 32B for the liver and lung, respectively. The mean mononuclear cell count in the liver (Fig. 32A) and the mean mononuclear cell count in the lung (Fig. 32B) are depicted for each. As depicted in Fig. 32B, for example, the masked IL-2 polypeptide constructs AK111 and AK168 did not result in detectable mononuclear cell counts in the lung, unlike the unmasked constructs AK081 and AK167.

Phenotype of infiltrating immune cells.

The phenotype of immune cells infiltrating tumors in vivo is assessed in mouse models treated with the masked IL-2 polypeptide constructs or the masked IL-15 polypeptide constructs obtained in Example 1. Mice are treated with the constructs and the phenotype of immune cells infiltrating the tumor is assessed. In some experiments, some mice are treated with controls for comparison. In some experiments, some mice are treated with aldesleukin as a control for treatment with the masked IL-2 polypeptide. Tumor-bearing mice are treated with the construct, aldesleukin, or another control, and tumors, tissues such as liver, lung, and spleen, and blood are collected at various time points after the initial dose, such as five days, seven days, or ten days after the initial dose. In some experiments, immune cells are isolated from the tumors, tissues, and blood and subjected to phenotypic assessment using flow cytometry. In some experiments, isolated immune cells are assessed using markers of interest, such as CD8 ⁺ T cells, CD8 ⁺ memory T cells, activated NK cells, CD4 ⁺ T cells, and CD4 ⁺ Treg cells.

In some studies, the phenotype of immune cells infiltrating tumors in vivo was assessed using the following method. Female C57BL/6 mice were purchased from Charles River

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Laboratories and were 8-10 weeks old at study entry. MC38 tumor cells (5^105 per mouse) were injected subcutaneously into the right flank of each mouse. When tumor size reached ~100 ^mm3 (day 0), mice received a single intravenous dose of 2 mg/kg of the construct of interest (e.g., unmasked IL-2 parental polypeptide construct, masked IL-2 polypeptide construct, or non-cleavable masked IL-2 polypeptide construct) in PBS. On day 5, mice were sacrificed by _CO2 asphyxiation and tumors, liver, spleen, and blood were collected. Cell suspensions were prepared from the spleen by mechanical disruption and passing through a 40 μm cell strainer. Tumor tissues were enzymatically digested using Miltenyi Tumor Dissociation Kit reagents (Miltenyi, catalog #130-096-730), and a gentleMACS Dissociator (Miltenyi) was used for mechanical dissociation steps. Splenic red blood cells, tumor cell suspensions, and blood were lysed using ACK buffer (Gibco catalog #A10492). Cell suspensions were stained with the following antibodies: CD45 (clone 30-F11, eBioscience), CD3 (clone 2C11, Biolegend), CD8 (clone 53-6.7, BD Biosciences), CD4 (clone RM-45, BD Biosciences), FOXP3 (MF-14, Biolegend), CD25 (3C7, Biolegend), CD44 (clone IM7, eBioscience), and NKp46 (29A1.4, eBioscience). Data were acquired on a MACSQuant Analyzer flow cytometer (Milenyi) and analyzed using FlowJo.

Results of in vivo studies examining the responses of CD4, CD8, NK, and Treg percentages in spleen, blood, and tumor, conducted as described above, using constructs AK032, AK081, AK111, AK167, and AK168, as well as an RSV IgG control, are shown in Figs. 25A-25L. AK111 and AK168 are exemplary masked IL-2 polypeptide constructs.

Results of in vivo studies examining the responses of CD4, CD8, NK, and Treg percentages in spleen, blood, and tumor, performed as described above, using constructs AK167, AK168, AK191, AK197, AK203, AK209, and AK211, as well as an RSV IgG control, are depicted in Figs. 26A-26L. AK168, AK191, AK197, AK203, and AK209 are exemplary masked IL-2 polypeptide constructs, each containing a different cleavable peptide sequence in the linker joining the IL-2 polypeptide to the half-life extension domain. Statistical analysis was performed using one-way ANOVA compared to the non-cleavable AK211 construct.

Results of in vivo studies examining the responses of CD4, CD8, NK, and Treg percentages in spleen, blood, and tumor, conducted as described above, using constructs AK235, AK191, AK192, AK193, AK210, AK189, AK190, and AK211, are depicted in Figs. 27A-27L. AK191, AK192, AK193, AK210, AK189, and AK190 are exemplary masked IL-2 polypeptide constructs, each of which contains a cleavable peptide sequence in a linker connecting the IL-2 polypeptide to the half-life extension domain. The linker sequence also varies among these constructs depending on the linker sequence used. AK189, AK190 and AK210 contain the IL-2 polypeptide having the C125A mutation, and AK191, AK192 and AK193 contain the IL-2 polypeptide having the C125A, R38A, F42A, Y45A and E62A mutations. Construct AK235 is an unmasked construct, and construct AK211 contains a non-cleavable linker sequence. Statistical analysis was performed using one-way ANOVA compared to the non-cleavable AK211 construct.

Results of in vivo T cell activation studies in spleen, blood, and tumor performed as described above using constructs AK235, AK191, AK192, AK193, AK210, AK189, AK190, and AK211 as described above are shown in Figs. 28A-28I. T cell activation was measured as the mean fluorescence intensity (MFI) of CD25 in CD8' T cells, CD4' T cells, or Foxp3 ⁺ cells in the spleen, blood, and tumor. Statistical analysis was performed using one-way ANOVA compared to the non-cleavable AK211 construct.

In vivo cleavage.

In vivo cleavage of masked cytokine constructs (e.g., masked IL-2 polypeptide constructs or masked IL15 polypeptide constructs) is assessed. In some studies, a control antibody is administered for comparison. In some studies, in vivo cleavage is assessed by administering the construct of interest to a mouse and, after a period of time, capturing human IgG and then measuring levels of, for example, human IgG, CD122, and IL-2 or IL-15.

In some in vivo degradation studies of masked IL-2 polypeptide constructs, drug levels (i.e., levels of the administered construct including cleavage byproducts) were determined by ELISA using anti-human IgG (clone M1310G05, Biolegend) as capture antibodies and various detection antibodies. HRP- or biotin-conjugated detection antibodies to human IgG (ab97225, Abcam) or CD122 (clone 9A2, Ancell) and IL-2 (Poly5176, Biolegend) were used to determine total and cleaved drug levels.

- 214 049880, respectively. The concentrations of cleaved and released IL-2 are calculated by subtracting the concentration of uncleaved (i.e., intact) drug from the total concentration. Figs. 29A-29D depict the results of in vivo cleavage studies of exemplary masked IL-2 polypeptide constructs AK168 (cleaved peptide sequence: MPYDLYHP; SEQ ID NO: 96) and AK209 (cleaved peptide sequence: VPLSLY; SEQ ID NO: 135). Construct AK167 is a cleavable unmasked IL-2 polypeptide construct that contains the same IL-2 polypeptide as the masked AK168 construct. As depicted in Fig. 29B-29D, both masked (AK168 and AK209) and unmasked (AK167) constructs were efficiently cleaved, and both cleavable peptide sequences were cleaved. Fig. 29E depicts the results of a pharmacokinetic study of total plasma IgG concentration (μg/mL) for total levels of the AK167, AK168, and AK209 constructs, as well as for levels of the uncleaved forms of each construct.

Tumor eradication and inhibition of metastasis.

The ability of the masked IL-2 polypeptide constructs and the masked IL-15 polypeptide constructs obtained in Example 1 to promote tumor eradication and inhibit metastasis was assessed in vivo using mouse models such as syngeneic MC38, CT26, and B16F10 tumor models.

Mice are implanted subcutaneously with tumor cells and the tumors are allowed to grow to a palpable size. Tumor-bearing mice are treated with masked IL-2 constructs or masked IL-15 polypeptide constructs and tumor volumes are measured during treatment. In some experiments, some mice are treated with controls for comparison. In some experiments, some mice are treated with aldesleukin as a control for masked IL-2 polypeptide treatment. Tumor volumes are measured periodically during the course of treatment. In some experiments, body weight is also measured periodically during the course of treatment. In some experiments, plasma samples are obtained during the course of treatment and analyzed for pharmacokinetics, pharmacodynamics, cleavage, and blood markers such as CD8+ T cell, CD8+ memory T cell, activated NK cell, CD4+ T cell, and CD4+ Treg cell markers.

The ability of the masked IL-2 polypeptide constructs and masked IL-15 polypeptide constructs to inhibit metastasis is also assessed in vivo using mouse models suitable for metastasis studies, such as syngeneic CT26 tumor models for assessing lung metastasis. Mice are implanted subcutaneously with tumor cells. In some experiments, tumors are allowed to grow to a palpable size before treatment. In some experiments, treatment is initiated before the tumor has grown to a palpable size. Tumor-bearing mice are treated with masked IL-2 constructs or masked IL-15 polypeptide constructs and assessed for tumor cell metastasis to tissues such as the lung, liver, and lymph nodes.

In some studies, a syngeneic tumor model was used to evaluate the ability of masked IL-2 polypeptide constructs to reduce tumor volume according to the following method. Female C57BL/6 mice were purchased from Charles River Laboratories and were 8-10 weeks old at the start of the study. MC38 tumor cells (5 × 10 ⁵ cells/mouse) were injected subcutaneously into the right flank of each mouse. When tumor size reached ~125 mm 3 (day 0), mice were randomized to receive 2 mg/kg doses of AK081, AK111, AK167, or AK168 or anti-RSV antibody as a control in PBS. Mice were injected intraperitoneally for 6 doses three times per week. Tumor volume was calculated (length (width ² )/2) using calipers and body weight was recorded twice per week. In Fig. 33A and 33B depict the results of a syngeneic tumor model study in which tumor volume and body weight were assessed over the course of treatment. As shown in Fig. 33A, treatment with representative IL-2 polypeptide constructs, including the masked AK111 and AK168 constructs, resulted in tumor growth inhibition over time compared to the RSV antibody control. As shown in Fig. 33B, no overall reduction in body weight was observed when mice were treated with the masked AK111 and AK168 constructs.

Bioactivity in cynomolgus macaques.

The in vivo bioactivity of the masked IL-2 polypeptide constructs and the masked IL-15 polypeptide constructs prepared in Example 1 is assessed in vivo in cynomolgus monkeys. Cynomolgus monkeys are treated with the constructs and assessed for bioactivity, pharmacokinetics, and in vivo degradation. In some experiments, some macaques are treated with controls for comparison. In some experiments, some macaques are treated with aldesleukin as a control for masked IL-2 polypeptide treatment. In some experiments, macaques are administered different doses of the construct, aldesleukin, or other control. Blood is collected from macaques at different time points and then assessed for specific cell types, such as CD8' T cells, CD8' memory T cells, activated NK cells, CD4' T cells, and CD4' Treg cells, and/or markers of interest, such as for a dose-response analysis of total lymphocytes.

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Ki67 ⁺ and soluble CD25. Some experiments assess the longitudinal kinetics of proliferation and expansion of specific circulating T and NK cell types. In some experiments, the pharmacokinetics and degradation of IL-2 masked polypeptide constructs and IL-15 masked polypeptide constructs are determined by ELISA, PAGE, and mass spectrometry.

To investigate the safety profile of exemplary IL-2 masked polypeptide constructs in non-human primates, a dose-ranging study was conducted according to the following method. Groups of 3 healthy male cynomolgus monkeys (Macaca fascicularis) were randomly assigned to receive a single intravenous bolus dose of 2 ml/kg of activatable (i.e., cleavable) masked IL-2 polypeptide proteins or non-cleavable masked IL-2 polypeptide proteins at 10, 30, and 100 nmol/kg in 100 mM sodium citrate buffer (pH 5.5). A third group received the parent unmasked cleavable protein at 3, 10, and 30 nmol/kg as a positive control. This third group received the lower range of drug due to the higher potency of the parent unmasked molecules. Doses are calculated in moles to account for differences in molecular weight. Blood samples are collected before and at 1, 24, 48, 72, 96, 168, 264, and 336 hours after drug administration. An automated hematology analyzer is used to monitor changes in lymphocyte subsets and serum chemistry. Total and intact (i.e., unbroken) drug levels are measured in plasma using a customized ELISA as described above. Soluble CD25 levels are measured using an ELISA (R&D systems, catalog # DR2A00) to monitor immune stimulation. Plasma inflammatory cytokine levels are quantified using a customized multiplexed electrochemiluminescence assay (Meso Scale Discovery). Blood pressure is monitored as an indicator of vascular leak syndrome. PK is analyzed using an ELISA that captures IL-2 and detects human Fc and an ELISA that captures human Fc and detects human Fc.

The present invention is not intended to be limited in scope to the specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the present invention. Various modifications of the compositions and methods described herein will become apparent from the description and teachings herein. Such modifications can be practiced without departing from the actual scope and spirit of the disclosure, and are intended to be within the scope of the present disclosure.

Sequences

SEQ ID AMINO ACID SEQUENCE 1 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLN RWITFAQSIISTLT 3 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQC LEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN RWITFAQSIISTLT 4 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTKKFYMPKKATELKHLQC LEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN RWITFAQSIISTLT 5 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFAFAMPKKATELKHLQCL EEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 6 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQC LEEALKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFL NRWITFAQSIISTLT 7 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTKKFYMPKKATELKHLQC LEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFL NRWITFAQSIISTLT 8 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQC LEEELKPLEEVLNGAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFL NRWITFAQSIISTLT 9 ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATE RIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICT

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10 AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQAS WACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVV HVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPD TQYEFQVRVKPLQ 11 GGSSPPMPYDLYHPSGP 12 GSPMPYDLYHP 13 GSPPMPYDLYHPSGP 14 GSPSMPYDLYHPSGP 15 GPPSGSSPMPYDLYHPSGGG 16 GSSGGPPGGMPYDLYHPSGGG 17 SGSPSGSGGGMPYDLYHPSGGG 18 GPPGPPGSSGMPYDLYHPSGGG 19 GSSSGPPGPPSMPYDLYHPSGGG 20 PA 21 GGGGSGGGGSGGGGS 22 PSGPSAGGAA 23 GGPPASAGS 24 GSPPAGGAP 25 GPGSGSGGAA 26 GGGGSGGGGS 27 GGGGSGGGGSGGGGSGGGGS 28 PGSGS 29 GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG 30 GGGGSGGGGSGGGGSGGGGSGGGGS 31 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 32 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 33 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 34 GGSSPP 35 SGP 36 GSP 37 GSPP 38 GSPS 39 GPPSGSSP 40 GSSGGPPGG 41 SGSPSGSGGG 42 GPPGPPGSSG 43 SGGG 44 GSSSGPPGPPS 45 GGS 46 GGGSSGGS 47 GGSGG 48 GGGS 49 GS 50 GSGGGSSGGS 51 GSSGGS 52 GGGSSGGSG 53 GGSAGGS 54 GHS 55 GPS 56 GAS 57 SGG 58 SGGSGG 59 SSG 60 GGGSGG 61 GG 62 GGG 63 SHGG 64 HGGG 65 SGAA

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66 SGPA 67 GGSGGS 68 GGSGGP 69 GGSGGG 70 GSGGPGPS 71 SGPPGSS 72 SSGGSGP 73 SSPSPSGG 74 SPGSSS 75 GGPGSSP 76 SGPPGGPSS 77 GPGPGSPPGGSS 78 SGPP 79 PGSPSSSS 80 PSPGGPS 81 GGPPS 82 PSPPSS 83 SGGPGP 84 GPSPGS 85 GSPGPSP 86 PSSGGSS 87 SGSSGP 88 GGSSSPP 89 GSPGSP 90 PPPS 91 APPPS 92 AAPPPS 93 SAPPPS 94 SSGP 95 SSPGP 96 MPYDLYHP 97 GGIGQLTA 98 DLGRFQTF 99 DSGGFMLT 100 TSVLMAAP 101 TSEFVFAPDQ 102 KLVLPVLP 103 KPILFFRL 104 ANQLKG 105 QSQLKE 106 HEQLTV 107 PANLVAPDP 108 PAPGVYPGP 109 APAGLIVPYN 110 PQALVA 111 VGNLNF 112 VANLYE 113 VYNLMD 114 TFNIKQ 115 DLWKLLP 116 PGSTKRA 117 QQYRALKS 118 YVPRAVL 119 GVNKWPT 120 LAQAVRSS 121 RAAAVKSP 122 DLLAVVAAS 123 VQTVTWPD 124 AIPMSIPP

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125 GYEVHHQK 126 VHHQKLVF 127 IRRVSYSF 128 MPYDLYHPILFFRL 129 GGIGQLTSVLMAAP 130 DSGGFMLTLVLPVLP 131 TSEFVFAPDLGRFQTF 132 TSTSGRSANPR 133 TSTSGRSANPG 134 TSTSGRSANPH 135 VPLSLY 136 TSASGASASAA 137 PSSSPGGGSSP 138 ISSGLLSGRSDNH 139 ISSGLLSGRSDDH 140 ISSGLLSGRSDIH 141 ISSGLLSGRSDQH 142 ISSGLLSGRSDTH 143 ISSGLLSGRSANP 144 ISSGLLSGRSDNP 145 ISSGLLSGRSANPRG 146 AVGLLAPPGGLSGRSDNH 147 AVGLLAPPGGLSGRSDDH 148 AVGLLAPPGGLSGRSDIH 149 AVGLLAPPGGLSGRSDQH 150 AVGLLAPPGGLSGRSDTH 151 AVGLLAPPGGLSGRSANP 152 AVGLLAPPGGLSGRSDNP 153 AVGLLAPPSGRSANPRG 154 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 155 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 156 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 157 EIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQKPDQSPKLWVHGTSNLASGVPS RFSGSGSGTDFTLTINSLEAEDAATYYCHHWSNTQWTFGGGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 158 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYFMNWVRQAPGQGLEWMGRVDPEQG RADYAEKFKKRVTITADKSTSTAYMELSSLRSEDTAVYYCARRAMDNYGFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 159 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGS ETTFMCEYADETATIVEFLNRWITFCQSIISTLT 160 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT 161 CGGHQYERRGGC

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162 CSGHQYERREGC 163 CGGHYFERHGGC 164 CSGHYFERHEGC 165 CSFHQYERHEGC 166 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIED LIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLS SNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 167 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 168 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 169 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 170 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 171 DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADES AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLV RPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLF FAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQGLKCASLQKFGERAFKA WAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKL K ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVGSKDVCKNYAEAKDVFLGMFLYEY AR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELF E QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDCLS VF LNQLCVLHEKTPVSDRVTKCCTESLVNGRPCFSALEVDETYVPKEFNAETFTFHADICTL SEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGK KLV AASQAALGL 172 LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP 173 DICLPRWGCLW 174 RLIEDICLPRWGCLWEDD 175 DCAWHLGELVWCT 176 NKFRGKYK 177 NARKFYKG 178 FYWHCLDE 179 FYCHWALE 180 FYCHTIDE 181 AQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPK 182 ADAQQNKFNKDQQSAFYEILNMPNLNEEQRNGFIQSLKDDPSQSTNVLGEAKKLNESQ APK 183 ADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNESQAPK 184 ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK 185 ADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK 186 VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK 187 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYGMHWVRQAPGKGLEWVTVIWYDGSN EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDWLGEADYGMDVWG QGTTVTVSS 188 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPTFGQGTKVEIKR 189 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYGMHWVRQAPGKGLEWVTVIWYDGSN

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EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGEQWRGFDYWGQGT TVTVSS 190 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPTFGQGTKVEIKR 191 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYGMHWVRQAPGKGLEWVTVIWYDGSN EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDQEQWRLAFDYWGQG TTVTVSS 192 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPTFGQGTKVEIKR 193 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYGMHWVRQAPGKGLEWVTVIWYDGSN EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGAVAGTGRDYYYYGM DVWGQGTTVTVSS 194 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRA SGVPDRFSGSGSGTDFTLKLISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIKR 195 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYGMHWVRQAPGKGLEWVTVIWYDGSN EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSYYDSSGYYYGEDFD YWGQGTTVTVSS 196 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQTSQFPTFGQGTKVEIKR 197 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYGMHWVRQAPGKGLEWVTVIWYDGSN EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREEWELEDYGMDVWGQ GTTVTVSS 198 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQTTQFPTFGQGTKVEIKR 199 QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVIWYDGSN KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDFDSHYGMDVWGQG TTVTVSS 200 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQTTQFPTFGQGTKVEIKR 201 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYGMHWVRQAPGKGLEWVTVIWYDGSN EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDNWGSDAFDIWGQGTT VTVSS 202 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQVTQFPTFGQGTKVEIKR 203 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYGMHWVRQAPGKGLEWVTVIWYDGSN EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDWFGEADYGMDVWG QGTTVTVSS 204 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQTTQFPTFGQGTKVEIKR 205 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYGMHWVRQAPGKGLEWVTVIWYDGSN EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRISITPFDYWGQGTTVT VSS 206 EIVLTQSPGTLLSLPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKR 207 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDT RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQQVAGMLDYWGQGTTVT VSS 208 EIVLTQSPGTLLSLPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKR 209 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYGMHWVRQAPGKGLEWVTVIWYDGSN EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDFWSDYPFDYWGQGT TVTVSS 210 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQTTQFPTFGQGTKVEIKR 211 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYGMHWVRQAPGKGLEWVTVIWYDGSN EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREEWFGEADYGMDVWG QGTTVTVSS 212 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQTTQFPTFGQGTKVEIKR 213 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYGMHWVRQAPGKGLEWVTVIWYDGSN

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EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGAVAGTGRDYYYYGM DVWGQGTTVTVSS 214 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRA SGVPDRFSGSGSGTDFTLKLISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIKR 215 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYGMHWVRQAPGKGLEWVTVIWYDGSN EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSYYDSSGYYFGEDFDY WGQGTTVTVSS 216 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFS GVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQVTQFPTFGQGTKVEIKR 217 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYGMHWVRQAPGKGLEWVTVIWYDGSN EYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGTVAGTGRDYYYYGM DVWGQGTTVTVSS 218 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRA SGVPDRFSGSGSGTDFTLKLISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIKR 219 EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSHTFGQGTKLEISR 220 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDT RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGNWNCFDYWGQGTLVT VSS 221 GMLSLAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLP VSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPIS LQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLE TLTPDTQYEFQVRVKPLQAFRTLTGH 222 QKLTTVDI 223 CQKLTTVDIC 224 SHYFER 225 CSHYFERC 226 AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQAS WACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDF 227 LNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQP TNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRR QATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLN HCLEHLVQYRTDWDHSWTEQSVDYRHKFSL PSVDGQKRYTFRVRSRFNPLCGSAQHWSEW SHPIHWGSNTSKENPFLFALEAV 228 TLPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEI TSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQL ELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRF NPLCGSAQHWSEWSHPIHWGSNT 229 TLPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEI TSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVI 230 SSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWIT FCQSIISTLT 231 ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATE RIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFP GEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETS 232 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPP 233 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDP 234 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIR 235 SGGSGGGGSGGGSGGGGSLQ 236 SGRSA 237 SGRSANA 238 SGRNAQ 239 SGRNAQVR 240 SGRSDN 241 SGRSDNPN

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242 GSGKSA 243 APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 244 APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLN RWITFAQSIISTLT 245 APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQC LEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN RWITFAQSIISTLT 246 APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTKKFYMPKKATELKHLQC LEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN RWITFAQSIISTLT 247 APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFAFAMPKKATELKHLQCL EEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 248 APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQC LEEALKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFL NRWITFAQSIISTLT 249 APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTKKFYMPKKATELKHLQC LEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFL NRWITFAQSIISTLT 250 APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMMLTAKFAMPKKATELKHLQC LEEELKPLEEVLNGAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFL NRWITFAQSIISTLT 251 APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT 252 EVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS 253 EVQLVESGGGLVQPGGSLRLSCAASGTFSSFGMSWVRQAPGKEPEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS 254 EVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLKPEDTA VYYCTIGGSLSRSSQGTQVTVSS 255 EVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTLVTVSS 256 EVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 257 EVQLVESGGGLVQPGNSLRLSCAASGTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRETISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 258 EVQLVESGGGLVQPGNSLRLSCAASGTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRETISRDNAKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 259 EVQLVESGGGLVQPGNSLRLSCAASGTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSGQGTLVTVSS 260 TSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLE EALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 261 AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQAS WACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVV HVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLTLKQKQEWICLETLTPD TQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 262 GSGPDSGGFMLTSGPAP 263 GSSPPGDSGGFMLTSGP 264 DSGGFMLTS 265 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 266 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

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KAKGQPREPQVYTLPPCRDELTKNQVS LWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGGSGPDSGGFMLTSGPAPTSSSTKKTQLQLEHLLLD LQMILNGINNYKNPKLTAMLTAKFAM PKKATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFAQSIISTLT 267 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGSAVN GTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVET HRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYE FQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 268 GSGP 269 GPAP 270 ISSGLLGGLSGRSDQP 271 ISSGLLSGRSDQG 272 ISSGLLSGRSDQA 273 ISSGLLSGRSDSP 274 ISSGLLSGRSDTP 275 ISSGLLSGRSDMP 276 ISSGLLSGRSD 277 ISSGLLSGRSDQP 278 ISSGLLGGLSGRSDNP 279 ISSGLLSSGGLSGRSDQP 280 ISSGLLSSGGLSGRSDNP 281 ISSGLLSGRS 282 ISSGLLSGRSESP 283 ISSGLLSGRSEQP 284 ISSGLLSGRSEQH 285 LSSGLLSGRSDQP 286 LSSGLLGGLSGRSDQP 287 LSSGLLSGRSDQG 288 LSSGLLSGRSDQA 289 LSSGLLSGRSDSP 290 LSSGLLSGRSDTP 291 LSSGLLSGRSDMP 292 LSSGLSGRSD 293 GKQLRVVNEYSSMDNMLLG 294 LSSGLLGGLSGRSDNP 295 LSSGLLSSGGLSGRSDQP 296 LSSGLLSSGGLSGRSDNP 297 GKQLRVVNEYSSEDNMLLG 298 LSSGLLSGRSESP 299 LSSGLLSGRSEQP 300 LSSGLLSGRSEQH 301 MPYDLYH 302 LSGRSDNH 303 GGGSSP 304 SGGP 305 SGPSGSPG 306 GSIPVSLRSG 307 GPSGPAGLKGAPG 308 GPPGPAGMKGLPG 309 GYVADAPK 310 KKLADEPE 311 GGSRPAHLRDSGK 312 SFTQARVVGG

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313 VHMPLGFLGPRQARVVN 314 LSGRSDNHSPLGLAGS 315 VPLSLYSG 316 IPESLRAG 317 IPVSLRSG 318 SGSGGSPVPLSLYSGGP 319 GGGSSPVPLSLYSGGP 320 GGSSPPVPLSLYSGPSGSPG 321 GGSGGGSGGGSGGGSGGGSG 322 GGSGGGSGGGSGGGSGGGSGGGSG 323 PGPGP 324 SGGCGGHQYERRGGC 325 SGGCSGHQYERREGC 326 SGGCGGHYFERHGGC 327 SGGCSGHYFERHEGC 328 SGGCSFHQYERHEGC 329 PSGS 330 GSPG 331 GGSPGG 332 GGPGGP 333 GGSG 334 GSPPGG 335 GPGSPG 336 GSSPPG 337 GGP 338 SGPGSGS 339 GGSSPPGGGSSGGGSGP 340 SGPGSGS 341 SGSGGSP 342 IYDQKT 343 AHNYKT 344 MMDQAN 345 MLGEFVSE 346 GLVALRGA 347 KEHKYKAE 348 GGSSPPGGGSSGGGSGPGSGS 349 GGSSPPVPLSLYSGPGSGS 350 GNPMGSEPVNFKQLRVVNGGP 351 GNPMGSEPVNFKLLRVVNGGP 352 GNPMGSDPVNFKQLRVVNGGP 353 GNPMGSDPVNFKLLRVVNGGP 354 GGSSPPMPYDLYHPSGPSGSPG 355 GGSSPPGGGSSGGGSGPSGSPG 356 RQARVVG 357 LGGSGRSNAQVRLE 358 LGGSGRKASLSLE 359 SGRIGFLRTA 360 SGAIGFLRTA 361 RPARSGRSAGGSVA 362 VTGRGDSPASS 363 PRFKIIGG 364 LSGRIGFLRTA 365 LSGRSNAMPYDLYHP 366 LSGRSNAGGIGQLTA 367 LSGRSNAVPLSLY 368 LSGRSNADSGGFMLT 369 LSGRSNAHEQLTA 370 LSGRSNARAAAVKSP 371 LSGRSNATSVLMAAP

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372 VPLSLYLSGRSNA 373 DSGGFMLTLSGRSNA 374 GGIGQLTALSGRSNA 375 MPYDLYHPLSGRSNA 376 HEQLTVLSGRSNA 377 RAAAVKSPLSGRSNA 378 TSVLMAAPLSGRSNA 379 IPVSLRSGRSNAQRLE 380 VPLSLYRQARVVG 381 DSGGFMLTRQARVVG 382 GGIGQLTARQARVVG 383 MPYDLYHPRQARVVG 384 HEQLTVRQARVVG 385 RAAAVKSPRQARVVG 386 TSVLMAAPRQARVVG 387 KQLRVVNEYSSMDNMLLG 388 KQLRVVNEYSSEDNMLLG 389 KQLRVVNGYSSEDNMLLG 390 KQLRVVGGLVHLKNTMET 391 TRDRLDEVNFKQLRVVNG 392 TRDRLDEVNFKLLRVVNG 393 TRDRLDPVNFKQLRVVNG 394 TRDRLDPVNFKLLRVVNG 395 NPMGSEPVNFKQLRVVNG 396 NPMGSEPVNFKLLRVVNG 397 NPMGSDPVNFKQLRVVNG 398 NPMGSDPVNFKLLRVVNG 399 TYSRSKYLATA 400 TYSRSRYLATA 401 KQLRVVNEYSSE 402 KQLRVVNGYSSE 403 KQLRVVGGLVAL 404 KQLRVVNGLVAL 405 SPGRVVGGLVAL 406 PQPRTYSRSRYL 407 PQPRTTSRSRYL 408 VVNEYSSRGPYH 409 VVNEYSSERGPYH 410 NKVSMSSSRGPYH 411 NKVSMSSTRGPYH 412 APAMMRGSVILTV 413 APAMMEGSVILTV 414 RGSVIITVQTVTW 415 RGSVILTVQTVTW 416 GTRDRLDEVNFKQLRVVNGGP 417 GTRDRLDEVNFKLLRVVNGGP 418 GTRDRLDPVNFKQLRVVNGGP 419 GTRDRLDPVNFKLLRVVNGGP 420 RKGKALAAYRLE 421 RKGKAGAAYRLE 422 RQARVVGGLVAL 423 GGVRGPRFKIIGG 424 GGVRGPRVKIIGG 425 VTGRGDSHSLTTN 426 VTGRGDSPSLTTN 427 TGHGQASQGLLDR 428 TGHGQASSGLLDR 429 KQLRVVNENLENY 430 KQLRVVNGNLENY

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431 SNVNDVANYNFF 432 SNVNDVSNYNFF 433 IDFNAAQNLYEK 434 IDFNAAYNLYEK 435 IQWNAGQPLQER 436 IQWNAPQPLQER 437 SMDNRLLGLFGE 438 SMDNMLLGLFGE 439 VPIDDPQDLLEG 440 VPIDDPEDLLEG 441 IPENLPPGLPLT 442 IPENLPPLLPLT 443 QPPSLTKNQVSL 444 QPPSLTRNQVSL 445 DSHSLTKNQVSL 446 DSHSLTTNQVSL 447 KAIQLTKNQVSL 448 KAIQLTYNQVSL 449 AEPWTNRNTDGS 450 AEPWTVRNTDGS 451 KQLRVVNG 452 KQLRVVTGRGDSP 453 KQLRVVNGRGDSP 454 PSSRRRVVRKGVS 455 PSSRRRVNRKGVS 456 SPGRVVTGRGDSP 457 SPGRVVGGRGDSP 458 NSGRAVTGRGDSP 459 NSGRAVTYRGDSP 460 TGHGQPSSRRRVN 461 TGHGQASSRRRVN 462 TGHGQSSRGPYH 463 TGHGQASSRGPYH 464 RGSVILTKNQVSL 465 RGSVILTVNQVSL 466 SPGRVVGINYWLA 467 SPGRVVGGNYWLA 468 SPGRVVGSNKGAI 469 SPGRVVGGNKGAI 470 PGARGRAPNHAVV 471 PGARGRAFNHAVV 472 PGARGNAFNNLDR 473 PGARGRAFNNLDR 474 VSNKYISNNEQLP 475 VSNKYFSNNEQLP 476 KVSNKALHVTNI 477 KVSNKALPVTNI 478 VTGRGPSPDVPLT 479 VTGRGDSPDVPLT 480 TGHGQRSSNIRTS 481 TGHGQASSNIRTS 482 TGHGQHSSNIANI 483 TGHGQASSNIANI 484 TGHGQASRNDYSY 485 TGHGQASSNDYSY 486 KALHVTNRNTDGS 487 KALHVTNINTDGS 488 RVVRKKVSNKALP 489 RVVRKGVSNKALP

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490 RQARVVGINYWLA 491 RQARVVGGNYWLA 492 GKQLRVVNGYS SEDNMLLGGP 493 GKQLRVVGGLVHLKNTMETGP 494 AGQPKQLRVVNG 495 AGQPLQLRVVNG 496 AGQPLQERVVNG 497 AGQPKQERVVNG 498 GTANKQLRVVNG 499 GTANKQLHVVNG 500 GTANIQLRVVNG 501 GTANIQLHVVNG 502 GKQLRVVNEYSSMDNMLLGGP 503 GKQLRVVNEYSSEDNMLLGGP 504 KQLRTVAGLAGK 505 KQLRTVNGLAGK 506 KQLRVVAGLAGK 507 KQLRVVNGLAGK 508 GIKYKQLRVVNG 509 GIKYKYLRVVNG 510 GIKYLQLRVVNG 511 GIKYLYLRVVNG 512 THLDLTYSRSKYLATA 513 THLDLTPSRSKYLATA 514 THLDLTYSRSRYLATA 515 THLDLTPSRSRYLATA 516 TYSRSKYLAPANGNAE 517 TYSRSKYLATANGNAE 518 TYSRSRYLAPANGNAE 519 TYSRSRYLATANGNAE 520 DPVNFKQLRVVNEYSSE 521 DPVNFKQLRVVNGYSSE 522 DPVNFKKLRVVNEYSSE 523 DPVNFKKLRVVNGYSSE 524 RKGKAGAAKNLNEKDY 525 RKGKAGAAKNLYEKDY 526 RKGKAGAAQNLNEKDY 527 RKGKAGAAQNLYEKDY 528 VTGRGDSHSLTKNQVSL 529 VTGRGDSHSLTTNQVSL 530 VTGRGDSPSLTKNQVSL 531 VTGRGDSPSLTTNQVSL 532 TGHGQASSERSSNIRTS 533 TGHGQASSERSSNSRTS 534 TGHGQASSERSSTIRTS 535 TGHGQASSERSSTSRTS 536 GISSGLLSGRSDQPSGP 537 GGSGISSGLLSGRSDQPSGP 538 DPVNFKLLRVVNEYSSE 539 DPVNFKLLRVVNGYSSE 540 DPVNFKQLRVVGGLVAL 541 DPVNFKQLRVVNGLVAL 542 DPVNFKLLRVVGGLVAL 543 DPVNFKLLRVVNGLVAL 544 KQLRVQNGDSTE 545 KQLRVVNNDATE 546 KQLRVVNGDSTE 547 ISNNKQLRVVNG 548 ISNNKQLPVVNG

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549 ISNNEQLRVVNG 550 ISNNEQLPVVNG 551 KVSNKQLRVVNG 552 KVSNKQLPVVNG 553 KVSNKALRVVNG 554 KVSNKALPVVNG 555 KQLRVQNNDATE 556 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLT 557 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGAPTSSSSTKKTQLQLEHLLLDLQMILNGINNYKNPK LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLE LKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT 558 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLTGGGGSGGGGSGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQS PMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVC KMTHGKTRWTQPQLICTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 559 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPL EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQS IISTLT 560 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTKKFYMPKKATELKHLQCLEEELKPL EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQS IISTLT 561 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFAFAMPKKATELKHLQCLEEELKPLE EVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSI ISTLT 562 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLTPSGPSAGGAAEIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQ KPDQSPKLWVHGTSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHHWSNTQW TFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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563 ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATE RIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGPPASAGS QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYFMNWVRQAPGQGLEWMGRVDPEQG RADYAEKFKKRVTITADKSTSTAYMELSSLRSEDTAVYYCARRAMDNYGFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 564 ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQ CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATE RIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGSPPAGGAP QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYFMNWVRQAPGQGLEWMGRVDPEQG RADYAEKFKKRVTITADKSTSTAYMELSSLRSEDTAVYYCARRAMDNYGFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 565 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLTGPGSGSGGAAEIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQ KPDQSPKLWVHGTSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHHWSNTQW TFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 566 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTGPPSGSSPMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQV HAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRV MAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTW EEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQ 567 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGAPTSSSSTKKTQLQLEHLLLDLQMILNGINNYKNPK LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLE LKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTGPPSGSSPMPYDLYHPSGGGAVN GTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVET HRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYE FQVRVKPLQ 568 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPL EEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFA QSIISTLT 569 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

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KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLLSSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTKKFYMPKKATELKHLQCLEEELKPL EEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFA QSIISTLT 570 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPL EEVLNGAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFA QSIISTLT 571 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGSGGGGSGGGG SGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPK KATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFAQSIISTLT 572 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTF MCEYADETATIVEFLNRWITFAQSIISTLT 573 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK GSETTFMCEYADETATIVEFLNRWITFAQSIISTLT 574 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYK NPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI VLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT 575 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPK LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVL

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ELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT 576 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGSGGGGSGGGG SGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPK KATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEY ADETATIVEFLNRWITFAQSIISTLT 577 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETT FMCEYADETATIVEFLNRWITFAQSIISTLT 578 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLEL KGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT 579 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYK NPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINV FVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT 580 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLTGGGGSGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRI KSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPV DQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHG KTRWTQPQLICTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 581 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLTGGGGSGGGGSGGGGSGGGGSELCDDDPPEIPHATFKAMAYKEGTML NCECKRGFRRIKSGSLYMLCTGNSSHHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKT TEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGP AESVCKMTHGKTRWTQPQLICTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG

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582 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLN RWITFAQSIISTLTGGGGSGGGGSGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCEC KRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEM QSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAES VCKMTHGKTRWTQPQLICTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG 583 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLN RWITFAQSIISTLTGGGGSGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFR RIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQ PVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMT HGKTRWTQPQLICTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 584 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLN RWITFAQSIISTLTGGGGSGGGGSGGGGSGGGGSELCDDDPPEIPHATFKAMAYKEGTM LNCECKRGFRRIKSGSLYMLCTGNSSHHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERK TTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRG PAESVCKMTHGKTRWTQPQLICTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG 585 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTGSSGGPPGGMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSC QVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRW RVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGH TWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQ 586 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTSGSPSGSGGGMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTS CQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVR WRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPG HTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQ 587 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTGPPGPPGSSGMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSC QVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRW RVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGH TWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQ 588 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

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DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTGSSSGPPGPPSMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTS CQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVR WRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPG HTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQ 589 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPL EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQS IISTLTGSSGGPPGGMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTS CQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVR WRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPG HTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQ 590 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPL EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQS nSTLTSGSPSGSGGGMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDT SCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVR WRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPG HTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQ 591 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPL EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQS IISTLTGPPGPPGSSGMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTS CQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVR WRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPG HTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQ 592 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPL EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQS IISTLTGSSSGPPGPPSMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDT SCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVR WRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPG HTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQ 593 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPL EEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFA QSIISTLTGPPSGSSPMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTS CQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVR WRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPG HTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQ 594 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

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KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLLSSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPL EEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFA QSIISTLTGSSGGPPGGMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQD TSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGV RWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSP GHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQ 595 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPL EEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFA QSIISTLTSGSPSGSGGGMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQ DTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREG VRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTL SPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQ 596 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPL EEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFA QSIISTLTGPPGPPGSSGMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQD TSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGV RWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSP GHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQ 597 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPL EEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFA QSIISTLTGSSSGPPGPPSMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQ DTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREG VRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTL SPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQ 598 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGAPTSSSSTKKTQLQLEHLLLDLQMILNGINNYKNPK LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLE LKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTGSSGGPPGGMPYDLYHPSGGGA VNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASW ACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHV ETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQ YEFQVRVKPLQ 599 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGAPTSSSSTKKTQLQLEHLLLDLQMILNGINNYKNPK LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLE

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LKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTSGSPSGSGGGMPYDLYHPSGGG AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQAS WACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVV HVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPD TQYEFQVRVKPLQ 600 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGAPTSSSSTKKTQLQLEHLLLDLQMILNGINNYKNPK LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLE LKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTGPPGPPGSSGMPYDLYHPSGGGA VNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASW ACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHV ETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQ YEFQVRVKPLQ 601 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPAELCDDD PPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSHSSWDNQCQCTSS ATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFV VGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGAPTSSSSTKKTQLQLEHLLLDLQMILNGINNYKNPK LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLE LKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTGSSSGPPGPPSMPYDLYHPSGGG AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQAS WACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVV HVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPD TQYEFQVRVKPLQ 602 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTGPPSGSSPMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQV HAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRV MAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTW EEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALG KD 603 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGAVNG TSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACN LILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETH RCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEF QVRVKPLQ 604 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSPMPYD LYHPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVE FLNRWITFAQSIISTLT 605 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

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KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSPPMPY DLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKAT ELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLT 606 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSPSMPY DLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKAT ELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLT 607 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFAQS IISTLT 608 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKK ATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 609 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGSAVN GTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVET HRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYE FQVRVKPLQ 610 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTGPPSGSSPMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQV HAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRV MAIQDF 611 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTGSSGGPPGGMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSC QVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRW RVMAIQDF 612 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTSGSPSGSGGGMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTS CQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVR WRVMAIQDF

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613 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTGPPGPPGSSGMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSC QVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRW RVMAIQDF 614 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTGSSSGPPGPPSMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTS CQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVR WRVMAIQDF 615 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLT 616 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 617 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGPPSGSSP MPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRW NQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFEN LRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQK QEWICLETLTPDTQYEFQVRVKPLQ 618 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPP TLT 619 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRKKLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 620 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRKKLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGPPSGSSP MPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRW NQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFEN LRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQK QEWICLETLTPDTQYEFQVRVKPLQ 621 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYDTTPP VLDSDGSFFLVSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE

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EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLT 622 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRKELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 623 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRKELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGPPSGSSP MPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRW NQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFEN LRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQK QEWICLETLTPDTQYEFQVRVKPLQ 624 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVEGFYPSDIAVEWESNGQPENNYKTTPP TLT 625 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRKKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 626 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRKKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGPPSGSSP MPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRW NQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFEN LRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQK QEWICLETLTPDTQYEFQVRVKPLQ 627 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTPGPGPMPYDLYHPSGGCGGHQYERRGGC 628 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTPGPGPMPYDLYHPSGGCSGHQYERREGC 629 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTPGPGPMPYDLYHPSGGCGGHYFERHGGC 630 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTPGPGPMPYDLYHPSGGCSGHYFERHEGC

- 239 049880

631 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTPGPGPMPYDLYHPSGGCSFHQYERHEGC 632 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTPSGSSSMPYDLYHPSGGCGGHQYERRGGC 633 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTPSGSSSMPYDLYHPSGGCSGHQYERREGC 634 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTPSGSSSMPYDLYHPSGGCGGHYFERHGGC 635 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTPSGSSSMPYDLYHPSGGCSGHYFERHEGC 636 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLTPSGSSSMPYDLYHPSGGCSFHQYERHEGC 637 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGSAVN GTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVET HRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYE FQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 638 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGPPSGSSP MPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRW NQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFEN LRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQK QEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 639 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRKKLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP

- 240 049880

VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGPPSGSSP MPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRW NQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFEN LRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQK QEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 640 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRKELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGPPSGSSP MPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRW NQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFEN LRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQK QEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 641 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRKKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGPPSGSSP MPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRW NQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFEN LRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQK QEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 642 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPL EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQS IISTLTGPPSGSSPMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQ VHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWR VMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHT WEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAAL GKD 643 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPL EEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFA QSIISTLTGPPSGSSPMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTS CQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVR WRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPG HTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPA ALGKD 644 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGSAVN GTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVET HRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYE FQVRVKPLQ 645 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEILCLLLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIS TLT 646 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP

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VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEILLLLLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIIS TLT 647 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEILCLFLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIS TLT 648 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEILLLFLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIIS TLT 649 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLCLLLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSII STLT 650 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLLLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSII STLT 651 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEILLLLLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIIS TLT 652 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEILLLFLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIIS TLT 653 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLLLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLT 654 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLCLFLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSII STLT 655 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

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DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLFLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSII STLT 656 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEHLLLFLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLT 657 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEILCLLLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSII STLT 658 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEILLLLLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSII STLT 659 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEILLLLLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSII STLT 660 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEILCLFLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSII STLT 661 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP TLT 662 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAPTSSSTK KTQLQLEILLLFLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIIS TLT 663 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYDTTPP VLDSDGSFFLVSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPMP YDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE

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TATIVEFLNRWITFAQSIISTLT 664 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRKELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGAVN GTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVET HRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYE FQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 665 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVEGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQESLSLSPGGGSSPPMP YDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE TATIVEFLNRWITFAQSIISTLT 666 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRKKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGAVN GTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVET HRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYE FQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 667 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKK ATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 668 GGSSPPMPYDLYHPSGPGSGS 669 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKK ATELKHLQCLEEALKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYA DETATIVEFLNRWITFAQSIISTLT 670 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPMP YDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE TATIVEFLNRWITFAQSIISTLT 671 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGAVN GTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVET HRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYE FQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 672 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGISSGLLS GRSDNHSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE TATIVEFLNRWITFAQSIISTLT

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673 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGAVGLL APPGGLSGRSDNHSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTF MCEYADETATIVEFLNRWITFAQSIISTLT 674 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSPGVPLS LYSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV EFLNRWITFAQSIISTLT 675 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPVP LSLYSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATE LKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETA TIVEFLNRWITFAQSIISTLT 676 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSPGGV PLSLYSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKAT ELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLT 677 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPR AAAVKSPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKK ATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 678 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGPGGPR AAAVKSPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKK ATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 679 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSGRAA AVKSPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKAT ELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLT 680 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPR AAAVKSPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPK KATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFAQSIISTLT 681 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGPGGPR AAAVKSPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPK

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KATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFAQSIISTLT 682 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSGHEQ LTVSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATEL KHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLT 683 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPHE QLTVSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATE LKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETA TIVEFLNRWITFAQSIISTLT 684 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSPPGGH EQLTVSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKAT ELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLT 685 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSGHEQ LTVSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATEL KHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETAT IVEFLNRWITFAQSIISTLT 686 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPHE QLTVSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATE LKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETA TIVEFLNRWITFAQSIISTLT 687 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSPPGGH EQLTVSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKAT ELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLT 688 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGPDSG GFMLTSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKAT ELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLT 689 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGPGSPGD SGGFMLTSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKK ATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 690 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP

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PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSSPPGDS GGFMLTSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE TATIVEFLNRWITFAQSIISTLT 691 GISSGLLSGRSDNHGGP 692 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGPGSPGD SGGFMLTSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKK ATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 693 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSSPPGDS GGFMLTSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKK ATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 694 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGISSGLLS GRSDNHSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKK ATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 695 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGAVGLL APPGGLSGRSDNHSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAK FAMPKKATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTF MCEYADETATIVEFLNRWITFAQSIISTLT 696 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSPGGV PLSLYSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKAT ELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLT 697 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSPGVPLS LYSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELK HLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLT 698 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSGRAA AVKSPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKAT ELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLT 699 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPG GGSSGGGSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPK KATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFAQSIISTLT

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700 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPG GGSSGGGSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKK ATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 701 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKK ATELKHLQCLEERLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 702 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKFYMPKK ATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 703 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFNMPKK ATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 704 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKK ATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 705 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKK ATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 706 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFRMPKK ATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 707 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFAFYMPKK ATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 708 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFAMPKK

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ATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 709 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFYMPKK ATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 710 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTGMLTFKFAMPKK ATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 711 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPVP LSLYSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATE LKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETA TIVEFLNRWITFAQSIISTLT 712 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGAVNG TSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACN LILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETH RCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEF QVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 713 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGISSGLLS GRSDNHGGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKK ATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 714 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGISSGLLS GRSDNHGGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKK ATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFAQSIISTLT 715 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPGSGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL ELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIV QMFINTS 716 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGPPSGSSP MPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRW NQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFEN LRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQK QEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 717 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

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DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSPGVPLS LYSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK HLQCLEERLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV EFLNRWITFAQSIISTLT 718 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSPGVPLS LYSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKFYMPKKATELK HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV EFLNRWITFAQSIISTLT 719 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSPGVPLS LYSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK HLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV EFLNRWITFAQSIISTLT 720 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSPGVPLS LYSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELK HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV EFLNRWITFAQSIISTLT 721 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQESLSLSPG 722 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSPGVPLS LYSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFRMPKKATELK HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV EFLNRWITFAQSIISTLT 723 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSPGVPLS LYSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFYMPKKATELK HLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV EFLNRWITFAQSIISTLT 724 GISSGLLSGRSDNPSGP 725 GGSGISSGLLSGRSDNPSGP 726 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSPGVPLS LYSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELK HLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLT 727 SGS 728 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSGRAA AVKSPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFYMPKKAT ELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET

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ATIVEFLNRWITFAQSIISTLT 729 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGISSGLLS GRSSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFYMPKKATEL KHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLT 730 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSGISSG LLSGRSSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFYMPKKA TELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE TATIVEFLNRWITFAQSIISTLT 731 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGISSGLLS GRSDNPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFYMPKKA TELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE TATIVEFLNRWITFAQSIISTLT 732 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSGISSG LLSGRSDNPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFYMP KKATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFAQSIISTLT 733 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGISSGLLS GRSDQPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFYMPKKA TELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADE TATIVEFLNRWITFAQSIISTLT 734 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSGISSG LLSGRSDQPSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFYMP KKATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFAQSIISTLT 735 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGKQLRVV NEYSSMDNMLLGGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFY MPKKATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFAQSIISTLT 736 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGKQLRVV NEYSSEDNMLLGGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFY MPKKATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFAQSIISTLT 737 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGKQLRVV

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NGYSSEDNMLLGGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFY MPKKATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFAQSIISTLT 738 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGKQLRVV GGLVHLKNTMETGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFY MPKKATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFAQSIISTLT 739 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGTRDRLD EVNFKQLRVVNGGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFY MPKKATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFAQSIISTLT 740 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGTRDRLD EVNFKLLRVVNGGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFY MPKKATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFAQSIISTLT 741 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGTRDRLD PVNFKQLRVVNGGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFY MPKKATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFAQSIISTLT 742 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGTRDRLD PVNFKLLRVVNGGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFY MPKKATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFAQSIISTLT 743 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGNPMGSE PVNFKQLRVVNGGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFY MPKKATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFAQSIISTLT 744 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGNPMGSE PVNFKLLRVVNGGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFY MPKKATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFAQSIISTLT 745 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGNPMGSD PVNFKQLRVVNGGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFY MPKKATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFAQSIISTLT 746 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

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KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGNPMGSD PVNFKLLRVVNGGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFY MPKKATELKHLQCLEESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC EYADETATIVEFLNRWITFAQSIISTLT 747 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPSGSPGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFL LELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHI VQMFINTS 748 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPG GGSSGGGSGPSGSPGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFL LELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHI VQMFINTS 749 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPSGSPGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFL LELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHI VQMFINTSGGGGSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNS GFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPE SLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKN WELTASASHQPPGVYPQGHSDTT 750 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPSGSPGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFL LELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHI VQMFINTSGGGGSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNS GFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPE SLSPSGKEPAAS 751 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPG GGSSGGGSGPGSGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL ELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIV QMFINTS 752 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPGSGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL ELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIV QMFINTSGGGGSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSG FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPES LSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKN WELTASASHQPPGVYPQGHSDTT 753 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPM PYDLYHPSGPGSGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL ELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIV

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QMFINTSGGGGSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSG FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPES LSPSGKEPAAS 754 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPVP LSLYSGPGSGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQ VISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTS 755 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGSAVN GTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVET HRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYE FQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDPGGPSPVPLSLYSGGPITCPPPMSVE HADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPAL VHQRPAPPSGGGGSGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESD VHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEEL EEKNIKEFLQSFVHIVQMFINTS 756 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGSAVN GTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVET HRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYE FQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDSGSGGSPVPLSLYSGGPITCPPPMSV EHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPA LVHQRPAPPSGGGGSGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTES DVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECE ELEEKNIKEFLQSFVHIVQMFINTS 757 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGSAVN GTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVET HRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYE FQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDGGGSSPVPLSLYSGGPITCPPPMSV EHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPA LVHQRPAPPSGGGGSGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTES DVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECE ELEEKNIKEFLQSFVHIVQMFINTS 758 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPVP LSLYSGPSGSPGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQ VISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSGGSGGGSGGGSGGGSGGGSGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKR KAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPS GKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELT ASASHQPPGVYPQGHSDTT 759 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPVP LSLYSGPSGSPGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQ

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VISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSGGSGGGSGGGSGGGSGGGSGGGSGITCPPPMSVEHADIWVKSYSLYSRERYICNS GFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPE SLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKN WELTASASHQPPGVYPQGHSDTT 760 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPVP LSLYSGPGSGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQ VISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSGGGGSGGGGSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICN SGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQP ESLSPSGKEPAAS 761 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSSPPVP LSLYSGPGSGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQ VISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSGGGGSGGGGSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICN SGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQP ESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAK NWELTASASHQPPGVYPQGHSDTT 762 PGPGPMPYDLYHPSGGCGGHQYERRGGC 763 PGPGPMPYDLYHPSGGCSGHQYERREGC 764 PGPGPMPYDLYHPSGGCGGHYFERHGGC 765 PGPGPMPYDLYHPSGGCSGHYFERHEGC 766 PGPGPMPYDLYHPSGGCSFHQYERHEGC 767 PSGSSMPYDLYHPSGGCGGHQYERRGGC 768 PSGSSMPYDLYHPSGGCSGHQYERREGC 769 PSGSSMPYDLYHPSGGCGGHYFERHGGC 770 PSGSSMPYDLYHPSGGCSGHYFERHEGC 771 PSGSSMPYDLYHPSGGCSFHQYERHEGC 772 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRKKLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 773 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRKELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 774 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRKKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 775 APTSSSTKKTQLQLEILCLLLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT 776 APTSSSTKKTQLQLEILLLLLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFIQSIISTLT 777 APTSSSTKKTQLQLEILCLFLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT 778 APTSSSTKKTQLQLEILLLFLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFIQSIISTLT 779 APTSSSTKKTQLQLEHLCLLLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT

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780 APTSSSTKKTQLQLEHLLLLLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFIQSIISTLT 781 APTSSSTKKTQLQLEILLLLLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 782 APTSSSTKKTQLQLEILLLFLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 783 APTSSSTKKTQLQLEHLLLLLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 784 APTSSSTKKTQLQLEHLCLFLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT 785 APTSSSTKKTQLQLEHLLLFLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFIQSIISTLT 786 APTSSSTKKTQLQLEHLLLFLQMILNGILNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 787 APTSSSTKKTQLQLEILCLLLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT 788 APTSSSTKKTQLQLEILLLLLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFIQSIISTLT 789 APTSSSTKKTQLQLEILLLLLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 790 APTSSSTKKTQLQLEILCLFLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT 791 APTSSSTKKTQLQLEILLLFLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFIQSIISTLT 792 APTSSSTKKTQLQLEILLLFLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 793 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYDTTPP VLDSDGSFFLVSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 794 SG 795 PGSG 796 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVEGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQESLSLSPG 797 GISSGLLSGRSSGP 798 GGSGISSGLLSGRSSGP 799 GSG 800 GISSGLLSGRSDNHSGP 801 GGAVGLLAPPGGLSGRSDNHSGP 802 GSPGVPLSLYSGP 803 GGSSPPVPLSLYSGP 804 GGSPGGVPLSLYSGP 805 GGSSPPRAAAVKSPSGP 806 GGPGGPRAAAVKSPSGP 807 GGSGRAAAVKSPSGP

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808 GGSGHEQLTVSGP 809 GGSSPPHEQLTVSGP 810 GSPPGGHEQLTVSGP 811 GSGPDSGGFMLTSGP 812 GPGSPGDSGGFMLTSGP 813 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTSKFYMPKKATELKHLQCL EESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 814 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTGMLTFKFAMPKKATELKHLQCL EESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 815 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EERLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 816 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 817 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFNMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 818 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 819 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQC LEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN RWITFAQSIISTLT 820 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFRMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 821 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFAFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 822 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFAMPKKATELKHLQCL EESLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT 823 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGS QLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT 824 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAAS 825 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPS 826 AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQAS WACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVV HVETHRSNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWISLETLTPD TQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 827 AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQAS WACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVV HVETHRSNISWEISQASHYFEDHLEFEARTLSPGHTWEEAPLLTLKWKQEWISLATLTPD TQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 828 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGSAVN GTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVET HRSNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWISLETLTPDTQYE FQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 829 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP

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VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGSAVN GTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVET HRSNISWEISQASHYFEDHLEFEARTLSPGHTWEEAPLLTLKWKQEWISLATLTPDTQYE FQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD 830 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPGSGSAVN GTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWAC NLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVET HRSNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWISLETLTPDTQYE FQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD

Claims (25)

CLAUSE OF THE INVENTION 1. Masked cytokine containing: (a) a first half-life extending domain and a second half-life extending domain, wherein the first half-life extending domain is a first Fc domain or fragment thereof, and the second half-life extending domain is a second Fc domain or fragment thereof; b) a masking fragment 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 a first half-life extension domain via a first linker, wherein the first linker comprises a cleavable peptide, wherein the cytokine is linked to a second half-life extension domain via a second linker, and wherein the first half-life extension domain and the second half-life extension domain comprise modifications that facilitate association of the first and second half-life extension domains. 2. The masked cytokine of claim 1, wherein the masking fragment comprises the amino acid sequence of SEQ ID NO: 826. 3. The masked cytokine of claim 1, 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. 4. The masked cytokine of claim 1, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 96. 5. The masked cytokine of claim 1, 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. 6. A pharmaceutical composition comprising a masked cytokine according to claim 1 and a pharmaceutically acceptable carrier. 7. A kit for the treatment or prevention of cancer, comprising a masked cytokine according to claim 1. 8. The masked cytokine 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. 9. The masked cytokine of claim 2, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 96. 10. The masked cytokine of claim 2, 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. 11. The masked cytokine of claim 1, wherein the masking fragment 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. 12. A pharmaceutical composition containing a masked cytokine according to claim 2 and a pharmaceutically acceptable carrier. 13. A kit for the treatment or prevention of cancer, comprising a masked cytokine according to item 2. 14. A pharmaceutical composition comprising a masked cytokine according to claim 11 and a pharmaceutically acceptable carrier. 15. A kit for the treatment or prevention of cancer, comprising a masked cytokine according to claim 11. - 258 049880 16. Masked cytokine containing: a) a first polypeptide comprising a first half-life extension 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 fragment thereof, and the second half-life extending domain is a second Fc domain or fragment thereof; wherein the masking fragment 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 linked 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 the first half-life extension domain and the second half-life extension domain comprise modifications that facilitate association of the first and second half-life extension domains. 17. The masked cytokine of claim 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 masked cytokine of claim 16, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 135. 19. The masked cytokine of claim 16, 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. 20. A pharmaceutical composition comprising a masked cytokine according to claim 16 and a pharmaceutically acceptable carrier. 21. A kit for the treatment or prevention of cancer, comprising a masked cytokine according to claim 16. 22. The masked cytokine of claim 16, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 96. 23. The masked cytokine of claim 16, wherein the masking fragment 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: 28 and the second linker comprises the amino acid sequence of SEQ ID NO: 35. 24. A pharmaceutical composition comprising a masked cytokine according to claim 23 and a pharmaceutically acceptable carrier. 25. A kit for the treatment or prevention of cancer, comprising a masked cytokine according to claim 23.