Engineered Cleavable Fc Domain as a Carrier and Methods of Use Thereof

Abstract

The present disclosure relates to a cleavable carrier and a cytokine prodrug conjugated to the cleavable carrier, wherein the cleavable carrier is an engineered Fc domain comprising at least one tumor-associated protease cleavage site. Upon cleavage at the cleavage site of the carrier Fc domain, the cytokine is released from the masking moiety. This platform provides enzymatically induced prodrug activation. The present disclosure further provides a pharmaceutical composition comprising the cytokine prodrug conjugated to the cleavable carrier, its use as a medicament, and methods of treating and administering the disease.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 389,476, filed on July 15, 2022, and U.S. Provisional Patent Application No. 63 / 448,943, filed on February 28, 2023, the entire contents of each of which are hereby incorporated by reference in their entirety.

[0002] Cross - Reference to Sequence Listing This application is being filed using a sequence listing that was electronically submitted in XML format. The sequence listing file, entitled XTX_UNIVFC_WO1_SL.XML, was created on July 12, 2023, and is 210,539 bytes in size. The information in the electronic form of the sequence listing is hereby incorporated by reference in its entirety.

Background Art

[0003] Cancer is the second leading cause of death in the United States, accounting for more deaths than the next five leading causes (chronic respiratory diseases, stroke, accidents, Alzheimer's disease, and diabetes). While significant progress has been made, particularly in targeted therapies, much research remains in this field. Immunotherapy and the sub - specialty of immuno - oncology in this field have created viable and exciting treatment options for treating malignancies. In particular, it is now recognized that one of the hallmarks of cancer is immune evasion, and that through significant efforts, targets have been identified to re - activate the immune system to recognize and treat cancer, and therapies against these targets have been developed.

[0004] One effective strategy for stimulating the immune system to induce anti-tumor cytotoxicity is cytokine therapy. Unfortunately, cytokines administered to patients generally have a very short half-life and thus require frequent administration. For example, the product label of Aldesleukin, marketed under the brand name Proleukin, states that the drug has a half-life of 85 minutes in patients who received a 5-minute intravenous (IV) infusion. Furthermore, administration of high doses of cytokines can cause health hazards such as vascular leakage due to systemic immune activation. To extend the half-life and reduce toxicity, carrier moieties such as the Fc domain, albumin, and PEG are fused to cytokines through cleavable linkers, allowing the release of the active cytokine when the fusion molecule reaches the tumor microenvironment.

Summary of the Invention

[0005] The present invention provides, inter alia, compositions and methods for use in precision cancer therapy based on cleavable carriers. Specifically, the present invention is based on a cleavable carrier (e.g., a cleavable Fc domain) that can be linked to a therapeutic agent, particularly a masked therapeutic agent, thereby delivering and activating the linked therapeutic agent at specific targets such as different tumor microenvironments. For example, the cleavable carrier may be an Fc domain engineered to contain a cleavage site cleavable by a tumor-specific protease. Such an engineered cleavable Fc domain is linked to a cytokine or a masking moiety of a cytokine such that the cytokine is released from its masking moiety only upon cleavage of the Fc domain by a tumor-specific protease. Prior to the present invention, existing tumor-specific therapeutic agents typically included a cleavable linker. The approach of the present invention described herein allows for more flexible linker design without the constraints of cleavage sites for more effective masking and activation. Thus, the present invention represents a significant improvement in the field of precision medicine, promising safer and more effective therapeutic agents for cancer patients.

[0006] In one aspect of the present invention, there is provided a masked therapeutic agent comprising a therapeutic molecule, a masking moiety, and a cleavable carrier moiety comprising a tumor-associated protease cleavage site engineered to be cleavable. The cleavable carrier moiety is fused to the therapeutic molecule and / or the masking moiety, and upon cleavage of the engineered tumor-associated protease site within the cleavable carrier moiety, the therapeutic molecule is released from the masking moiety. In some embodiments, the cleavable carrier moiety is fused to the therapeutic moiety and / or the masking moiety via a non-cleavable linker. In other embodiments, the cleavable carrier moiety is fused to the therapeutic moiety and / or the masking moiety via a cleavable linker.

[0007] In some embodiments, the cleavable carrier moiety is a cleavable Fc domain. The cleavable Fc domain comprises a first Fc domain and a second Fc domain, and one of the first or second Fc domains comprises an engineered tumor-associated protease cleavage site. In some embodiments, the carrier moiety may be a half-life extension domain selected from the group consisting of albumin, transferrin, and tissue factor. In other embodiments, the carrier moiety may be an antigen targeting domain selected from the group consisting of immunoglobulin, Fab, F(ab)2, scFv, VHH, ScAb, nanobody, VH, VL, single domain antibody, CL, and CK, and the tumor-associated protease cleavage site is engineered within the antigen targeting domain.

[0008] In one aspect, the present invention provides, inter alia, an engineered Fc domain comprising one or more amino acid substitutions compared to the wild-type Fc domain, the engineered Fc domain comprising a protease cleavage site.

[0009] In some embodiments, the protease cleavage site is a tissue-selective protease cleavage site. In some embodiments, the protease cleavage site is a tumor-associated protease cleavage site. In some embodiments, the protease cleavage site is an inflammation-associated protease cleavage site.

[0010] In one embodiment, the cleavable carrier moiety is an engineered cleavable Fc domain that includes at least one tumor-associated protease cleavage site. The Fc domain is a human Fc domain.

[0011] The therapeutic molecule can be any agent having a therapeutic effect such as suppression of tumor growth, invasion, and progression. In some embodiments, the therapeutic molecule is a cytokine, its variant, or a functional fragment thereof.

[0012] In one embodiment, a masked cytokine is provided by the present disclosure. The masked cytokine includes a cytokine molecule, a masking moiety, and a carrier moiety that includes an engineered tumor-associated protease cleavage site, where the carrier moiety is fused to the cytokine and / or the masking moiety, and upon cleavage of the engineered tumor-associated protease site, the masked cytokine is released from the masking moiety. In a preferred embodiment, the present invention provides a masked cytokine that includes a cytokine molecule, a masking moiety, and an engineered Fc domain that includes a tumor-associated cleavage site, where the engineered Fc domain is fused to the cytokine moiety or the masking moiety such that when the masking moiety binds to the cytokine moiety and the tumor-associated protease cleavage site on the engineered Fc domain is cleaved, the cytokine molecule is released from the masking moiety. The engineered Fc domain is fused to the cytokine molecule and / or the masking moiety via a non-cleavable linker. In some embodiments, the engineered Fc domain is directly fused to the cytokine molecule and / or the masking moiety.

[0013] In some embodiments, the protease cleavage site is a tissue-selective protease cleavage site. In some embodiments, the protease cleavage site is an inflammation-associated protease cleavage site.

[0014] In some embodiments, the protease cleavage site is a tumor-associated protease cleavage site. Thus, in some embodiments, the protease cleavage site within the engineered Fc domain is a tumor-associated protease cleavage site. The tumor-associated protease cleavage site can be recognized by a tumor-associated protease such that the Fc domain is cleaved. By way of non-limiting example, the tumor-associated protease can be a matrix metalloproteinase (MMP) selected from the group consisting of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP27, and MMP28. In one embodiment, the tumor-associated protease is MMP2. In another embodiment, the tumor-associated protease is MMP3. In yet another embodiment, the tumor-associated protease is MMP9. In yet another embodiment, the tumor-associated protease is MMP10. Other disease-related and tissue-selective proteases include, but are not limited to, cathepsin (cathepsin B, cathepsin D, cathepsin F, cathepsin K, cathepsin L, cathepsin V, cathepsin S, cathepsin W), ADAM, ADAMTS, kallikrein 1-15, HTRA1-2-3, HGFAc, PRSS, TMPRSS, elastase, PR-3, granzyme (granzyme A, B, M, H, and K), fibroblast activation protein (FAP), plasmin, urokinase plasminogen activator (uPA), tryptase, caspase, thrombin, legumain, chymase, collagenase, napsin A, and matriptase 1-2. In one embodiment, the disease-related and tissue-selective protease is cathepsin B. In another embodiment, the disease-related and tissue-selective protease is matriptase.

[0015] According to the present disclosure, the cleavable Fc domain is engineered to include at least one protease cleavage site. By way of non-limiting example, the engineered Fc domain includes one or more amino acid substitutions compared to the wild-type Fc domain, and the substitution generates a protease cleavage site. In some embodiments, the engineered Fc domain includes one or more amino acid substitutions in the hinge region, CH2 domain, CH3 domain, and / or CH2-CH3 linker region such that the engineered Fc domain includes a protease cleavage site in the hinge region, CH2 domain, CH3 domain, and / or CH2-CH3 linker region. In some embodiments, the engineered Fc domain includes one or amino acid substitutions at positions 439-446 according to EU numbering such that the substitution generates a protease cleavage site at positions 439-446 of the Fc domain.

[0016] In one aspect, the invention provides, inter alia, an engineered Fc domain that includes one or more amino acid substitutions in the hinge region, CH2 domain, CH3 domain, and / or CH2-CH3 linker such that the engineered Fc domain includes a protease cleavage site.

[0017] In some embodiments, one or more amino acid substitutions are within the hinge region. In some embodiments, one or more amino acid substitutions are within the hinge region. In some embodiments, one or more amino acid substitutions are within the CH2 domain. In some embodiments, one or more amino acid substitutions are within the CH3 domain. In some embodiments, one or more amino acid substitutions are within the CH2-CH3 linker.

[0018] In one aspect, the invention provides, inter alia, an engineered Fc domain that includes one or more amino acid substitutions at positions 436-447 according to EU numbering such that the engineered Fc domain includes a protease cleavage site.

[0019] In some embodiments, the protease cleavage site is located between positions 438 and 445, between positions 439 and 446, between positions 440 and 447, between positions 438 and 447, between positions 437 and 444, between positions 440 and 443, between positions 441 and 444, between positions 442 and 445, between positions 444 and 447, between positions 441 and 447, between positions 443 and 447, between positions 436 and 443, or between positions 442 and 447 according to the EU numbering.

[0020] In some embodiments, the engineered Fc domain comprises at least one set of combined mutations including the N434I, Y346V, and Q438L mutations; the H435S, Q438I, and S440G mutations; the T437S, Q438N, and K438E mutations; the K439V and L441S mutations; the Q438P, K439T, and L441T mutations; the K439E, P445E, and G446E mutations; and the K439A, S444A, and G446V mutations.

[0021] In some embodiments, the engineered Fc domain comprises a cleavage substrate in the G-chain region. In some embodiments, the engineered Fc domain comprises a cleavage substrate in the F-chain region. In some embodiments, the engineered Fc domain comprises a cleavage substrate in the FG-loop region.

[0022] In another aspect, the present invention provides, inter alia, an engineered Fc domain comprising one or more amino acid substitutions at positions 416 - 425 according to the EU numbering such that the engineered Fc domain comprises a protease cleavage site.

[0023] In yet another aspect, the present invention provides, inter alia, an engineered Fc domain, wherein the engineered Fc domain comprises one or more amino acid substitutions at positions 426 to 437 according to EU numbering so as to include a protease cleavage site. As a non-limiting example, an engineered cleavable Fc domain comprising a tumor-associated protease cleavage site having the amino acid sequence of VPLSLYSG (SEQ ID NO: 95) is provided by the present disclosure. As a non-limiting example, an engineered cleavable Fc domain comprising a tumor-associated protease cleavage site having the amino acid sequence of VPLSLYSGP (SEQ ID NO: 209) is provided by the present disclosure. In one embodiment, the engineered Fc domain comprises a tumor-associated protease cleavage site having the amino acid sequence of IHVTLKSL (SEQ ID NO: 99). In one embodiment, the engineered Fc domain comprises a tumor-associated protease cleavage site having the amino acid sequence of NSYTIKGL (SEQ ID NO: 100). In one embodiment, the engineered Fc domain comprises a tumor-associated protease cleavage site having the amino acid sequence of SNESLSLS (SEQ ID NO: 102). In one embodiment, the engineered Fc domain comprises a tumor-associated protease cleavage site having the amino acid sequence of QVSSSLSP (SEQ ID NO: 104). In one embodiment, the engineered Fc domain comprises a tumor-associated protease cleavage site having the amino acid sequence of PTSTSLSP (SEQ ID NO: 105). In one embodiment, the engineered Fc domain comprises a tumor-associated protease cleavage site having the amino acid sequence of ESLSLSEE (SEQ ID NO: 107). In one embodiment, the engineered Fc domain comprises a tumor-associated protease cleavage site having the amino acid sequence of ASLSLAPV (SEQ ID NO: 108). In one embodiment, the engineered Fc domain comprises a tumor-associated protease cleavage site having the amino acid sequence of SQESLSLS (SEQ ID NO: 109). In one embodiment, the engineered Fc domain comprises a tumor-associated protease cleavage site having the amino acid sequence of PLGL (SEQ ID NO: 110).

[0024] In some embodiments, the protease site comprises any one of SEQ ID NOs: 95, 97, 99-100, 102, 104, 105, 107-110, 119, 122, 123, 135-139, 143-208, and 209.

[0025] In some embodiments, the engineered Fc domain recited in any one of the preceding claims comprises any one of the sets of amino acid substitutions listed in Table 5, Table 8a, Table 11, Table 11a, Table 11b, or Table 11c.

[0026] In some embodiments, the engineered Fc domain comprises at least one set of combined substitutions in Table 5.

[0027] In some embodiments, the engineered Fc domain comprises at least one set of combined substitutions in Table 8a.

[0028] In some embodiments, the engineered Fc domain comprises at least one set of combined substitutions in Table 11.

[0029] In some embodiments, the engineered Fc domain comprises at least one set of combined substitutions in Table 11a.

[0030] In some embodiments, the engineered Fc domain comprises at least one set of combined substitutions in Table 11b.

[0031] In some embodiments, the engineered Fc domain comprises at least one set of combined substitutions in Table 11c.

[0032] In some embodiments, the engineered cleavable Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, wherein one of the first or second Fc polypeptides comprises a protease cleavage site. In some examples, one of the first or second Fc polypeptides comprises a tumor-associated protease cleavage site described herein.

[0033] In some embodiments, each of the first and / or second Fc polypeptides comprises one or more modifications that promote non-covalent binding of the first and second Fc polypeptides.

[0034] In some embodiments, the first Fc polypeptide comprises a first IgG1, IgG2, or IgG4 Fc domain, or a fragment thereof. In some embodiments, the second Fc polypeptide comprises a second IgG1, IgG2, or IgG4 Fc domain, or a fragment thereof.

[0035] In some embodiments, the cytokine molecule of the mask type and / or the targeted cytokine is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-22, IL-34, TNF-α, TNF-β, CXCL8 (IL-8), G-CSF, GM-CSF, LIF, OSM, IFN-α, IFN-β, IFN-γ, CD154, LT-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β, M-CSF, or MSP, or a variant thereof, or a fragment thereof. As a non-limiting example, the cytokine molecule of the mask type or the targeted cytokine is IL-2, IL-15, or IL-12, or a variant thereof, or a fragment thereof.

[0036] In one embodiment, the cytokine molecule is IL-2, and the IL-2 is a modified IL-2 cytokine or a functional fragment thereof as compared to the sequence of mature IL-2 having the amino acid sequence of SEQ ID NO: 13. As a non-limiting example, the modified IL-2 cytokine or a functional fragment thereof includes modified R38A, F42A, Y45A, and E62A with respect to the sequence of mature IL-2 having the amino acid sequence of SEQ ID NO: 13. In some examples, the modified IL-2 cytokine or a functional fragment thereof includes modified C125A with respect to the sequence of mature IL-2 having SEQ ID NO: 13. In other examples, the modified IL-2 cytokine or a functional fragment thereof includes R38A, F42A, Y45A, E62A, and C125A with respect to the sequence of mature IL-2 having SEQ ID NO: 13.

[0037] In another embodiment, the cytokine molecule is IL-15, and the IL-15 polypeptide includes the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO: 16.

[0038] In another embodiment, the cytokine molecule is IL-12, and the IL-12 polypeptide or a functional fragment thereof includes an IL-12p40 polypeptide or a functional fragment thereof covalently linked to an IL-12p35 polypeptide or a functional fragment thereof. The IL-12p40-IL-12p35 linker is 5 to 20 amino acids in length. In some examples, the IL-12p40-IL-12p35 linker is rich in amino acid residues G and S.

[0039] According to the present disclosure, the mask-type cytokine includes a masking moiety that binds to the cytokine molecule.

[0040] In some embodiments, the masking moiety is a cytokine receptor, or a variant thereof, or a fragment thereof. By way of non-limiting example, the masking moiety is CD121, IL-18Rα, IL-18Rβ, CD25, CD122, CD132, CD124, CD213a13, CD132, CD127, IL-9R, CD213a1, CD213a2, CD1243, CD132, IL-15Ra, CDw131, CDw125, CD131, CD116, CD126, CD130, IL-11Ra, CD114, CD212, LIFR, OSMR, IL-20Rα, IL-20Rβ, IL-14R, CD4, CDw127, CD118, CDw119, CD40, LTβR, CD120a, CD120b, CDw137, BCMA, TACI, CD27, CD30, CD95, GITR, LTbR, HVEM, OX40, TRAILR1-4, Apo3, RANK, OPG, TGF-βR1, TGF-βR2, TGF-βR3, CD115, or CDw136, or a variant thereof, or a fragment thereof.

[0041] In one embodiment, the masking moiety is CD122. In some examples, CD122 is an engineered CD122 polypeptide or a fragment thereof that contains one or more mutations relative to the wild-type CD122 amino acid sequence. The engineered CD122 contains one or more mutations selected from the group consisting of F8C, A94C, L106C, C122S, C122V, C122A, N123C, N123Q, C168V, C168A, C168S, L169C, Q177C, V184C, S195C, and R204C.

[0042] In some embodiments, the masking moiety is a peptide, polypeptide, protein, ligand, or agent that specifically binds to a cytokine, or a variant thereof, or a fragment thereof.

[0043] In some embodiments, the masking moiety comprises a Fab, a single-chain Fv (scFv), a single-domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibody (bsFab), a single-domain antibody-binding Fab (s-Fab), a single heavy-chain antibody (HcAb), an antibody, or a combination thereof.

[0044] In some embodiments, the masking moiety comprises an anti-IL-2 scFv. In some embodiments, the masking moiety comprises an scFv having SEQ ID NO: 124. In some embodiments, the masking moiety comprises an scFv having SEQ ID NO: 125. In some embodiments, the masking moiety comprises scFvs having SEQ ID NO: 124 and SEQ ID NO: 125. In some embodiments, the masking moiety comprises an scFv having SEQ ID NO: 124 linked to SEQ ID NO: 125 by a linker. In some embodiments, the masking moiety comprises an scFv having SEQ ID NO: 142.

[0045] In some embodiments, the masking moiety comprises an anti-IL-2 VHH. In some embodiments, the masking moiety comprises a VHH having hCDR1 of SEQ ID NO: 132, hCDR2 of SEQ ID NO: 133, and hCDR3 of SEQ ID NO: 134. In some embodiments, the masking moiety comprises a VHH having SEQ ID NO: 121.

[0046] In some embodiments, the mask-type cytokine of the present disclosure further comprises an engineered cleavable Fc domain portion that is further modified. In some embodiments, the first and second Fc polypeptides of the engineered cleavable Fc domain portion further comprise the same amino acid substitutions described herein. In other embodiments, the first and second Fc polypeptides of the engineered cleavable Fc domain portion comprise different amino acid substitutions described herein. By way of non-limiting example, the engineered Fc domain comprises a first Fc polypeptide comprising the Y349C, T366S, L368A, Y407V, and N297A mutations, and a second Fc polypeptide comprising the S354C, T366W, and N297A mutations. Alternatively, the engineered Fc domain comprises a first Fc polypeptide comprising the S354C, T366W, and N297A mutations, and a second Fc polynucleotide comprising the Y349C, T366S, L368A, Y407V, and N297A mutations. In another example, the engineered Fc domain comprises a first Fc polypeptide comprising the Y349C, T366S, L368A, Y407V, N297A, and I253A mutations, and a second Fc polypeptide comprising the S354C, T366W, N297A, and I253A mutations. Alternatively, the engineered Fc domain comprises a first Fc polypeptide comprising the S354C, T366W, N297A, and I253A mutations, and a second Fc polynucleotide comprising the Y349C, T366S, L368A, Y407V, N297A, and I253A mutations.

[0047] The masked cytokine of the present disclosure includes a further modified and operably cleavable Fc domain portion. In some embodiments, the first Fc polypeptide of the operably modified Fc domain includes a CH3 domain with modifications that reduce or eliminate binding to Protein A, and the second Fc domain includes a CH3 domain that binds to Protein A. In some embodiments, the CH3 domain that binds to Protein A is a human IgG1, IgG2, or IgG4 sequence. The second CH3 domain may be a human IgG1, IgG2, or IgG4 sequence that includes modifications at position H435 and / or Y436 according to Kabat numbering. As a non-limiting example, the second CH3 domain includes the modifications H435R and Y436F according to Kabat numbering.

[0048] In one embodiment, the first CH3 domain includes a human IgG3 sequence.

[0049] In some embodiments, the masked cytokine further includes a targeting moiety, which includes one or more antigen-binding domains, peptides, polypeptides, proteins, ligands, or agents that specifically bind to an antigen or ligand. As non-limiting examples, the targeting moiety includes antigen-binding domains selected from the group consisting of Fab, single-chain Fv (scFv), single-domain antibodies (VHH), one or more CDRs, variable heavy chains (VH), variable light chains (VL), Fab-like bispecific antibodies (bsFab), single-domain antibody-binding Fab (s-Fab), antibodies, and combinations thereof.

[0050] In one embodiment, the targeting moiety includes a first antigen-binding domain and a second antigen-binding domain. In some examples, the first and second antigen-binding domains specifically bind to the same target. The first and second antigen-binding domains may include the same amino acid sequence. In other examples, the first and second antigen-binding domains specifically bind to different targets. The first and second antigen-binding domains include different amino acid sequences.

[0051] The targeting moiety may bind to a molecule specific for the target, and thus targets a masked cytokine to a target, for example, a targeted cell, tissue, and organ. In some embodiments, the targeting moiety is specific for PD-1, PD-L1, PD-L2, CTLA-4, TIGIT, TIM-3, LAG-3, CD25, CD16a, CD16b, NKG2A, NKG2D, NKP44, NKP30, CD19, CD20, CD30, CD38, BCMA, human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epidermal growth factor receptor (EGFR), glypican-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), nectin-4, Liv-1, glycoprotein NMB (GPNMB), prostate-specific membrane antigen (PSMA), Trop-2, carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), prostate six-transmembrane epithelial antigen 1 (STEAP1), folate receptor alpha (FR-α), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), ectonucleotide pyrophosphatase / phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG), CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD94, CD96, CD98, CD123, CD138, CD352, CD47, signal regulatory protein alpha (SIRPα), claudin 18.2, claudin 6, 5T4, fibroblast activation protein alpha (FAPα), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epithelial cell adhesion molecule (EPCAM), or a combination thereof.

[0052] In one preferred embodiment, the targeting moiety binds to PD-1.

[0053] According to the present disclosure, the portions of the masked cytokine are covalently linked. In some embodiments, the targeting portion is linked to the engineered Fc domain via one or both of the first and second Fc polypeptides. In one example, the targeting portion is linked to the N-terminus of the first Fc polypeptide of the engineered Fc domain, and the C-terminus of the first Fc polypeptide is linked to the N-terminus of the cytokine or a fragment thereof. In another example, the C-terminus of the targeting portion is linked to the N-terminus of the second Fc polypeptide of the engineered Fc domain, and the C-terminus of the second Fc polypeptide is linked to the N-terminus of the masking portion.

[0054] According to the present disclosure, the protease cleavage site has an in vitro cleavage efficiency that results in at least 10% active cytokine. In some embodiments, about 10% - 30% of the active cytokine is released after cleavage.

[0055] In some embodiments, the present disclosure provides a nucleic acid molecule encoding an engineered Fc domain comprising a protease cleavage site. The present disclosure also provides a nucleic acid molecule encoding a masked cytokine.

[0056] In some embodiments, the present disclosure provides a vector, a host cell, comprising a nucleic acid molecule encoding an engineered Fc domain comprising the protease cleavage site and the masked cytokine described herein. A method of producing the masked cytokine is provided herein, the method comprising culturing a host cell comprising a nucleic acid molecule encoding an engineered Fc domain or a masked cytokine comprising a protease cleavage site or a masked cytokine under conditions that produce the engineered Fc domain or the masked cytokine.

[0057] Also provided in the present disclosure are compositions, pharmaceutical compositions, and kits that include an engineered Fc domain or a masked cytokine. A pharmaceutical composition that includes an engineered cleavable Fc domain and / or a masked cytokine further includes a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition that includes an engineered cleavable Fc domain and / or a masked cytokine is formulated for administration via a defined route, such as intravenous (IV) infusion.

[0058] In another aspect of the present disclosure, methods of using an engineered cleavable Fc domain and a masked cytokine are provided. In some embodiments, the present disclosure provides a method for treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of a masked cytokine, a composition comprising the masked cytokine, or a pharmaceutical composition.

[0059] In some embodiments, the masked cytokine, the composition comprising the masked cytokine, or the pharmaceutical composition is used in a method for treating or preventing an inflammatory disease or an autoimmune disease in a subject, the method comprising administering to the subject an effective amount of a masked cytokine, a composition comprising the masked cytokine, or a pharmaceutical composition.

[0060] In some embodiments, the engineered cleavable Fc domain is used alone or in combination with other therapeutic agents for treating neoplastic diseases, inflammatory diseases, and / or autoimmune diseases.

[0061] In one aspect, the present invention provides a masked cytokine that includes, inter alia, an engineered Fc domain that includes a cytokine portion, a masking portion, and a tumor-associated protease cleavage site, wherein the engineered Fc domain is fused to the cytokine portion or the masking portion such that the masking portion binds to the cytokine portion and the cytokine portion is released from the masking portion upon cleavage of the tumor-associated protease cleavage site on the engineered Fc domain.

[0062] In one aspect, the present invention provides a masked therapeutic active molecule comprising an engineered Fc domain comprising a therapeutic active domain, a masking moiety, and a tumor-related cleavage site, wherein the engineered Fc domain is such that the masking moiety binds to the therapeutic active domain and the therapeutic active domain is released from the masking moiety upon cleavage of the tumor-related protease cleavage site on the engineered Fc domain, and is fused to the therapeutic active domain or the masking moiety.

[0063] In some embodiments, the engineered Fc domain is fused to the therapeutic active domain or the masking moiety via a non-cleavable linker. In some embodiments, the engineered Fc domain is directly fused to the therapeutic active domain or the masking moiety.

[0064] In some embodiments, the therapeutic active domain is a cell engager. In some embodiments, the therapeutic active domain is a co-stimulatory domain. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The drawings are for illustrative purposes only and not for purposes of limitation.

[0066]

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[0067] Definitions To more readily understand the present invention, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout this specification. Publications and other references cited herein to explain the background of the invention and to provide additional detail regarding its practice are incorporated herein by reference.

[0068] It should be understood that the present invention is not limited to particular compositions or biological systems which may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0069] As used herein in the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "IL-2 polypeptide" optionally includes combinations of two or more such polypeptides, and the like.

[0070] As used herein, the term "about" refers to the normal error range for each value readily known to those of ordinary skill in the art. Reference to "about" values or parameters herein includes (and describes) embodiments that essentially target such values or parameters.

[0071] It is understood that the aspects and embodiments of the invention described herein include aspects and embodiments "comprising", "consisting of", and "consisting essentially of".

[0072] As used herein, the term "and / or" refers to any one of the items with which the term is associated, any combination of the items, or all of the items. For example, the phrase "A, B, and / or C" is intended to encompass 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 (alone); B (alone); and C (alone).

[0073] Antibody: As used herein, the term "antibody" is used in its broadest sense and includes polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions having polyepitope specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules (e.g., scFv and heavy-chain-only antibodies (HcAb)), and antibody fragments (e.g., Fab, F(ab’)2, and Fv). The term "immunoglobulin (Ig)" is used interchangeably with "antibody" herein. Antibodies can be natural or artificial.

[0074] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units together with an additional polypeptide called the J chain and contain ten antigen-binding sites, while IgA antibodies consist of two to five basic four-chain units that can polymerize to form multivalent aggregates in combination with the J chain. In the case of IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H chain and L chain also have regularly spaced intra-chain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the a and y chains, and four CH domains for the p and s isotypes. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at the other end. VL aligns with VH, and CL aligns with the first constant domain of the heavy chain (CHI). Specific amino acid residues are thought to form the interface between the light and heavy chain variable domains. The pairing of VH and VL together forms a single antigen-binding site. For the structure and properties of different 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, pages 71 and Chapter 6.

[0075] The light chains from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Immunoglobulins can be assigned to different classes or isotypes according to the amino acid sequence of their heavy chain constant domains (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each having heavy chains designated as a, 8, y, and p, respectively. The classes of y and a are further divided into subclasses based on relatively small differences in CH sequences and functions. For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies may exist in multiple polymorphic variants called allotypes (Jefferis and Lefranc 2009. mAbs Vol 1 Issue(4):1-7), any of which are suitable for use in the present invention. Common allotype variants in the human population are those designated by the letters a, f, n, and z.

[0076] An "isolated" antibody is one that has been identified, separated, and / or recovered from the components of its production environment (e.g., natural or recombinant). In some embodiments, the isolated polypeptide is free of all other components from its production environment. Contaminating components of its production environment, such as those arising from recombinant transfected cells, are materials that typically interfere with the research, diagnostic, or therapeutic use of the antibody and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide is purified to (1) greater than 95% by weight of the antibody, as determined, for example, by the Lowry method, and in some embodiments greater than 99% by weight, (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver staining. Because an isolated antibody is not to contain at least one component of the antibody's natural environment, an isolated antibody is one that contains the antibody in situ within recombinant cells. However, usually, an isolated polypeptide or antibody is prepared by at least one purification step.

[0077] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. In some embodiments, the monoclonal antibody has a C-terminal truncation in the heavy chain and / or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are truncated at the C-terminus of the heavy chain and / or light chain. In some embodiments, the C-terminal truncation removes the C-terminal lysine from the heavy chain. In some embodiments, the monoclonal antibody has an N-terminal truncation in the heavy chain and / or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are truncated at the N-terminus of the heavy chain and / or light chain. In some embodiments, the shortened form of the monoclonal antibody can be produced by recombinant techniques. In some embodiments, the monoclonal antibody is highly specific and directed against a single antigenic site. In some embodiments, the monoclonal antibody is highly specific and directed against multiple antigenic sites (e.g., bispecific antibodies or multispecific antibodies, etc.). The modifier "monoclonal" indicates the characteristics of the antibody when obtained from a substantially homogeneous antibody population and is not to be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques including, for example, the hybridoma method, recombinant DNA methods, phage display techniques, and techniques for producing human antibodies or human-like antibodies in animals having part or all of the human immunoglobulin locus or genes encoding human immunoglobulin sequences.

[0078] The terms "full-length antibody", "intact antibody", or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form as contrasted with antibody fragments. Specifically, whole antibodies include those having heavy and light chains that include an Fc region. The constant domain may be a naive sequence constant domain (e.g., a human naive sequence constant domain) or an amino acid sequence variant thereof. In some cases, the intact antibody may have one or more effector functions.

[0079] An "antibody fragment" includes a portion of an intact antibody, for example, an antigen-binding region and / or a variable region of an intact antibody, and / or a constant region of an intact antibody. Examples of antibody fragments include the Fc region of an antibody, a portion of the Fc region, or a portion of an antibody that includes the Fc region. Examples of antigen-binding antibody fragments include domain antibodies (dAbs), Fab, Fab’, F(ab’)2 and Fv fragments, diabodies, linear antibodies (see, e.g., 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-chain Fv (scFv), single-domain antibodies (VHH), one or more CDRs, variable heavy chain (VH), variable light chain (VL), Fab-like bispecific antibodies (bsFab), single-domain antibody-binding Fab (s-Fab), and combinations thereof. A single heavy-chain antibody or single light-chain antibody can be engineered or, in the case of a heavy chain, isolated from a camelid, shark, library, or mouse engineered to produce a single heavy-chain molecule.

[0080] Papain digestion of an antibody produced two identical antigen-binding fragments called "Fab" fragments and a remaining "Fc" fragment, a name reflecting the ability to crystallize readily. The Fab fragment consists of the entire L chain combined with the variable domain of the H chain (VH) and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment that approximately corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and can still cross-link antigens. The Fab' fragment differs from the Fab fragment in having several additional residues at the carboxy terminus of the CHI domain that include one or more cysteines from the antibody hinge region. Fab'-SH is the name herein for the Fab' where the cysteine residue of the constant domain has a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that had hinge cysteines between them. Other chemical conjugates of antibody fragments are also known. The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of an antibody are determined by sequences and glycans within the Fc region, and the region recognized by Fc receptors (FcR) is found on certain cell types.

[0081] The term "diabody" refers to a small antibody fragment with two antigen-binding sites that contains a heavy chain variable (VH) domain connected to a light chain variable (VL) domain in the same polypeptide chain (VH-VL).

[0082] The "percent amino acid sequence identity (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning those sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and does not take into account any conservative substitutions as part of the sequence identity. The alignment for determining the percent amino acid sequence identity can be accomplished using various methods within the skill in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, etc. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared. For example, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, a given amino acid sequence A that may be referred to as having or containing a particular percent amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows. 100 × fraction X / Y Where X is the number of amino acid residues scored as perfect matches by the sequences in the alignment of A and B by the program, and Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B is not equal to the percent amino acid sequence identity of B to A.

[0083] The "effector function" of an antibody refers to the biological activity resulting from the Fc region of the antibody (Fc region of the native sequence or Fc region of an amino acid sequence variant), and varies depending on the antibody isotype. Examples of antibody effector functions include Clq 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.

[0084] Amino acid: As used herein, the term "amino acid" refers, in its broadest sense, to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, the amino acid is a natural amino acid. In some embodiments, the amino acid is a synthetic amino acid, and in some embodiments, the amino acid is a d-amino acid, and in some embodiments, the amino acid is an l-amino acid. "Standard amino acid" refers to any of the 20 standard l-amino acids commonly found in native peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether prepared synthetically or obtained from natural sources. As used herein, "synthetic amino acid" includes, but is not limited to, salts, amino acid derivatives (such as amides), and / or chemically modified amino acids including substitutions. Amino acids including the carboxy-terminal amino acid and / or the amino-terminal amino acid in a peptide can be modified by substitution with methylation, amidation, acetylation, protecting groups, and / or other chemical groups that can change the circulating half-life of the peptide without adversely affecting their activity. Amino acids may be involved in disulfide bonds. Amino acids may include one or post-translational modifications such as association with one or more chemical entities (e.g., methyl group, acetate group, acetyl group, phosphate group, formyl moiety, isoprenoid group, sulfate group, polyethylene glycol moiety, lipid moiety, carbohydrate moiety, biotin moiety, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and can refer to free amino acids and / or amino acid residues of a peptide. Whether the term refers to a free amino acid or a residue of a peptide will be apparent from the context in which the term is used.

[0085] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0086] At risk: As used herein, an "at risk" individual for developing a disorder may or may not have a detectable disease or symptom of a disease and may or may not exhibit a detectable disease or symptom of a disease prior to the treatment methods described herein. "At risk" indicates that, as is known in the art, an individual has one or more risk factors that are measurable parameters correlated with the development of a disease. An individual having one or more of these risk factors has a higher probability of developing a disorder than an individual not having one or more of these risk factors.

[0087] Biological activity: As used herein, the phrase "biological activity" refers to the characteristics of any agent that is active in a biological system, particularly in a living organism. For example, an agent that has a biological effect on an organism when administered to that organism is considered to be biologically active.

[0088] Binding affinity: As used herein, the term "binding affinity" refers to the strength of the non-covalent interaction between a single 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 generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0089] Pharmaceutical carrier: As used herein, the term "pharmaceutical carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals to which it is exposed at the dosages and concentrations employed. Physiologically acceptable carriers are often aqueous pH buffer solutions. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; 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 dextrin; 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™.

[0090] Chronic: As used herein, "chronic" administration refers to the administration of a drug in a continuous fashion, as opposed to an acute mode, such that the initial therapeutic effect (activity) predominates over an extended period of time. "Intermittent" administration is not continuous without interruption, but rather is a treatment that is inherently periodic.

[0091] Disease: As used herein, the term "disease" is anything that can be identified as deviating from a healthy or normal state by a pathological condition, e.g., symptoms or other distinguishing factors. The term "disease" includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative diseases, inflammatory diseases, immune diseases, metabolic diseases, infectious diseases, and ischemic diseases.

[0092] Delivery: As used herein, the term "delivery" encompasses both local delivery and systemic delivery. For example, delivery of mRNA includes situations where the mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as "local distribution" or "local delivery"), and situations where the mRNA is delivered to a target tissue, the encoded protein is expressed, secreted into the patient's circulatory system (e.g., serum), systemically distributed, and absorbed by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[0093] Dosing interval: As used herein, in the context of a method for treating a disease, the term "dosing interval" is the frequency at which a therapeutic composition, e.g., an mRNA composition, is administered to a subject (mammal) in need thereof, at an effective dose of mRNA such that over at least the period of the dosing interval, one or more symptoms associated with the disease are reduced or one or more biomarkers associated with the disease are reduced. Dosing frequency and dosing interval may be used interchangeably in the present disclosure.

[0094] Effective amount: As used herein, "effective amount" refers to an amount effective at the required dosage and for the required period to achieve a desired or indicated effect, including at least a therapeutic or prophylactic result. The effective amount may be provided in one or more administrations. A "therapeutically effective amount" is at least the minimum concentration required to effect a measurable improvement in a particular disorder. The therapeutically effective amount herein may vary according to factors such as the patient's disease state, age, sex, and weight, as well as the ability of the antibody to elicit a desired response in an individual. The therapeutically effective amount may also be an amount at which any toxic or detrimental effects of the targeted cytokine are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective at the required dosage and for the required period to achieve a desired prophylactic result. Typically, although not necessarily, since prophylactic dosages are used in subjects at a pre- or early stage of a disease, the prophylactically effective amount may be less than the therapeutically effective amount.

[0095] Effective dose: As used herein, an effective dose is the amount of mRNA in a pharmaceutical composition that, when administered to a subject in need thereof, is effective to cause a mammalian subject to achieve an expected result, such as reduction of symptoms associated with a disease, according to the methods of the present invention.

[0096] Host cell: As used herein, the term "host cell" can be a recipient of any recombinant vector or isolated polynucleotide, such as a vector or polynucleotide of an engineered cleavable Fc domain or a masked cytokine of the present disclosure, or an individual cell or cell culture that was a recipient. Host cells include cells transfected or infected in vivo or in vitro with a recombinant vector or polynucleotide of the present invention. In some cases, the host cell is a mammalian host cell.

[0097] Improve, increase, or decrease: As used herein, the terms "improve", "increase", or "decrease", or grammatical equivalents thereof, refer to a value relative to a baseline measurement, such as a measurement in the same individual prior to initiation of treatment described herein, or a measurement in a control subject (or control subjects) without the treatment described herein. A "control subject" is a subject suffering from the same type of disease as the subject being treated and is approximately the same age as the subject being treated.

[0098] In combination with: As used herein, "in combination with" or "in combination" refers to the administration of one therapeutic modality in addition to another therapeutic modality. Thus, "in combination with" or "in combination" refers to the administration of one therapeutic modality before, during, or after the administration of another therapeutic modality to an individual.

[0099] Subject: As used herein, an "individual" or "subject" is a mammal. For purposes of treatment, "mammals" include humans, domestic and farm animals, and zoo, sports, or pet animals such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, and cats. In some embodiments, the individual or subject is a human.

[0100] In vitro: As used herein, the term "in vitro" refers to events that occur not within a multicellular organism, but rather in an artificial environment, such as in a test tube or reaction vessel, within cell culture, and the like.

[0101] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms such as humans and non-human animals. In the context of cell-based systems, this term can be used to refer to events that occur within living cells (e.g., rather than in an in vitro system).

[0102] Isolated: As used herein, an "isolated" nucleic acid molecule encoding a cytokine polypeptide described herein is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in the environment in which it is produced. In some embodiments, the isolated nucleic acid is unassociated with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and cytokine polypeptides herein are in a form other than that in which they are naturally found or in their natural environment. Thus, an isolated nucleic acid molecule is distinct from the nucleic acids encoding the polypeptides and cytokine polypeptides herein that are naturally present within a cell.

[0103] Pharmaceutical formulation: As used herein, the term "pharmaceutical formulation" refers to a preparation that is in a form that enables the biological activity of the active ingredient to be effective and that contains no additional components that have unacceptable toxicity to the subject to whom the formulation is administered.

[0104] Moiety: As used herein, the term "moiety" refers to a substructure that is part of a molecule.

[0105] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to substances that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic reaction, or other problems or complications within the scope of sound medical judgment and comparable to a reasonable benefit / risk ratio.

[0106] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. The pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic acids and organic acids as well as bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed by inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N+(C1-4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc.Still other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium cations, quaternary ammonium cations, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Still other pharmaceutically acceptable salts include salts formed from the quaternization of amines using suitable electrophiles, such as alkyl halides, to form quaternized alkylated amino salts.

[0107] Polypeptide: As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, e.g., a polymer of at least 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. Throughout the present disclosure, standard three-letter or one-letter symbols for amino acids are used. As used herein, this term encompasses amino acid chains of any length, including full-length proteins, and the amino acid residues are linked by covalent peptide bonds.

[0108] Polynucleotide: As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide, having a length of at least 5, 6, 7, 8, 9, or 10 bases or base pairs, and is intended to include single-stranded and double-stranded forms of DNA and / or RNA. Throughout the present disclosure, this term is used interchangeably with “nucleic acid molecule.”

[0109] Prevention: As used herein, the term “prevention” includes providing prophylaxis against the occurrence or recurrence of a disease in an individual. The individual may be predisposed to, susceptible to, or at risk of developing a disorder, but has not been diagnosed with the disorder. In some embodiments, the targeted cytokines described herein are used to delay the onset of the disorder.

[0110] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). A human includes both prenatal and postnatal forms. In many embodiments, the subject is a human. A subject can be a patient, where a patient refers to a human presented to a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient". A subject may or may not be suffering from a disease or disorder or be susceptible to a disease or disorder and may or may not exhibit symptoms of the disease or disorder.

[0111] Substantially: As used herein, the term "substantially" refers to a qualitative condition indicating an amount that is the same as, or nearly the same as, the total or total range or degree of a desired feature or characteristic. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, achieve completion and / or progress to completeness, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0112] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by a disease being treated. In some embodiments, the target tissue includes a tissue that exhibits a disease-related pathology, symptom, or characteristic.

[0113] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount sufficient to treat, diagnose, prevent, and / or delay the onset of symptoms of a disease, disorder, and / or condition when administered to a subject suffering from, or susceptible to, the disease, disorder, and / or condition. One of ordinary skill in the art will understand that a therapeutically effective amount is typically administered via a dosing regimen that includes at least one unit dose.

[0114] To treat: As used herein, the terms "treat", "treatment", or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the occurrence, reduce the severity, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. This can refer to any clinical intervention designed to alter the natural course of an individual or cell being treated during the course of a clinical pathology. Treatment can be administered to a subject that does not exhibit symptoms of a disease and / or exhibits only early signs of a disease for the purpose of reducing the risk of developing lesions associated with the disease. An individual is successfully "treated" if, for example, one or more symptoms associated with a disorder (e.g., a neoplastic disease) are alleviated or eliminated. For example, an individual is successfully "treated" if the treatment results in an improvement in the quality of life of the individual suffering from the disease, a reduction in the dosage of other drugs required for the treatment of the disease, a reduction in the frequency of recurrence of the disease, a reduction in the severity of the disease, a delay in the onset or progression of the disease, and / or an extension of the survival of the individual.

[0115] Vector: As used herein, the term "vector" refers to a macromolecule or molecular complex that includes a polynucleotide that is delivered to a host cell or organism, typically a virus or plasmid, either in vitro or in vivo.

[0116] Various aspects of the invention are described in detail in the following sections. The use of the sections is not intended to limit the invention. Each section can be applied to any aspect of the invention. In this application, unless otherwise specified, the use of "or" means "and / or".

Modes for Carrying Out the Invention

[0117] I. Introduction The specific delivery and activation of therapeutic agents such as cytokines to tumor cells is desirable to enhance the safety of such treatments (e.g., to avoid targeting normal cells). The present invention provides compositions and methods for such precision treatment of cancer. Specifically, the present invention relates to a universal and cleavable carrier platform, among other things, for the purpose of precision treatment of tumors, together with the masking portion of the present invention. Such cleavable carriers can be linked to therapeutic agents (e.g., cytokines) for tumor-specific delivery and activation.

[0118] Generally, the present disclosure relates to a cleavable carrier portion comprising an engineered tumor-associated protease cleavage site such that the carrier portion is cleavable. The cleavable carrier portion is fused to a therapeutic molecule and / or a masking portion to form a masked therapeutic agent. For example, the carrier portion is fused to a therapeutic molecule and / or a masking portion via a non-cleavable linker. A masked therapeutic agent comprising such a cleavable carrier portion is capable of releasing an active therapeutic molecule from the masking portion upon cleavage of the engineered tumor-associated protease site within the cleavable carrier portion.

[0119] As used herein, the term "cleavable carrier" refers to any agent that is enzymatically or non-enzymatically cleavable from the present composition. For example, the cleavable carrier may be a protein, polypeptide, and their domains. In the context of the present disclosure, the terms "cleavable carrier" and "cleavable domain" are used interchangeably. Such cleavable carrier portion provides enzymatically induced prodrug activation, characterized in that cleavage of the carrier portion controls the release of the active therapeutic agent.

[0120] The cleavable carrier moiety may be a half-life extension domain derived from albumin, transferrin, or tissue factor. The cleavable carrier moiety may be an antigen targeting domain selected from the group consisting of immunoglobulins, Fab, F(ab)2, scFv, VHH, ScAb, and nanobodies. The tumor-associated protease cleavage site is engineered within the antigen targeting domain. The carrier moiety may be an Fc domain in which at least one tumor-associated protease cleavage site is engineered, for example, by amino acid substitution at a specific position in the Fc domain.

[0121] In addition to the aforementioned cytokines, the therapeutic molecule may include any therapeutic agent having a functional effect on the disease, such as a therapeutic protein, a cell engager, and a co-stimulatory domain.

[0122] The masking moiety within the masked therapeutic agent binds to the therapeutic molecule and masks it from release and / or activation. The masking moiety can be any agent that binds to the therapeutic molecule. For example, the masking moiety is a peptide, polypeptide, protein, ligand, or agent that specifically binds to a cytokine, or a variant thereof, or a fragment thereof.

[0123] The masked therapeutic agent may further include a targeting moiety, which can specifically bind to an antigen, ligand, or biomarker of a targeted cell, tissue, and / or organ. For example, the targeting moiety includes one or more antigen-binding domains, peptides, polypeptides, proteins, ligands, or agents that specifically bind to an antigen, ligand, or biomarker. In this context, the cytokine is also referred to as a targeted cytokine.

[0124] According to the present disclosure, the masked therapeutic agent may be interpreted as a fusion polypeptide. Upon cleavage of the engineered tumor-related protease site in the carrier portion, the polypeptide is cleaved, and then the masked therapeutic molecule is released and activated for its function in targeted cells, tissues, and / or organs (e.g., in tumor tissue). The fusion polypeptide may be produced by recombinant techniques known in the art.

[0125] Specifically, the present disclosure provides, inter alia, an engineered Fc domain comprising a tumor-related protease cleavage site. The engineered Fc domain having a tumor-related protease cleavage site provides a universal and cleavable platform that can be linked to therapeutic agents such as masked cytokines to control their activation. The engineered cleavable Fc domain of the present invention can be fused to a cytokine, a masking moiety, and / or a targeting moiety to produce a prodrug. The prodrug comprising the engineered Fc domain of the present disclosure can be activated at the disease site and specifically target the cells of interest for effective treatment of cancer without causing undesirable side effects.

[0126] The present disclosure also provides, inter alia, therapeutic agents, as well as pharmaceutical compositions and formulations thereof, comprising an engineered Fc domain and / or a targeted cytokine. Methods of using such agents, compositions, and formulations for the treatment of diseases (e.g., cancer) are also provided.

[0127] II. Compositions Provided by the present invention are, inter alia, a cleavable carrier, and a composition comprising the cleavable carrier. As a non-limiting example, the cleavable carrier is a cleavable Fc domain. The composition comprises a cleavable Fc domain. Among the compositions described herein, masked cytokines are provided. The masked cytokine comprises a cytokine moiety, a masking moiety, and an engineered cleavable Fc domain comprising a tumor-associated cleavage site, wherein the engineered cleavable Fc domain is such that the masking moiety is bound to the cytokine moiety and the cytokine moiety is released from the masking moiety upon cleavage of the tumor-associated protease cleavage site on the engineered Fc domain, and is fused to the cytokine moiety or the masking moiety. The engineered cleavable Fc domain is fused to the cytokine molecule and / or the masking moiety via a non-cleavable linker. The cleavable Fc domain-binding cytokine prodrug enhances the therapeutic efficacy of the cytokine in vivo.

[0128] Cleavable carrier As used herein, the term "cleavable carrier" refers to any agent that is enzymatically or non-enzymatically cleavable from the composition. For example, the cleavable carrier may be a protein, polypeptide, and their domains. In the context of the present disclosure, the terms "cleavable carrier" and "cleavable domain" are used interchangeably. The cleavable carrier moiety provides enzymatically induced prodrug activation, characterized in that cleavage of the carrier moiety controls the release of the active therapeutic agent.

[0129] The cleavable carrier moiety may be a half-life extension domain derived from albumin, transferrin, or tissue factor. The cleavable carrier moiety may be an antigen targeting domain selected from the group consisting of immunoglobulins, Fab, F(ab)2, scFv, VHH, ScAb, and nanobodies. The tumor-associated protease cleavage site is engineered within the antigen targeting domain. The carrier moiety may be an Fc domain in which at least one tumor-associated protease cleavage site is engineered, for example, by amino acid substitution at a specific position in the Fc domain.

[0130] Engineered cleavable Fc domain According to the present disclosure, the carrier moiety is an Fc domain derived from an immunoglobulin, such as IgM, IgG1, IgG2, IgG3, IgG4, IgD, IgE, and IgA, or variants thereof, or fragments thereof. Thus, the Fc domain is genetically engineered to be enzymatically cleavable. The engineered Fc domain is referred to as a "cleavable Fc domain".

[0131] As used herein, the term "Fc domain" refers to a polypeptide or fragment derived from an immunoglobulin. The Fc domain may be any antibody or fragment thereof. The Fc domain is derived from an antibody or fragment thereof that occurs naturally in an animal (e.g., a mammal), or is produced by any technique known in the art. In some embodiments, the Fc domain is a human Fc domain.

[0132] In some embodiments, the Fc domain comprises a first Fc polypeptide and a second Fc polypeptide. In some embodiments, the Fc domain is derived from any antibody or fragment thereof that comprises either a heavy chain Fc polypeptide or a light chain Fc polypeptide. In some embodiments, the Fc domain 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 a fragment thereof. In some embodiments, the Fc domain derived from an antibody or fragment thereof comprises a hinge region, a CH2 domain, and a CH3 domain, or fragments thereof. In some embodiments, the Fc domain comprises only the constant domain of the heavy chain polypeptide. In some embodiments, the Fc domain comprises only the constant domain of the light chain polypeptide. In some embodiments, the Fc domain comprises a first Fc polypeptide having CH2 and CH3 domains, and a second Fc polypeptide having CH2 and CH3 domains. In some embodiments, the Fc domain comprises a first Fc polypeptide having a CH3 domain, and a second Fc polypeptide having a CH3 domain. In some embodiments, the Fc domain comprises a first Fc polypeptide having CH2 and CH3 domains, and a second Fc polypeptide having a CH3 domain. In some embodiments, the Fc domain comprises a first Fc polypeptide having a CH3 domain, and a second Fc polypeptide having CH2 and CH3 domains.

[0133] In some embodiments, the first Fc polypeptide and the second Fc polypeptide are linked via a linker, such as a short peptide linker. In some embodiments, the linker is non-cleavable.

[0134] The engineered cleavable Fc domain contains one or more cleavable sites. As used herein, "cleavage site" refers to a recognizable site for cleavage of a portion of a cleavable peptide found anywhere within the Fc domain. A "cleavage site" can be an amino acid sequence such as a short peptide motif that is recognized and cleaved by a cleavage agent. The cleavage site may be a naturally occurring amino acid sequence within the Fc domain. Additionally and / or alternatively, the cleavage site may be introduced into the cleavable portion of the Fc domain by mutation (e.g., amino acid insertion, substitution, and deletion). The mutation does not alter other activities of the Fc domain. In some embodiments, one or more cleavage peptide motifs can be engineered within the cleavable Fc domain described herein. The cleavage agent may be an enzyme, e.g., a protease. In some embodiments, the cleavage site is a protease cleavage site such that the cleavable Fc domain is proteolytically cleavable. A protease is an enzyme that cleaves and hydrolyzes the peptide bond between two specific amino acid residues of a target substrate protein. Proteases generally recognize specific peptide motifs and cleave the peptide bond between two specific amino acid residues within the short peptide motif.

[0135] The Fc domain can be engineered to include one or more peptide motifs that can be recognized by one or more proteases, such as tumor - associated proteases, tissue - selective proteases, and disease (e.g., inflammation) - associated proteases. As a non - limiting example, the cleavable Fc domains described herein can be cleaved by matrix metalloproteases (MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP27 and MMP28), cathepsins (cathepsin B, cathepsin D, cathepsin F, cathepsin K, cathepsin L, cathepsin V, cathepsin S, and cathepsin W), ADAM, ADAMTS, kallikrein 1 - 15, HTRA1 - 2 - 3, HGFAc, PRSS, TMPRSS, elastase, PR - 3, granzyme (granzyme A, B, M, H, and K), fibroblast activation protein (FAP), plasmin, urokinase plasminogen activator (uPA), tryptase, caspase, thrombin, legumain, chymase, collagenase, napsin A, and matriptase 1 - 2, which are disease - associated and tissue - selective proteases selected therefrom.

[0136] In some embodiments, the cleavable Fc domain described herein comprises at least one engineered tumor-associated protease cleavage site. As used herein, a "tumor-associated protease cleavage site" is an amino acid sequence recognized by a protease, the expression of which is specific to or upregulated in tumor cells or their tumor cell environment. In some embodiments, the tumor-associated protease is a matrix metalloprotease (MMP) selected from the group consisting of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP27, and MMP28. In one embodiment, the tumor-associated protease is MMP2. In another embodiment, the tumor-associated protease is MMP3. In yet another embodiment, the tumor-associated protease is MMP9. In yet another embodiment, the tumor-associated protease is MMP10. In some embodiments, the protease is cathepsin B. In other embodiments, the protease is matriptase.

[0137] The advantage of such engineered cleavable Fc domains is that they provide a universal design construct such that fusion of the engineered cleavable Fc domain to a therapeutic agent and subsequent cleavage at the cleavage site enables release of the agent in a specific environment, e.g., the tumor microenvironment.

[0138] The tumor cell environment is complex and may contain multiple different proteases. Therefore, the exact site at which the Fc domain is cleaved in the tumor cell environment can vary between tumor types, between patients with the same tumor type, and even between cleavage products formed within the same tumor, depending on the specific tumor cell environment. Furthermore, even after cleavage, further modification of the initial cleavage product may occur, for example, by removal of one or two terminal amino acids, due to the action of yet another protease in the tumor cell environment. The distribution of cleavage products can be expected to be formed within the patient's tumor cell environment after administration of the single-structured, targeted cytokine as described herein.

[0139] As referred to herein, it will be understood that the cleavage site refers to the site between two specific amino acid residues within a cleavable peptide that is a target of a protease known to be associated with the tumor cell environment. In this sense, there may be more than one cleavage site within the cleavable peptides described herein, and different proteases cleave the cleavable peptide at different cleavage sites. Also, more than one protease may act on the same cleavage site within the cleavable peptide. Considerations regarding protease cleavage sites can be found in the art.

[0140] Accordingly, the cleavable Fc domain disclosed herein may be cleaved by one or more proteases. In some embodiments, the cleavage site of the cleavable Fc domain is the cleavage site of one or more tumor-associated proteases. In some embodiments, the cleavage site of the cleavable Fc domain is the cleavage site of one or more tissue-selective proteases. In some embodiments, the cleavage site of the cleavable Fc domain is the cleavage site of one or more inflammation-associated proteases. In other embodiments, the cleavable Fc domain comprises one or more cleavage sites of other disease-associated proteases.

[0141] In some embodiments, the cleavable Fc domain is a substrate of a protease that is co-localized with a region or tissue or organ that expresses a cytokine receptor.

[0142] The cleavable peptide motif in the Fc domain contains at least three amino acid residues. In some embodiments, the cleavable peptide motif is a 3mer (i.e., a peptide 3 amino acids in length), 4mer (i.e., a peptide 4 amino acids in length), 5mer (i.e., a peptide 5 amino acids in length), 6mer (i.e., a peptide 6 amino acids in length), 7mer (i.e., a peptide 7 amino acids in length), 8mer (i.e., a peptide 8 amino acids in length), 9mer (i.e., a peptide 9 amino acids in length), 10mer (i.e., a peptide 10 amino acids in length), 11mer (i.e., a peptide 11 amino acids in length), 12mer (i.e., a peptide 12 amino acids in length), 13mer (i.e., a peptide 13 amino acids in length), 14mer (i.e., a peptide 14 amino acids in length), 15mer (i.e., a peptide 15 amino acids in length), 16mer (i.e., a peptide 16 amino acids in length), 17mer (i.e., a peptide 17 amino acids in length), or 18mer (i.e., a peptide 18 amino acids in length).

[0143] In some embodiments, the cleavable peptide motif in the Fc domain is 3 to 18 amino acids in length. In some embodiments, the cleavable peptide motif in the Fc domain is 5 to 10 amino acids in length, or 5 to 8 amino acids in length, or 6 to 10 amino acids in length, or 7 to 10 amino acids in length, or 6 to 12 amino acids in length. In some embodiments, the cleavable peptide motif in the Fc domain is 3 amino acids in length. In some embodiments, the cleavable peptide motif in the Fc domain is 4 amino acids in length. In some embodiments, the cleavable peptide motif in the Fc domain is 5 amino acids in length. In some embodiments, the cleavable peptide motif in the Fc domain is 6 amino acids in length. In some embodiments, the cleavable peptide motif in the Fc domain is 7 amino acids in length. In some embodiments, the cleavable peptide motif in the Fc domain is 8 amino acids in length. In some embodiments, the cleavable peptide motif in the Fc domain is 9 amino acids in length. In some embodiments, the cleavable peptide motif in the Fc domain is 10 amino acids in length.

[0144] In some embodiments, the protease cleavage site within the engineered cleavable Fc domain is within the hinge region, within the CH2 domain, within the CH3 domain, and / or within the CH2-CH3 domain linker region. In some embodiments, the protease cleavage site within the engineered cleavable Fc domain is within the F strand region. In some embodiments, the protease cleavage site within the engineered cleavable Fc domain is within the FG loop region. In some embodiments, the protease cleavage site within the engineered cleavable Fc domain is within the G strand region.

[0145] In some embodiments, the cleavable site within the engineered cleavable Fc domain can be generated based on a short amino acid motif that is proximal to the protease cleavage site.

[0146] Based on the short peptide sequence of the Fc domain, one or more mutations (e.g., amino acid substitutions) may be introduced into the peptide sequence to generate a protease cleavage site. As a non-limiting example, one or more protease cleavage sites may be generated based on the short peptide sequences of SEQ ID NOs: 98, 101, 103, and 106.

[0147] In some embodiments, a protease cleavage site that can be recognized by MMP2 is generated. In some embodiments, a protease cleavage site that can be recognized by MMP3 is generated. In some embodiments, a protease cleavage site that can be recognized by MMP9 is generated. In some embodiments, a protease cleavage site that can be recognized by MMP10 is generated. In some embodiments, a protease cleavage site that can be recognized by cathepsin B is generated. In some embodiments, a protease cleavage site that can be recognized by matriptase is generated.

[0148] As a non-limiting example, the cleavable peptide motif within the engineered cleavable Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 95, 97, 99, 100, 102, 104, 105, 107, 108, 109, 110, 119, 122, 123, 135-139, 143-208, and 209. In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises VPLSLYSG (SEQ ID NO: 95). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises VPLSLYSGP (SEQ ID NO: 209). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises QQGNVFSC (SEQ ID NO: 97). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises IHVTLKSL (SEQ ID NO: 99). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises NSYTIKGL (SEQ ID NO: 100). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SNESLSLS (SEQ ID NO: 102). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises QVSSSLSP (SEQ ID NO: 104). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises PTSTSLSP (SEQ ID NO: 105). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises ESLSLSEE (SEQ ID NO: 107). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises ASLSLAPV (SEQ ID NO: 108). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SQESLSLS (SEQ ID NO: 109). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises PLGL (SEQ ID NO: 110). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises MPYDLYHP (SEQ ID NO: 135).In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises ISSGLLSGRS (SEQ ID NO: 136). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises RAAAVKSP (SEQ ID NO: 137). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises RPLALWRS (SEQ ID NO: 138). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises TQKPLGLS (SEQ ID NO: 139). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises APAGLIVPYN (SEQ ID NO: 119). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises PVSLRSGS (SEQ ID NO: 122). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises PANLVAPDP (SEQ ID NO: 123). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises RSKYLATA (SEQ ID NO: 143). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises GRPRHQGV (SEQ ID NO: 144). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises GLFG (SEQ ID NO: 145). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises GFLG (SEQ ID NO: 146). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises AGRRAAK (SEQ ID NO: 147). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises FRLWA (SEQ ID NO: 148). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises FRLWS (SEQ ID NO: 149). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises NFFGVGGE (SEQ ID NO: 150).In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes PMKRLTLA (SEQ ID NO: 151). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes FPLATYAP (SEQ ID NO: 152). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes FLVGGASL (SEQ ID NO: 153). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes KPMQFLGD (SEQ ID NO: 154). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes GIVRAKGV (SEQ ID NO: 155). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes ALFKSSFP (SEQ ID NO: 156). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes SGRRSPGG (SEQ ID NO: 157). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes SLGRRPGG (SEQ ID NO: 158). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes SLSGRRGG (SEQ ID NO: 159). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes SLSLGRRG (SEQ ID NO: 160). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes SLSLSGRR (SEQ ID NO: 161). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes GGPRRL (SEQ ID NO: 162). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes GGPLRL (SEQ ID NO: 163). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes GGPKLL (SEQ ID NO: 164). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes GGPRNL (SEQ ID NO: 165).In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises GGPRML (SEQ ID NO: 166). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises EHLRSPGG (SEQ ID NO: 167). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises FRSGVPGG (SEQ ID NO: 168). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SLLLRTGN (SEQ ID NO: 169). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises AGLRSPGG (SEQ ID NO: 170). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SLFRSAGP (SEQ ID NO: 171). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SLFRAPGP (SEQ ID NO: 172). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises WLFRSPLG (SEQ ID NO: 173). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SRLRSPQG (SEQ ID NO: 174). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SLVLSGRR (SEQ ID NO: 175). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises KQLRHMRG (SEQ ID NO: 176). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises LSGRSDNH (SEQ ID NO: 177). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises LSGK (SEQ ID NO: 178). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises LSGR (SEQ ID NO: 179). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises RQARVVGG (SEQ ID NO: 180).In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises RQRRVVGG (SEQ ID NO: 181). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises RQYRVVGG (SEQ ID NO: 182). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SKGRSLIG (SEQ ID NO: 183). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises PRFKIIGG (SEQ ID NO: 184). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises KQLRVVNG (SEQ ID NO: 185). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises IQPRITGG (SEQ ID NO: 186). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises KQSRKFVP (SEQ ID NO: 187). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises GRQSRAGG (SEQ ID NO: 188). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SGRSSPGG (SEQ ID NO: 189). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SSGRSPGG (SEQ ID NO: 190). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SLSGRSGG (SEQ ID NO: 191). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SLSSGRSG (SEQ ID NO: 192). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises KLSLSGRS (SEQ ID NO: 193). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises PLRLSRA (SEQ ID NO: 194). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises. It includes PLKLSRA (SEQ ID NO: 195). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes PLGLSGRS (SEQ ID NO: 196). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes PLGLRSRA (SEQ ID NO: 197). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes PLGLKSRA (SEQ ID NO: 198). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes RGSRAG (SEQ ID NO: 199). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes RLSRGK (SEQ ID NO: 200). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes RGSRGG (SEQ ID NO: 201). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes KGSRAG (SEQ ID NO: 202). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes KLSRGK (SEQ ID NO: 203). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes GRSRAG (SEQ ID NO: 204). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes LRSRGK (SEQ ID NO: 205). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes GRSRGG (SEQ ID NO: 206). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes GKSRAG (SEQ ID NO: 207). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes LKSRGK (SEQ ID NO: 208). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes VPLSLYSGP (SEQ ID NO: 209).

[0149] Incorporation of a Cleavable Substrate into the G Chain of the Fc Domain In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions in the CH3 domain. In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions in the C-terminal region within the Fc domain. In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions in the G strand within the Fc domain. In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions between positions 436 and 447 according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions between positions 438 and 447 according to EU numbering.

[0150] In some embodiments, the cleavable peptide motif is located between positions 438 and 445 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 439 and 446 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 440 and 447 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 438 and 447 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 437 and 444 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 440 and 443 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 441 and 444 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 442 and 445 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 444 and 447 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 441 and 447 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 443 and 447 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 436 and 443 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 442 and 447 according to the EU numbering.

[0151] In some embodiments, the engineered cleavable Fc domain includes the Q438V, K439P, S440L, L441S, S442L, L443Y, P445G substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain includes the K439V, S440P, S444Y, P445S substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain includes the S440V, L441P, S442L, L443S, S444L, P445Y, G446S substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain includes the Q438M, K439P, S440Y, L441D, S442L, L443Y, S444H substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain includes the K439M, S440P, L441Y, S442D, S444Y, P445H, G446P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain includes the S440M, L441P, S442Y, L443D, S444L, P445Y, G446H, G447P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain includes the Q438R, K439A, S440A, L441A, S442V, L443K substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain includes the K439R, S440A, L441A, S442A, L443V, S444K, P445S, G446P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain includes the S440R, L441A, S442A, L443A, S444V, P445K, G446S, G447P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain includes the K439R, S440P, S442A, S444W, P445R, G446S substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain includes the Q438A, K439P, S440A, L441G, S442L, L443I, S444V, G446Y, G447N substitutions according to the EU numbering.In some embodiments, the engineered cleavable Fc domain comprises the K439P, S440V, L441S, S442L, L443R, P445G, G446S substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the Q438P, K439A, S440N, S442V, L443A, S444P, P445D, G446P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440P, S442G substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the K439G, S440P, S442G substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L441P, S442L, L443G, S444L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440G, L441P, S442L, L443G, S444L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S442P, S444G, P445L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S444P, P445L, G447L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L443G, S444P, P445L, G447L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L443G, S444P, P445L, G447L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440M, L441P, S442Y, L443D, S444L, P445Y, G446H, G447P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S442G, L443G, S444P, P445L, G447L substitutions according to the EU numbering.

[0152] In some embodiments, the engineered cleavable Fc domain comprises the S440R, L441S, S442K, L443Y, S444L, P445A, G446T, G447A substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440G, L441R, S442P, L443R, S444H, P445Q, G447V substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S444G, P445L, G446F substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S444G, P445F, G446L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L441A, S442G, L443R, S444R, P445A, G446A, G447K substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L443F, S444R, P445L, G446W, G447A substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L443F, S444R, P445L, G446W, G447S substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440N, L441F, S442F, L443G, S444V, P445G, G447E substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440P, L441M, S442K, L443R, S444L, P445T, G446L, G447A substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the K439P, S440M, L441K, S442R, S444T, P445L, G446A substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the Q438P, K439M, S440K, L441R, S442L, L443T, S444L, P445A substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440F, L441P, S442L, L443A, S444T, P445Y, G446A, G447P substitutions according to the EU numbering.In some embodiments, the engineered cleavable Fc domain comprises the S440F, S442V, L443G, S444G, P445A, G446S, G447L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440K, L441P, S442M, L443Q, S444F, P445L, G447D substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440G, L441I, S442V, L443R, S444A, P445K, G447V substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440A, S442F, L443K, P445S, G446F, G447P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L441G, S442R, L443R substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S442G, L443R, S444R substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L443G, S444R, P445R substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S444G, P445R, G446R substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the P445G, G446R, G447R substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S442G, L443G, S444P, P445R, G446R, G447L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S442G, L443G, S444P, P445L, G446R, G447L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S442G, L443G, S444P, P445K, G446L, G447L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S442G, L443G, S444P, P445R, G446N, G447L substitutions according to the EU numbering.In some embodiments, the engineered cleavable Fc domain comprises S442G, L443G, S444P, P445R, G446M, G447L substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440E, L441H, S442L, L443R substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440F, L441R, L443G, S444V substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442L, S444R, P445T, G447N substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440A, L441G, S442L, L443R substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442F, L443R, P445A, G447P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442F, L443R, S444A, G447P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440W, S442F, L443R, G446L substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L441R, S442L, L443R, G446Q substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442V, P445G, G446R, G447R substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440K, L441Q, S442L, L443R, S444H, P445M, G446R substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440L, L441S, S442G, L443R, P445D, G446N, G447H substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S444L, P445S, G447K substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S444L, P445S, G447R substitutions according to EU numbering.In some embodiments, the engineered cleavable Fc domain comprises the S440R, L441Q, S442A, L443R, S444V, P445V substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440R, L441Q, S442R, L443R, S444V, P445V substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the K439R, S440Q, L441R, S442R, L443V, S444V, P445G substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the Q438R, K439Q, S440R, L441R, S442V, L443V, S444G, P445G substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440R, L441Q, S442Y, L443R, S444V, P445V substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L441K, S442G, L443R, P445L, G446I substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440P, L441R, S442F, L443K, S444I, P445I substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440K, L441Q, S442L, L443R, S444V, P445V, G446N substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440I, L441Q, S442P, L443R, S444I, P445T substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440K, L441Q, L443R, S444K, P445F, G446V, G447P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440G, L441R, S442Q, L443S, S444R, P445A substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L441G, S442R, L443S substitutions according to the EU numbering.In some embodiments, the engineered cleavable Fc domain comprises the L441S, S442G, L443R substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L443G, S444R, P445S substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L443S, S444G, P445R, G446S substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the P445G, G446R, G447S substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L441P, S442L, L443R, S444L, P445S, G446R, G447A substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L441P, S442L, L443K, S444L, P445S, G446R, G447A substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440P, S442G, P445G, G446R, G447S substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440P, S442G, S444R, P445S, G446R, G447A substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S440P, S442G, S444K, P445S, G446R, G447A substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S442R, L443G, P445R, G446A substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the S442R, according to the EU numbering. It includes the P445R and G447K substitutions. In some embodiments, the engineered cleavable Fc domain includes the S442R, L443G, P445R substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain includes the S442K, L443G, P445R, G446A substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain includes the S442K, P445R, G447K substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain includes the S442G, L443R, P445R, G446A substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain includes the S442L, L443R, P445R, G447K substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain includes the S442G, L443R, P445R substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain includes the S442G, L443K, P445R, G446A substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain includes the S442L, L443K, P445R, G447K substitutions according to EU numbering.

[0153] In some embodiments, the engineered cleavable Fc domain includes one or more substitutions shown in Table 5, Table 8a, or Table 11.

Table 1

[0154] In some embodiments, the engineered cleavable Fc domain further comprises one or more substitutions that enable heterodimerization of two Fc polypeptides. In some embodiments, the engineered cleavable Fc domain further comprises the Y349C; T366S; L368A; and Y407V substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the S354C and T366W substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the Y349C; T366S; L368A; Y407V, and N297A substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the S354C, T366W, and N297A substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the Y349C; T366S; L368A; Y407V, N297A, and I253A substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the S354C, T366W, N297A, and I253A substitutions.

[0155] In some embodiments, the engineered cleavable Fc domain is used as a carrier moiety for a cytokine for cancer treatment. The engineered cleavable Fc domain is fused to a masked cytokine molecule such that upon cleavage of an engineered protease cleavage site, such as an engineered tumor-associated protease cleavage site in the cleavable Fc domain, the masked cytokine is released from the masking moiety.

[0156] In some embodiments, the cleavable Fc domain may comprise additional mutations described herein in addition to the incorporation of one or more protease cleavage sites.

[0157] Incorporation of a Cleavable Substrate into the F Chain of the Fc Domain In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions in the F chain of the CH3 domain. In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions in the F chain within the Fc domain. In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions between positions 416 and 425 according to EU numbering.

[0158] In some embodiments, the cleavable peptide motif is located between positions 416 and 423 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 416 and 425 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 416 and 423 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 417 and 425 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 417 and 424 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 418 and 425 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 419 and 422 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 419 and 426 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 420 and 423 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 421 and 424 according to the EU numbering.

[0159] In some embodiments, the engineered cleavable Fc domain comprises the substitutions R416A, W417P, Q418A, Q419G, G420L, N421I, F423P, S424Y, C425N according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the substitutions R416I, W417S, Q418S, Q419G, G420L, N421L, V422S, F423G, S424R, C425S according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the substitutions R416V, W417P, Q418L, Q419S, G420L, N421Y, V422S, F423G, C425G according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the substitutions W417P, Q418A, Q419N, G420L, N421V, V422A, F423P, S424D, C425P according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the substitutions W417V, Q418P, Q419L, G420S, N421L, V422Y, F423S, S424G, C425G according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the substitutions Q418M, Q419P, G420Y, N421D, V422L, F423Y, S424H, C425P according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the substitutions Q418P, Q419V, G420S, N421L, V422R, F423S, S424G, C425S according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the substitutions Q418R, Q419A, G420A, N421A, F423K, C425P according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the substitutions Q418R, Q419P, G420L, N421A, V422L, F423W, S424R, C425S according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the substitutions Q418V, Q419P, G420L, N421S, V422L, F423Y, C425G according to the EU numbering.

[0160] In some embodiments, the engineered cleavable Fc domain comprises the Q419P, G420L, N421G, V422L, C425G substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the Q418V, Q419P, G420L, N421S, V422L, F423Y, C425G substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the G420P, N421L, V422G, F423L, C425G substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the N421P, V422L, F423G, S424L, C425G substitutions according to the EU numbering.

[0161] In some embodiments, the engineered cleavable Fc domain comprises the substitutions shown in Table 11a. [Table 2] TIFF2025524631000007.tif28170

[0162] In some embodiments, the engineered cleavable Fc domain further comprises substitutions that enable heterodimerization of two Fc polypeptides. In some embodiments, the engineered cleavable Fc domain comprises the substitutions shown in Table 11a and comprises serine at position 367 according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the substitutions shown in Table 11a and further comprises a knob mutation. In some embodiments, the engineered cleavable Fc domain comprises the substitutions shown in Table 11a and further comprises a hole mutation.

[0163] In some embodiments, the engineered cleavable Fc domain comprises the mutations listed in Table 11a and combinations of mutations that stabilize the Fc domain. In some embodiments, the engineered cleavable Fc domain comprises a combination of the mutations listed in Table 11a and C367S. In some embodiments, the engineered cleavable Fc domain comprises a combination of the mutations listed in Table 11a and C425G. In some embodiments, the engineered cleavable Fc domain comprises a combination of the mutations listed in Table 11a and S375C. In some embodiments, the engineered cleavable Fc domain comprises a combination of the mutations listed in Table 11a and P396C. In some embodiments, the engineered cleavable Fc domain comprises a combination of the mutations listed in Table 11a and 432C. In some embodiments, the engineered cleavable Fc domain comprises a combination of the mutations listed in Table 11a and T437C. In some embodiments, the engineered cleavable Fc domain comprises a combination of the mutations listed in Table 11a and S408C. In some embodiments, the engineered cleavable Fc domain comprises a combination of the mutations listed in Table 11a and 379C. In some embodiments, the engineered cleavable Fc domain comprises a combination of the mutations listed in Table 11a and W381C. In some embodiments, the engineered cleavable Fc domain comprises a combination of the mutations listed in Table 11a and L410C. In some embodiments, the engineered cleavable Fc domain comprises a combination of the mutations listed in Table 11a and K370C. In some embodiments, the engineered cleavable Fc domain comprises a combination of the mutations listed in Table 11a and F405C. In some embodiments, the engineered cleavable Fc domain comprises a combination of the mutations listed in Table 11a and 371C. In some embodiments, the engineered cleavable Fc domain comprises a combination of the mutations listed in Table 11a and S403C.

[0164] In some embodiments, the engineered cleavable Fc domain further comprises the Y349C; T366S; L368A; and Y407V substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the S354C and T366W substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the Y349C; T366S; L368A; Y407V, and N297A substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the S354C, T366W, and N297A substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the Y349C; T366S; L368A; Y407V, N297A, and I253A substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the S354C, T366W, N297A, and I253A substitutions.

[0165] Incorporation of Cleavable Substrates into the FG Loop of the Fc Domain In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions in the FG loop within the Fc domain. In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions between positions 426 and 437 according to EU numbering.

[0166] In some embodiments, the cleavable peptide motif is located between positions 430 and 437 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 428 and 434 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 428 and 433 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 428 and 437 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 429 and 437 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 434 and 437 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 433 and 436 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 432 and 435 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 431 and 434 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 430 and 433 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 429 and 432 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 428 and 431 according to the EU numbering.

[0167] In some embodiments, the engineered cleavable Fc domain comprises the E430V, A431P, H433S, N434L, H435Y, Y436S, T437G substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the E430R, L432A, H433A, N434V, H435K, Y436S, T437P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the E430M, A431P, L432Y, H433D, N434L, H435Y, Y436H, T437P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the 428-ins-VP, H429L, E430S, A431L, L432Y, H433S, N434G substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the 428-ins-RA, H429A, E430A, A431V, L432K, H433S, N434P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the 428-ins-MP, H429Y, E430D, A431L, L432Y, N434P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the 428-ins-VPL, H429S, E430L, A431Y, L432S, H433G substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the 428-ins-RAA, H429A, E430V, A431K, L432S, H433P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the 428-ins-MPY, H429D, E430L, A431Y, L432H, H433P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the 428-ins-I, H429S, E430S, A431G, H433L, N434S, H435G, Y436R, T437S substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the E430R, A431P, H433A, N434L, H435W, Y436R, T437S substitutions according to the EU numbering.In some embodiments, the engineered cleavable Fc domain comprises the E430T, A431Q, L432K, H433P, N434L, H435G, Y436L, T437S substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the E430A, A431G, H433I, N434V, H435P, T437N 428-ins-A, H429P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the E430P, A431V, L432S, H433L, N434R, H435S, Y436G, T437S substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the H429P, E430A, A431N, H433V, N434A, H435P, Y436D, T437P substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the N434P, H435L, Y436G, T437L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the H433P, N434L, H435G, Y436L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the L432P, H433L, N434G, H435L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the A431P, H433G, N434L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the E430G, A431P, H433G, N434L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the E430P, A431L, L432G, H433L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the H429G, E430P, A431L, L432G, H433L substitutions according to the EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the 428-ins-G, H429P, E430L, A431G, H433G substitutions according to the EU numbering.In some embodiments, the engineered cleavable Fc domain comprises the 428-ins-GGP, H429L, E430G, A431L, L432G substitutions according to EU numbering.

[0168] In some embodiments, the engineered cleavable Fc domain comprises the protease cleavage site of Table 11b. The engineered cleavable Fc domain may comprise the substitutions shown in Table 11b.

Table 3

[0169] In some embodiments, the engineered cleavable Fc domain further comprises substitutions that allow for heterodimerization of two Fc polypeptides. In some embodiments, the engineered cleavable Fc domain further comprises substitutions that can extend the half-life of a polypeptide comprising the engineered cleavable Fc domain.

[0170] In some embodiments, the engineered cleavable Fc domain comprises the substitutions shown in Table 11b and comprises serine at position 367 according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the substitutions shown in Table 11b and further comprises a knob mutation. In some embodiments, the engineered cleavable Fc domain comprises the substitutions shown in Table 11b and further comprises a hole mutation.

[0171] In some embodiments, the engineered cleavable Fc domain further comprises the Y349C; T366S; L368A; and Y407V substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the S354C and T366W substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the Y349C; T366S; L368A; Y407V, and N297A substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the S354C, T366W, and N297A substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the Y349C; T366S; L368A; Y407V, N297A, and I253A substitutions. In some embodiments, the engineered cleavable Fc domain further comprises the S354C, T366W, N297A, and I253A substitutions.

[0172] In some embodiments, an engineered cleavable Fc domain that comprises one or more substitutions between positions 416 and 416, or between positions 428 and 437, has reduced binding to Protein A.

[0173] Exemplary engineered cleavable Fc domains having cathepsin B or matriptase substrates In some embodiments, the engineered cleavable Fc domain comprises a non-MMP protease cleavage site of Table 11c. In one embodiment, the non-MMP protease is cathepsin B. In another embodiment, the non-MMP protease is matriptase. The engineered cleavable Fc domain may comprise any one set of the substitutions of Table 11c.

[0174] In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions between positions 436 and 447 according to EU numbering to incorporate a cleavage site for cathepsin B or matriptase.

[0175] In some embodiments, the cleavable peptide motif is located between positions 440 and 447 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 444 and 447 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 441 and 447 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 443 and 447 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 438 and 445 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 436 and 443 according to the EU numbering. In some embodiments, the cleavable peptide motif is located between positions 442 and 447 according to the EU numbering.

Table 4

[0176] In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions between positions 416 and 425 according to the EU numbering to incorporate the cleavage site of Table 11c. In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions between positions 426 and 437 according to the EU numbering to incorporate the cleavage site of Table 11c.

[0177] In some embodiments, the engineered cleavable Fc domain further comprises substitutions that enable heterodimerization of two Fc polypeptides. In some embodiments, the engineered cleavable Fc domain further comprises substitutions that can extend the half-life of the polypeptide comprising the engineered cleavable Fc domain.

[0178] In some embodiments, the engineered cleavable Fc domain contains the substitutions shown in Table 11c and includes serine at position 367 according to EU numbering. In some embodiments, the engineered cleavable Fc domain contains the substitutions shown in Table 11c and further includes a knob mutation. In some embodiments, the engineered cleavable Fc domain contains the substitutions shown in Table 11c and further includes a hole mutation.

[0179] In some embodiments, the engineered cleavable Fc domain further includes the Y349C; T366S; L368A; and Y407V substitutions. In some embodiments, the engineered cleavable Fc domain further includes the S354C and T366W substitutions. In some embodiments, the engineered cleavable Fc domain further includes the Y349C; T366S; L368A; Y407V, and N297A substitutions. In some embodiments, the engineered cleavable Fc domain further includes the S354C, T366W, and N297A substitutions. In some embodiments, the engineered cleavable Fc domain further includes the Y349C; T366S; L368A; Y407V, N297A, and I253A substitutions. In some embodiments, the engineered cleavable Fc domain further includes the S354C, T366W, N297A, and I253A substitutions.

[0180] Fc engineering for promoting heterodimerization or increasing half-life An Fc domain or fragment thereof capable of FcRn-mediated recycling can reduce or otherwise delay the clearance of a targeted cytokine from a subject, thereby extending the half-life of the administered targeted cytokine. In some embodiments, the cleavable Fc domain 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.

[0181] The cleavable Fc domain or fragment thereof may be derived from any antibody or fragment thereof. However, in some embodiments, either the first Fc polypeptide or the second Fc polypeptide may not bind to the FcRn receptor, such as a light chain polypeptide. For example, in some embodiments, the first Fc polypeptide does not directly interact with the FcRn receptor, yet the targeted cytokine has an extended half-life because it includes a second Fc polypeptide that can interact with the FcRn receptor, for example by including a heavy chain polypeptide. It is recognized in the art that FcRn-mediated recycling requires binding of an antibody or fragment thereof to the Fc region of the FcRn receptor. For example, studies have shown that residues I253, S254, H435, and Y436 (numbering 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) have also been investigated and reported. Yeung, Y.A., et al. (J. Immunol. 182 (2009) 7667-7671).

[0182] In some embodiments, the first and / or second Fc polypeptides of the cleavable Fc domain each include one or more modifications that promote non-covalent binding of the first and second Fc polypeptides. In some embodiments, the first Fc polypeptide includes an IgG1 Fc domain or fragment thereof that includes the mutations Y349C, T366S, L368A, and Y407V to form a "hole" in the first half-life extension domain, and the second Fc polypeptide includes an IgG1 Fc domain or fragment thereof that includes the mutations S354C and T366W to form a "knob" in the second half-life extension portion.

[0183] In some embodiments, the first and second Fc polypeptides are each an Fc domain or a fragment thereof of IgG1, IgG2, or IgG4. In some embodiments, the first and second Fc polypeptides are each an IgG1 Fc domain or a fragment thereof. Human IgG1 immunoglobulin heavy chain constant gamma 1 has the following sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 1)

[0184] In some embodiments, the first and second Fc polypeptides are derived from the sequence of human IgG1 immunoglobulin heavy chain constant gamma 1 having SEQ ID NO: 1 (the "parent sequence"), such that the first and second Fc polypeptides each comprise SEQ ID NO: 1 or a fragment thereof having one or more amino acid modifications.

[0185] In some embodiments, the first and Fc polypeptides each comprise the portion of SEQ ID NO: 1 shown in bold above, optionally having one or more amino acid modifications, namely, the following. DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2)

[0186] In some embodiments, the first and second Fc polypeptides comprise SEQ ID NO: 2 with amino substitutions that promote the association of the first and second Fc polypeptides according to the "knob-into-hole" approach. In some embodiments, the sequence of SEQ ID NO: 2 comprises the mutations Y349C; T366S; L368A; and Y407V (numbering according to the Kabat EU numbering system) to form a "hole" in the first Fc polypeptide, and the mutations S354C and T366W (numbering according to the Kabat EU numbering system) to form a "knob" in the second Fc polypeptide. These modified sequences have SEQ ID NOs: 3 and 4 shown below:

[0187] First Fc polypeptide (Y349C; T366S; L368A; and Y407V) SEQ ID NO: 3: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0188] Second Fc polypeptide (S354C and T366W) SEQ ID NO: 4: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0189] In some embodiments, the first and second half-life extension domains each further comprise an amino substitution N297A numbered by the Kabat EU numbering system.

[0190] The first Fc polypeptide (Y349C; T366S; L368A; Y407V, and N297A) SEQ ID NO: 5: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0191] The second Fc polypeptide (S354C, T366W, and N297A) SEQ ID NO: 6: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0192] In some embodiments, the first and second Fc polypeptides each further comprise an amino substitution I253A numbered by the Kabat EU numbering system.

[0193] In some embodiments, the first and second Fc polypeptides each further comprise both amino substitutions N297A and I253A numbered by the Kabat EU numbering system.

[0194] First Fc polypeptide (Y349C; T366S; L368A; Y407V, N297A, and I253A) SEQ ID NO: 7: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0195] Second Fc polypeptide (S354C, T366W, N297A, and I253A) SEQ ID NO: 8: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0196] In some embodiments, the first Fc 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 any one of the amino acid sequences of any one of SEQ ID NOs: 2-8.

[0197] In some embodiments, the second Fc 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 any one of the amino acid sequences of any one of SEQ ID NOs: 2-8.

[0198] In some embodiments, the first Fc polypeptide comprises an amino acid sequence having one or more modifications such as one or more amino acid substitutions, additions, or deletions as compared to any one of the amino acid sequences of any one of SEQ ID NOs: 2-8. In some embodiments, the second Fc polypeptide comprises an amino acid sequence having one or more modifications such as one or more amino acid substitutions, additions, or deletions as compared to any one of the amino acid sequences of any one of SEQ ID NOs: 2-8. The one or more modifications may be any modification or alteration described herein, and in some embodiments, include any modification or alteration that promotes heterodimerization of the polypeptide chains and / or inhibits homodimerization of the polypeptide chains, changes effector function, or enhances effector function.

[0199] In some embodiments, the protease cleavage site described herein can be introduced into any one of SEQ ID NOs: 2-8.

[0200] In some embodiments, the cleavable Fc domain may further comprise one or more amino acid substitutions that alter effector function. 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 following amino substitutions: 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 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 the following amino 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 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.

[0201] In some embodiments, the cleavable Fc domain further comprises one or more amino acid substitutions that enhance effector function. In some embodiments, the half-life extension domain is an IgG1 Fc domain or a fragment thereof and comprises the following amino acid substitutions: S298A, E333A, and K334A numbered according to the Kabat EU numbering system; 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; either K334V, Q311M, and A330M or A330F; K334V, S298T, and either A330M or A330F; K334V, S239D, and either A330M or S298T; L234Y, Y296W, and K290Y, F243V, or E294L; either Y296W and 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 any of S239D, A330Y, I332E, and K326E or K326T. In some embodiments, the cleavable Fc domain is an IgG1 Fc domain or a 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.;

[0202] In some embodiments, the cleavable Fc domain further 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 the Fc-containing polypeptide (e.g., the heavy chain polypeptide or the Fc domain or a 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: M428F; T250Q and M428F; M252Y, S254T, and T256E; P257I and N434H; D376V and N434H; P257I and Q3111; N434A; N434W; M428F and N434S; V259I and V308F; M252Y, S254T, and T256E; V259I, V308F and M428F; T307Q and N434A; T307Q and N434S; T307Q, E380A, and N434A; V308P and N434A; N434H; and V308P.

[0203] Knob-into-hole approach One strategy for promoting the heterodimerization of two Fc polypeptides is an approach called "knob-into-hole".

[0204] In some embodiments, the cleavable Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, each of which comprises a CH3 domain. In some embodiments, the Fc polypeptide comprising the CH3 domain is a heavy chain polypeptide or a fragment thereof (e.g., an Fc domain or a fragment thereof). The CH3 domains of the two Fc polypeptides can be altered by the "knob-into-hole" technique, which is described in detail in, for example, several examples of WO1996 / 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 the two CH3 domains are modified to increase the heterodimerization of two half-life extension domains containing the two modified CH3 domains. This occurs by introducing bulky residues within the CH3 domain of one of the half-life extension domains to act as a "knob". Next, a "hole" is formed in the other half-life extension domain that can accommodate the knob to accommodate the bulky residues. Either of the modified CH3 domains can be the "knob" and the other can be the "hole". The introduction of a disulfide bridge further stabilizes the heterodimer (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.

[0205] By introducing the S354C and T366W mutations into the heavy chain to create a "knob" and the Y349C, T366S, L368A, and Y407V mutations into the heavy chain to create a "hole", it has been reported that a heterodimerization yield exceeding 97% can be achieved (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.

[0206] In some embodiments comprising a first Fc polypeptide and a second Fc polypeptide, the first half-life Fc polypeptide comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) comprising the amino acid mutations S354C and T366W (numbered according to the Kabat EU numbering system), and the second Fc polypeptide comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) comprising the amino acid mutations Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU numbering system). In some embodiments comprising a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) comprising the amino acid mutations Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU numbering system), and the second Fc polypeptide comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) comprising the amino acid mutations S354C and T366W (numbered according to the Kabat EU numbering system).

[0207] As yet another example of substitutions that can be made to form the knob and hole, there is the one described in US20140302037A1, the content of which is incorporated herein by reference. For example, in some embodiments, any of the following amino acid substitutions can be made to a first Fc polypeptide (the "first domain") and a pairing second Fc polypeptide (the "second domain"), each containing 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 according to the Kabat EU numbering system.

[0208] In some embodiments, any of the following amino acid substitutions can be made to a first Fc polypeptide (the "first domain") and a paired second Fc polypeptide (the "second domain"), each containing an Fc domain: (a) Y407T of the second domain and T366Y of the first domain, (b) Y407A of the second domain and T366W of the first domain, (c) F405A of the second domain and T394W of the first domain, (d) F405W of the second domain and T394S of the first domain, (e) Y407T of the second domain and T366Y of the first domain, (f) T366Y and F405A of the second domain and T394W and Y407T of the first domain, (g) T366W and F405W of the second domain and T394S and Y407A of the first domain, (h) F405W and Y407A of the second domain and T366W and T394S of the first domain, or (i) T366W of the second domain and T366S, L368A, and Y407V of the first domain, numbered according to the Kabat EU numbering system.

[0209] In embodiments comprising a first Fc polypeptide and a second Fc polypeptide, each containing an Fc domain, any of the heterodimerization modifications described herein can be used in the Fc domain to promote heterodimerization of any of the targeted cytokines described herein.

[0210] RF mutations or CH3 domain swapping for heterodimeric protein purification Two immunoglobulin heavy chains that differ by at least one amino acid enable the isolation of antigen-binding proteins based on the differential affinity of the immunoglobulin heavy chain and the modified or mutant immunoglobulin heavy chain for an affinity reagent. Antigen-binding proteins with IgG CH2 and CH3 regions having different affinities for Protein A enable rapid isolation by the different binding of these IgG regions to Protein A.

[0211] In one embodiment, the second Fc polypeptide comprises a 95R modification in the CH3 region (according to IMGT exon numbering; 435R in EU numbering). In another embodiment, the second Fc polypeptide further comprises a 96F modification (according to IMGT; 436F in EU). In some embodiments, the first Fc polypeptide comprises wild-type CH2 and CH3 domains derived from IgG1 or IgG4, and the second Fc polypeptide comprises a 95R / 96F modification according to IMGT exon numbering. In some embodiments, the first Fc polypeptide comprises wild-type CH2 and CH3 domains derived from IgG1 or IgG4, and the second Fc polypeptide comprises a 435R / 436F modification according to EU numbering.

[0212] In some embodiments, the first Fc polypeptide comprises wild-type CH2 and CH3 domains derived from IgG1 or IgG4, and the second Fc polypeptide comprises a CH3 domain derived from IgG3.

[0213] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 9. SEQ ID NO: 9 comprises a "knob mutation" together with the CH3 domain derived from IgG3. DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREELTKNQVSLWCLVKGFYPSDIAVEWESSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGGSPG (SEQ ID NO: 9)

[0214] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 10. SEQ ID NO: 10 comprises a "knob mutation" together with an "RF mutation (435R / 436F). DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGSPG (SEQ ID NO: 10)

[0215] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 11. DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 11)

[0216] In some embodiments, the protease cleavage site described herein may be introduced into any one of SEQ ID NOs: 9-11.

[0217] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 91% identity to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 92% identity to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 93% identity to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 94% identity to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 11.

[0218] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 91% identity to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 92% identity to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 93% identity to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 94% identity to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 5.

[0219] Masked or targeted cytokine According to the present disclosure, an engineered cleavable Fc domain comprising a tumor-associated protease cleavage site can be fused to a masked cytokine comprising a cytokine molecule and a masking moiety. Alternatively, an engineered cleavable Fc domain comprising a tumor-associated protease cleavage site can be fused to a targeted cytokine comprising a cytokine molecule, a masking moiety, and a targeting moiety. For example, upon cleavage of the Fc domain at the protease cleavage site, the masked and targeted cytokines are released and activated at the site of the disease, and can specifically target the cells of interest for effective treatment of cancer without causing unwanted side effects.

[0220] In some embodiments, the cytokine is linked to an engineered cleavable Fc domain comprising a tumor-associated protease cleavage site via a non-cleavable linker. In some embodiments, the masking moiety is linked to the engineered cleavable Fc domain via a non-cleavable linker. In some embodiments, the targeting moiety is linked to the engineered Fc domain, with or without a non-cleavable linker.

[0221] In other embodiments, the engineered cleavable Fc domain of the invention is directly linked to a cytokine, a masking moiety, or a targeting moiety without a linker.

[0222] Cytokine The immune system is long-range in communication and is designed to respond rapidly, specifically, and comprehensively to protect organisms from foreign invaders and diseases. The protein cytokine superfamily is an integral part of the intercellular signaling network and is essential for the generation and regulation of the immune system. These interacting biological signals have significant capabilities, such as affecting growth and development, hematopoiesis, lymphocyte recruitment, differentiation of T cell subsets, and inflammation.

[0223] Cytokines can be part of a larger immune program, such as part of T cell subset differentiation. Mature CD4 and CD8 T cells leave the thymus with a naive phenotype and produce various cytokines. In the periphery, these T cells encounter antigen-presenting cells (APCs) that present either major histocompatibility complex (MHC) class I molecules (which present peptides generated in the cytosol to CD8 T cells) or MHC class II molecules (which present peptides degraded in intracellular vesicles to CD4 T cells). After activation, CD4 T helper (Th) cells can be classified into two major subpopulations in mice and humans based on their characteristic cytokine and chemokine secretion profiles. Th1 cells mainly secrete IL-2, interferon-γ (IFN-γ), and tumor necrosis factor-β (TNF-β), while Th2 cells mainly secrete IL-4, IL-5, IL-6, IL-10, and IL-13. Th1 cells support cell-mediated immunity and as a result promote inflammation, cytotoxicity, and delayed-type hypersensitivity (DTH). Th2 cells support humoral immunity and play a role in downregulating the inflammatory effects of Th1 cells. This paradigm is an excellent example of an integrated biological network and is very useful for simplifying the understanding of typical immune responses and immune responses that alter pathogenicity. For example, a failure in the transmission of "self" can lead to a loss of tolerance to our own antigens and potentially promote destructive immune responses against self-tissues and autoimmune diseases. Autoimmunity, which is the main focus of this document, is the mechanism underlying a series of conditions such as type 1 diabetes, multiple sclerosis, and rheumatoid arthritis. Autoimmune diseases can be caused in part by cytokine- and chemokine-mediated dysregulation of Th cell subset differentiation. Except for the situation in which antigen and co-stimulatory signals are presented, the main factors affecting the development of Th subsets are cytokines and chemokines in the stimulatory environment. A better understanding of the properties and interactions of individual cytokines and chemokines that play a role in Th cell activation can lead to more advanced treatments for autoimmune diseases.

[0224] The targeted cytokine of the present invention may include any cytokine or its variant known in the art. For example, reference is made to Cameron MJ, Kelvin DJ. Cytokines, Chemokines and Their Receptors. Madame Curie Bioscience Database. Austin (TX): Landes Bioscience; 2000 - 2013, the content of which is incorporated in its entirety. For example, the cytokine incorporated into the targeted cytokine may be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-33, TNF-α, TNF-β, CXCL8 (IL-8), G-CSF, GM-CSF, LIF, OSM, IFN-α, IFN-β, IFN-γ, CD154, LT-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β, M-CSF, or MSP, or a fragment thereof.

[0225] In some embodiments, the cytokine is IL-1 or a variant thereof. In some embodiments, the cytokine is IL-2 or a variant thereof. In some embodiments, the cytokine is IL-3 or a variant thereof. In some embodiments, the cytokine is IL-4 or a variant thereof. In some embodiments, the cytokine is IL-5 or a variant thereof. In some embodiments, the cytokine is IL-6 or a variant thereof. In some embodiments, the cytokine is IL-7 or a variant thereof. In some embodiments, the cytokine is IL-9 or a variant thereof. In some embodiments, the cytokine is IL-10 or a variant thereof. In some embodiments, the cytokine is IL-11 or a variant thereof. In some embodiments, the cytokine is IL-12 or a variant thereof. In some embodiments, the cytokine is IL-13 or a variant thereof. In some embodiments, the cytokine is IL-14 or a variant thereof. In some embodiments, the cytokine is IL-15 or a variant thereof. In some embodiments, the cytokine is IL-16 or a variant thereof. In some embodiments, the cytokine is IL-17 or a variant thereof. In some embodiments, the cytokine is IL-18 or a variant thereof. In some embodiments, the cytokine is IL-20 or a variant thereof. In some embodiments, the cytokine is TNF-α or a variant thereof. In some embodiments, the cytokine is TNF-β or a variant thereof. In some embodiments, the cytokine is CXCL8 (IL-8) or a variant thereof. In some embodiments, the cytokine is G-CSF or a variant thereof. In some embodiments, the cytokine is GM-CSF or a variant thereof. In some embodiments, the cytokine is LIF or a variant thereof. In some embodiments, the cytokine is OSM or a variant thereof. In some embodiments, the cytokine is IFN-α or a variant thereof. In some embodiments, the cytokine is IFN-β or a variant thereof. In some embodiments, the cytokine is IFN-γ or a variant thereof.In some embodiments, the cytokine is CD154 or a variant thereof. In some embodiments, the cytokine is LT-β or a variant thereof. In some embodiments, the cytokine is 4-1BBL or a variant thereof. In some embodiments, the cytokine is APRIL or a variant thereof. In some embodiments, the cytokine is CD153 or a variant thereof. In some embodiments, the cytokine is CD70 or a variant thereof. In some embodiments, the cytokine is CD178 or a variant thereof. In some embodiments, the cytokine is GITRL or a variant thereof. In some embodiments, the cytokine is LIGHT or a variant thereof. In some embodiments, the cytokine is OX40L or a variant thereof. In some embodiments, the cytokine is TALL-1 or a variant thereof. In some embodiments, the cytokine is TRAIL or a variant thereof. In some embodiments, the cytokine is TWEAK or a variant thereof. In some embodiments, the cytokine is TRANCE or a variant thereof. In some embodiments, the cytokine is TGF-β or a variant thereof. In some embodiments, the cytokine is M-CSF or a variant thereof. In some embodiments, the cytokine is MSP or a variant thereof.

[0226] Interleukin 2 (IL-2) IL-2 cytokines or functional fragments thereof for use with a targeted cytokine or a cleavage product thereof are provided herein. Cytokines play a role in cell signaling, particularly in cells of the immune system. IL-2 is an interleukin and is one of the cytokine signaling molecules of the immune system that regulate the activity of white blood cells. Suitable IL-2 cytokines for use in the present invention can be any IL-2 or a functional fragment thereof. In some embodiments, the IL-2 is native IL-2, IL-2 containing one or more substitutions (e.g., an IL-2 mutein, or an IL-2 variant), or a truncated IL-2. In some embodiments, the IL-2 is a polypeptide that retains at least one property of IL-2 biological activity.

[0227] In some embodiments, the IL-2 is native IL-2. In some embodiments, the IL-2 contains a C125A substitution of mature IL-2 (SEQ ID NO: 13).

[0228] In some embodiments, the amino acid substitution reduces the affinity of the IL-2 polypeptide or a functional fragment thereof for CD25 (IL-2Rα).

[0229] In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence that results from introducing into the amino acid sequence of the IL-2 polypeptide or a functional fragment thereof one or more amino acid substitutions that increase the affinity of the IL-2 polypeptide or a functional fragment thereof for IL-2Rβ or IL-2Rγ. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions that enhance the affinity of the IL-2 polypeptide or a functional fragment thereof for IL-2Rβ (CD122) as compared to the amino acid sequence of wild-type IL-2. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions that reduce the affinity of the IL-2 polypeptide or a functional fragment thereof for IL-2Rα (CD25) as compared to the amino acid sequence of wild-type IL-2, and one or more amino acid substitutions that enhance the affinity of the IL-2 polypeptide or a functional fragment thereof for IL-2R (CD122) as compared to the amino acid sequence of wild-type IL-2.

[0230] In some embodiments, IL-2 binds to IL-2Rα with an affinity equal to or greater than that of wild-type IL-2. In some embodiments, IL-2 preferentially binds to CD25 (e.g., alpha-biased). In some embodiments, IL-2 has a reduced affinity for CD122 and / or CD132. In some embodiments, IL-2 comprises N88D and C125A relative to SEQ ID NO: 13.

[0231] In eukaryotic cells, native IL-2 is synthesized as a 153 amino acid precursor polypeptide having SEQ ID NO: 12. MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 12)

[0232] Next, it is processed into mature IL-2 by removing amino acid residues 1 to 20. Thereby, a mature form of IL-2 consisting of 133 amino acids (amino acid residues 21 to 153) having the sequence shown by SEQ ID NO: 13 is obtained. APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 13)

[0233] A "functional fragment" of an IL-2 cytokine comprises a portion of a full-length cytokine protein that retains or has an altered cytokine receptor binding ability (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the full-length cytokine protein). Cytokine receptor binding ability can be indicated, for example, by the ability of a cytokine to bind to its cognate receptor or a component thereof (e.g., one or more chains of a heterotrimeric receptor complex).

[0234] In some embodiments, the IL-2 cytokine or a functional fragment thereof is any native interleukin-2 (IL-2) protein or a modified variant thereof that can bind to the interleukin-2 receptor, particularly the IL-2Rα chain. In the context of IL-2 cytokine binding, the target protein can be IL-2R (including the IL-2Rα chain, IL-2Rβ chain, and IL-2Rγ chain), the IL-2Rα chain, the IL-2Rβ chain, or an IL-2Rα / β dimer complex. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises the amino acid sequence of amino acid residues 21 to 153 of SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises SEQ ID NO: 13, which is the amino acid sequence of mature IL-2.

[0235] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO: 13. Each of the at least one amino acid modification can be any amino acid modification such as a substitution, insertion, or deletion. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having 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 as compared to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 5 amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 13.

[0236] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 91% sequence identity with SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 92% sequence identity with SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 93% sequence identity with SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 94% sequence identity with SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 96% sequence identity with SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 13.

[0237] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions that decrease the affinity of the IL-2 peptide or a functional fragment thereof for IL-2Rα (CD25) as compared to the amino acid sequence of wild-type IL-2 of SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 13 such that one or more amino acid residues 38, 42, 45, and 62 are alanine (A). In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 13 such that amino acid residues 38, 42, 45, and 62 are alanine (A).

[0238] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises the amino acid sequence substitution C125A as compared to the amino acid sequence of SEQ ID NO: 13.

[0239] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 13 such that amino acid residues 38, 42, 45, and 62 are alanine (A) and amino acid residue 125 is alanine (A). In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid residues R38, F42, Y45, and E62 substituted with alanine in the amino acid sequence of SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid residues R38, F42, Y45, and E62 substituted with alanine (A) and amino acid residue C125 substituted with alanine (A) in the amino acid sequence of SEQ ID NO: 13.

[0240] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 14. APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 14)

[0241] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 92% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 14.

[0242] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 13, such that amino acid residue 88 is aspartic acid (D) and amino acid residue 125 is alanine (A). In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid residue N88 substituted for the aspartic acid in the amino acid sequence of SEQ ID NO: 13.

[0243] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 118. APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 118)

[0244] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 118. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 118. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 118. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 118. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 92% identity to SEQ ID NO: 118. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 118. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 118. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 118.

[0245] In some embodiments, the IL-2 cytokine or a functional fragment thereof has one or more amino acid residues removed, e.g., residues 1-3, compared to the amino acid sequence of mature IL-2 of SEQ ID NO: 13, for the purpose of removing O-glycosylation sites. In some embodiments, the IL-2 cytokine or a functional fragment thereof has one or more amino acid residues substituted compared to the amino acid sequence of mature IL-2 of SEQ ID NO: 13 for the purpose of removing O-glycosylation sites. In some embodiments, the IL-2 cytokine or a functional fragment thereof has one or more amino acid residues inserted, e.g., in the region of residues 1-3, compared to the amino acid sequence of mature IL-2 of SEQ ID NO: 13 for the purpose of removing O-glycosylation sites. In some embodiments, the IL-2 cytokine or a functional fragment thereof does not have an O-glycosylation site within residues 1-3.

[0246] Interleukin 15 (IL-15) Provided herein is an IL-15 cytokine or a functional fragment thereof for use in a targeted cytokine or a cleavage product thereof. Cytokines play a role in cell signaling, particularly in cells of the immune system. IL-15 is an interleukin and is a type of cytokine signaling molecule of the immune system that regulates the activity of white blood cells.

[0247] In eukaryotic cells, IL-15 is synthesized as a 162-amino acid precursor polypeptide (SEQ ID NO: 15) and is then processed to mature IL-15 by removal of amino acid residues 1-48. This results in the mature form of IL-15 consisting of 114 amino acids (amino acid residues 49-162) that is secreted in its mature, active form (see SEQ ID NO: 16).

[0248] IL-15 precursor polypeptide (SEQ ID NO: 15): MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0249] IL-15 mature polypeptide (SEQ ID NO: 16): NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0250] As used herein, the term "IL-15" or "IL-15 polypeptide" refers to any interleukin-15 (IL-15) protein, or a functional fragment or variant thereof. This term encompasses any native IL-15 derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., rats and mice). This 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 resulting from intracellular processing (e.g., the mature form of IL-15 consisting of amino acid residues 49-162). Thus, this term encompasses the protein encoded by the amino acid sequence of SEQ ID NO: 16, as well as sequence variants thereof. This term also encompasses natural variants of IL-15. This term also encompasses non-natural variants of IL-15, such as variants caused by cleavage, deletion, forms in which IL-15 is linked to another molecule, and at least one amino acid change (e.g., by substitution, addition, or deletion) to the amino acid sequence. In some embodiments, a variant or homolog has 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., a 50-, 100-, or 114-amino acid contiguous portion), compared to a native IL-15 polypeptide such as the IL-15 polypeptide encoded by the amino acid sequence of SEQ ID NO: 15 or 16. Thus, the term "IL-15" or "IL-15 polypeptide" includes an IL-15 protein comprising the amino acid sequence of SEQ ID NO: 15 or 16, and variants thereof, such as variants generated by one or more amino acid substitutions to the amino acid sequence of SEQ ID NO: 15 or 16.

[0251] A "functional fragment" of an IL-15 cytokine comprises a portion of a full-length cytokine protein that retains or has an altered cytokine receptor binding ability (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity as compared to the full-length cytokine protein). Cytokine receptor binding ability can be shown, for example, by the ability of the cytokine to bind to its cognate receptor or a component thereof (e.g., one or more chains of a heterotrimeric receptor complex).

[0252] In some embodiments, an IL-15 cytokine or a functional fragment thereof is any natural interleukin-2 (IL-15) protein or a modified variant thereof that can bind to the interleukin-2 receptor, particularly the IL-15Rα chain.

[0253] In some embodiments, an IL-15 cytokine or a fragment thereof comprises SEQ ID NO: 16 or a functional fragment thereof.

[0254] In some embodiments, an IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 16.

[0255] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO: 16. Each of the at least one amino acid modification can be any amino acid modification such as a substitution, insertion, or deletion. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having 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 as compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 5 amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 16.

[0256] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 16.

[0257] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 16.

[0258] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22, E46, and E53 as compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22, E46, E53, N71, N79, and N112 as compared to the amino acid sequence of SEQ ID NO: 16.

[0259] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position D22 as compared to the amino acid sequence of SEQ ID NO: 16.

[0260] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position E46 as compared to the amino acid sequence of SEQ ID NO: 16.

[0261] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position E53 as compared to the amino acid sequence of SEQ ID NO: 16.

[0262] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N71 as compared to the amino acid sequence of SEQ ID NO: 16.

[0263] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N79 as compared to the amino acid sequence of SEQ ID NO: 16.

[0264] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N112 as compared to the amino acid sequence of SEQ ID NO: 16.

[0265] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions E46 and E53 as compared to the amino acid sequence of SEQ ID NO: 16.

[0266] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71 and N79 as compared to the amino acid sequence of SEQ ID NO: 16.

[0267] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71 and N112 as compared to the amino acid sequence of SEQ ID NO: 16.

[0268] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N79 and N112 as compared to the amino acid sequence of SEQ ID NO: 16.

[0269] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71, N79, and N112 as compared to the amino acid sequence of SEQ ID NO: 16.

[0270] In some embodiments, the amino acid substitution at position D22 is D22A. In some embodiments, the amino acid substitution at position E46 is E46A. In some embodiments, the amino acid substitution at position E46 is E46R. In some embodiments, the amino acid substitution at position E46 is E46S. In some embodiments, the amino acid substitution at position E53 is E53A, E53R, or E53S. In some embodiments, the amino acid substitution at position N71 is N71Q. In some embodiments, the amino acid substitution at position N79 is N79Q. In some embodiments, the amino acid substitution at position N112 is N112Q.

[0271] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having the amino acid substitution D22A as compared to the amino acid sequence of SEQ ID NO: 16.

[0272] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 17. NWVNVISDLKKIEDLIQSMHIAATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 17)

[0273] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 17.

[0274] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having the amino acid substitution E46A compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 18. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLALQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 18)

[0275] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0276] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46A and E53A as compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 19. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLALQVISLASGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 19)

[0277] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46R and E53R as compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 20. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLRLQVISLRSGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 20)

[0278] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46S and E53S as compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLSLQVISLSSGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 21)

[0279] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitution E53A as compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLASGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 22)

[0280] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 23 and the amino acid sequence of SEQ ID NO: 24. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23, 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: 24. NWVNVISDLKKIEDLIQS (SEQ ID NO: 23) KVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 24)

[0281] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having the amino acid substitution N71Q as compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 25. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAQNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 25)

[0282] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution N79Q as compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 26. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 26)

[0283] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution N112Q as compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 27. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS (SEQ ID NO: 27)

[0284] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions N71Q and N79Q as compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 28. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAQNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 28)

[0285] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions N71Q and N112Q as compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 29. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAQNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS (SEQ ID NO: 29)

[0286] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions N79Q and N112Q as compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 30. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS (SEQ ID NO: 30)

[0287] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions N71Q, N79Q, and N112Q as compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 31. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAQNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS (SEQ ID NO: 31)

[0288] In some embodiments, additional mutations may be included in any of the above sequences at position N71. In some embodiments, the mutation is N71A, N71R, N71W, N71F, N71P, N71M, N71L, N71T, N71S, or N71Y.

[0289] In some embodiments, additional mutations may be included in any of the above sequences at position S73. In some embodiments, the mutation is S73A, S73W, S73V, or S73M.

[0290] In some embodiments, additional mutations may be included in any of the above sequences at one or more amino acid positions N72, N79, V80, T81, and N112. In some embodiments, one or more additional mutations selected from N72A, N79A, V80A, T81A, and N112R may be included in any of the above sequences.

[0291] In some embodiments, additional mutations may be included in any of the above sequences at one or more amino acid positions N72, S73, N79, V80, T81, and N112. In some embodiments, one or more additional mutations N72A, S73A, N79A, V80A, T81A, and N112 may be included in any of the above sequences.

[0292] In some embodiments, the IL-15 cytokine or a functional fragment thereof has one or more amino acid residues removed, e.g., residues 1-3, compared to the amino acid sequence of mature IL-15 of SEQ ID NO: 16, for the purpose of removing O-glycosylation sites. In some embodiments, the IL-15 cytokine or a functional fragment thereof has one or more amino acid residues substituted compared to the amino acid sequence of mature IL-15 of SEQ ID NO: 16 for the purpose of removing O-glycosylation sites. In some embodiments, the IL-15 cytokine or a functional fragment thereof has one or more amino acid residues inserted, e.g., in the region of residues 1-3, compared to the amino acid sequence of mature IL-15 of SEQ ID NO: 16 for the purpose of removing O-glycosylation sites. In some embodiments, the IL-15 cytokine or a functional fragment thereof does not have an O-glycosylation site within residues 1-3.

[0293] Interleukin 12 (IL-12) Provided herein is an IL-12 cytokine or a functional fragment thereof for use in a targeted cytokine or a cleavage product thereof. Cytokines play a role in cell signaling, particularly in cells of the immune system. IL-12 is an interleukin and is a type of cytokine signaling molecule of the immune system that regulates the activity of white blood cells.

[0294] Endogenous IL-12 exists as two different molecules, IL-12 p40 and IL-12 p35, which dimerize intracellularly during biosynthesis.

[0295] The full sequences of IL-12 p40 and IL-12 p35 are as follows (the propeptides cleaved during biosynthesis are shown in bold): IL-12 p40 subunit: MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 32) IL-12 p35 subunit: MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 33)

[0296] The mature form is as follows. IL-12 p40 subunit: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 34) IL-12 p35 subunit: RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 35)

[0297] These are expressed as two chains that dimerize covalently during biosynthesis via a disulfide bond between the two subunits: Cysteine C199 of the p40 subunit is associated with Cysteine C96 of the p35 subunit.

[0298] A "functional fragment" of an IL-12 cytokine comprises a portion of the full-length cytokine protein that retains or has an altered cytokine receptor binding ability (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity compared to the full-length cytokine protein). Cytokine receptor binding ability can be shown, for example, by the ability of the cytokine to bind to its cognate receptor or a component thereof.

[0299] In some embodiments, the IL-12 cytokine or a functional fragment thereof is any natural interleukin-2 (IL-12) protein or a modified variant thereof that is capable of binding to the interleukin-12 receptor.

[0300] In some embodiments, the IL-12 polypeptide or a functional fragment thereof comprises an IL-12p40 polypeptide or a functional fragment thereof covalently attached to an IL-12p35 polypeptide or a functional fragment thereof.

[0301] The IL-12p40 polypeptide or a functional fragment thereof may be attached to a first half-life extension domain such that the first polypeptide chain has the formula: N’HL1-L1-MM C’ and the second polypeptide chain has the formula: N’HL2-L2-[IL-12p40-linker-IL-12p35]C’ wherein “IL-12p40” is an IL-12p40 polypeptide or a functional fragment thereof and “IL-12p35” is an IL-12p35 polypeptide or a functional fragment thereof.

[0302] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 34. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having at least one amino acid modification compared to the amino acid sequence of SEQ ID NO: 34. Each of the at least one amino acid modification can be any amino acid modification such as a substitution, insertion, or deletion. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having 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 compared to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 5 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 34.

[0303] The IL-12p40 polypeptide contains a glycosaminoglycan (GAG) binding domain. GAGs such as heparin and heparan sulfate have been shown to bind to a number of growth factors and cytokines including IL-12. The physiological significance of this binding consists of two elements. First, GAGs can serve as a coreceptor on the cell surface and maintain a local high concentration of cytokines. Second, GAGs can regulate the biological activities of growth factors and cytokines through multiple mechanisms including dimerization and protection from proteolysis.

[0304] The GAG binding domain of the mature form of the IL-12 p40 subunit is shown in bold below. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 36)

[0305] Modifications to the GAG binding domain KSKREKKDRV (SEQ ID NO: 37) have been shown herein to increase the PK profile of constructs containing an IL-12 cytokine with a mutated GAG binding domain without any accompanying decrease in cytokine activity. Thus, in some embodiments, the IL-12p40 polypeptide contains at least one amino acid modification to the GAG binding domain. In some embodiments, the modification to the GAG binding domain is a deletion mutation. In some embodiments, the modification to the GAG binding domain is a deletion mutation and at least one substitution mutation.

[0306] In some embodiments, the GAG binding domain comprises the amino acid sequence KDNTERV. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 38. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTERVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 38)

[0307] In some embodiments, the GAG binding domain comprises the amino acid sequence KDNTEGRV. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 39. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 39)

[0308] In some embodiments, the GAG binding domain consists of the amino acid sequence KDNTERV. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the GAG binding domain consists of the amino acid sequence KDNTEGRV. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 39.

[0309] In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having one or more cysteine substitutions as compared to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having an amino acid substitution at position C252 as compared to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the amino acid substitution at position C252 is C252S. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the IL-12p40 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: 40. In some embodiments, the IL-12p40 polypeptide consists of the amino acid sequence of SEQ ID NO: 40. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEF GDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTC WWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAA EESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTW STPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEW ASVPCS (SEQ ID NO: 40)

[0310] In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having one or more cysteine substitutions compared to the amino acid sequence of SEQ ID NO: 34 and at least one amino acid modification to the GAG-binding domain. In some embodiments, the IL-12p40 polypeptide comprises an amino acid substitution at the C252S position compared to the amino acid sequence of SEQ ID NO: 34, and the GAG-binding domain comprises the amino acid sequence KDNTERV. In some embodiments, the IL-12p40 polypeptide comprises an amino acid substitution at the C252S position compared to the amino acid sequence of SEQ ID NO: 34, and the GAG-binding domain comprises the amino acid sequence KDNTEGRV (SEQ ID NO: 41). In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, the IL-12p40 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: 42. In some embodiments, the IL-12p40 polypeptide consists of the amino acid sequence of SEQ ID NO: 42. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 42)

[0311] In some embodiments, the IL-12p35 polypeptide comprises SEQ ID NO: 35. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having at least one amino acid modification compared to the amino acid sequence of SEQ ID NO: 35. Each of the at least one amino acid modification can be any amino acid modification such as substitution, insertion, or deletion. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having 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 compared to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 5 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 35.

[0312] In some embodiments, the IL-12p40-IL-12p35 linker is 5 to 20 amino acids in length.

[0313] In some embodiments, the IL-12p40-IL-12p35 linker is rich in amino acid residues G and S.

[0314] In some embodiments, the IL-12p40-IL-12p35 linker comprises only amino acid residue types selected from the group consisting of G and S.

[0315] In some embodiments, the IL-12p40-IL-12p35 linker comprises [(G)nS], wherein n = 4 or 5.

[0316] In some embodiments, the IL-12p40-IL-12p35 linker comprises the (GGGGS) (SEQ ID NO: 43) repeat.

[0317] In some embodiments, the IL-12p40-IL-12p35 linker comprises SEQ ID NO: 44. (GGGGSGGGGSGGGGS)

[0318] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO: 45. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having at least one amino acid modification as compared to the amino acid sequences of SEQ ID NOs: 34 and 35. Each of the at least one amino acid modification can be any amino acid modification such as a substitution, insertion, or deletion. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having 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 as compared to the amino acid sequences of SEQ ID NOs: 34 and 35. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 5 amino acid substitutions as compared to the amino acid sequences of SEQ ID NOs: 34 and 35. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMA (SEQ ID NO: 45)

[0319] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 45.

[0320] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 46. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTERVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 46)

[0321] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 47. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 47)

[0322] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 48. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 48)

[0323] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 49. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALC (SEQ ID NO: 49)

[0324] Masking portion Masking portions for use with masked or targeted cytokines, or any other masked therapeutic active molecule, are provided herein. It will be understood that the masking portion is cleaved from the targeted cytokine to form its cleavage product. The masking portion binds to the cytokine portion and inhibits the biological activity of the cytokine. Upon cleavage, the masking portion is released from the cytokine and activates the function of the cytokine at the desired target.

[0325] In some embodiments, the masking portion comprises an agent, peptide, or polypeptide that binds to the cytokine. In some embodiments, the masking portion comprises a cyclic peptide that binds to the cytokine. In some embodiments, the masking portion comprises a linear peptide that binds to the cytokine.

[0326] In some embodiments, the masking moiety comprises a Fab, a single-chain Fv (scFv), a single-domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibody (bsFab), a single-domain antibody-binding Fab (s-Fab), an antibody, or a combination thereof. In some embodiments, the masking moiety comprises a Fab that binds to a cytokine. In some embodiments, the masking moiety comprises a single-chain Fv (scFv) that binds to a cytokine. In some embodiments, the masking moiety comprises a single-domain antibody (VHH) that binds to a cytokine. In some embodiments, the masking moiety comprises one or more CDRs that bind to a cytokine. In some embodiments, the masking moiety comprises a variable heavy chain (VH) that binds to a cytokine. In some embodiments, the masking moiety comprises a variable light chain (VL) that binds to a cytokine. In some embodiments, the masking moiety comprises a Fab-like bispecific antibody (bsFab) that binds to a cytokine. In some embodiments, the masking moiety comprises a single-domain antibody-binding Fab (s-Fab) that binds to a cytokine. In some embodiments, the masking moiety comprises an antibody or a fragment thereof that binds to a cytokine. In some embodiments, the masking moiety comprises an antibody against a cytokine, or a binding fragment of such an antibody.

[0327] In some embodiments, the masking moiety is a receptor for the cytokine. In some embodiments, the masking moiety is a fragment of a receptor for the cytokine. In some embodiments, the masking moiety comprises the extracellular domain (ECD) of a receptor for the cytokine.

[0328] In some embodiments, the cytokine is IL-1α or IL-1β, and the masking moiety is a Fab, a single-chain Fv (scFv), or a single-domain antibody against IL-1α or IL-1β. In some embodiments, the cytokine is IL-1α or IL-1β, and the masking moiety is CD121a, CDw121b, or a fragment thereof.

[0329] In some embodiments, the cytokine is IL-2, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-2. In some embodiments, the cytokine is IL-2, and the masking moiety is IL-2Rα, IL-2Rβ, CD25, CD122, CD132, or a fragment thereof.

[0330] In some embodiments, the cytokine is IL-18, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-18. In some embodiments, the cytokine is IL-18, and the masking moiety is IL-18Rα, IL-18Rβ, or a fragment thereof.

[0331] In some embodiments, the cytokine is IL-4, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-4. In some embodiments, the cytokine is IL-4, and the masking moiety is CD124, CD213a13, CD132, or a fragment thereof.

[0332] In some embodiments, the cytokine is IL-7, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-7. In some embodiments, the cytokine is IL-7, and the masking moiety is CD127, CD132, or a fragment thereof.

[0333] In some embodiments, the cytokine is IL-9, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-9. In some embodiments, the cytokine is IL-9, and the masking moiety is IL-9R, CD132, or a fragment thereof.

[0334] In some embodiments, the cytokine is IL-13 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-13. In some embodiments, the cytokine is IL-13 and the masking moiety is CD213a1, CD213a2, or a fragment thereof.

[0335] In some embodiments, the cytokine is IL-15 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-15. In some embodiments, the cytokine is IL-15 and the masking moiety is IL-15Ra, CD122, CD132, or a fragment thereof.

[0336] In some embodiments, the cytokine is IL-3 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-3. In some embodiments, the cytokine is IL-3 and the masking moiety is CD123, CDw131, or a fragment thereof.

[0337] In some embodiments, the cytokine is IL-5 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-5. In some embodiments, the cytokine is IL-5 and the masking moiety is CDw125, CD131, or a fragment thereof.

[0338] In some embodiments, the cytokine is GM-CSF and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against GM-CSF. In some embodiments, the cytokine is GM-CSF and the masking moiety is CD116, CDw131, or a fragment thereof.

[0339] In some embodiments, the cytokine is IL-6 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-6. In some embodiments, the cytokine is IL-6 and the masking moiety is CD126, CD130, or a fragment thereof.

[0340] In some embodiments, the cytokine is IL-11, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-11. In some embodiments, the cytokine is IL-11, and the masking moiety is IL-11Ra, CD130, or a fragment thereof.

[0341] In some embodiments, the cytokine is G-CSF, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against G-CSF. In some embodiments, the cytokine is G-CSF, and the masking moiety is CD114 or a fragment thereof.

[0342] In some embodiments, the cytokine is IL-12, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-12. In some embodiments, the cytokine is IL-12, and the masking moiety is CD212 or a fragment thereof.

[0343] In some embodiments, the cytokine is LIF, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against LIF. In some embodiments, the cytokine is LIF, and the masking moiety is LIFR, CD130, or a fragment thereof.

[0344] In some embodiments, the cytokine is OSM, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against OSM. In some embodiments, the cytokine is OSM, and the masking moiety is OSMR, CD130, or a fragment thereof.

[0345] In some embodiments, the cytokine is IL-10, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-10. In some embodiments, the cytokine is IL-10, and the masking moiety is CDw210 or a fragment thereof.

[0346] In some embodiments, the cytokine is IL-20, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-20. In some embodiments, the cytokine is IL-20, and the masking moiety is IL-20Rα, IL-20Rβ, or a fragment thereof.

[0347] In some embodiments, the cytokine is IL-14, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-14. In some embodiments, the cytokine is IL-14, and the masking moiety is IL-14R or a fragment thereof.

[0348] In some embodiments, the cytokine is IL-16, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-16. In some embodiments, the cytokine is IL-16, and the masking moiety is CD4 or a fragment thereof.

[0349] In some embodiments, the cytokine is IL-17, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-17. In some embodiments, the cytokine is IL-17, and the masking moiety is CDw217 or a fragment thereof.

[0350] In some embodiments, the cytokine is IFN-α, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IFN-α. In some embodiments, the cytokine is IFN-α, and the masking moiety is CD118 or a fragment thereof.

[0351] In some embodiments, the cytokine is IFN-β, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IFN-β. In some embodiments, the cytokine is IFN-β, and the masking moiety is CD118 or a fragment thereof.

[0352] In some embodiments, the cytokine is IFN-γ and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IFN-γ. In some embodiments, the cytokine is IFN-γ and the masking moiety is CDw119 or a fragment thereof.

[0353] In some embodiments, the cytokine is CD154 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against CD154. In some embodiments, the cytokine is CD154 and the masking moiety is CD40 or a fragment thereof.

[0354] In some embodiments, the cytokine is LT-β and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against LT-β. In some embodiments, the cytokine is LT-β and the masking moiety is LT-βR or a fragment thereof.

[0355] In some embodiments, the cytokine is TNF-α and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TNF-α. In some embodiments, the cytokine is TNF-α and the masking moiety is CD120a, CD120b, or a fragment thereof.

[0356] In some embodiments, the cytokine is TNF-β and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TMF-β. In some embodiments, the cytokine is TNF-β and the masking moiety is CD120a, CD120b, or a fragment thereof.

[0357] In some embodiments, the cytokine is 4-1BBL and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against 4-1BBL. In some embodiments, the cytokine is 4-1BBL and the masking moiety is CDw137, 4-1BB, or a fragment thereof.

[0358] In some embodiments, the cytokine is APRIL and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against APRIL. In some embodiments, the cytokine is APRIL and the masking moiety is BCMA, TACI, or a fragment thereof.

[0359] In some embodiments, the cytokine is CD70 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against CD70. In some embodiments, the cytokine is CD70 and the masking moiety is CD27 or a fragment thereof.

[0360] In some embodiments, the cytokine is CD153 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against CD153. In some embodiments, the cytokine is CD153 and the masking moiety is CD30 or a fragment thereof.

[0361] In some embodiments, the cytokine is CD178 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against CD178. In some embodiments, the cytokine is CD178 and the masking moiety is CD95 or a fragment thereof.

[0362] In some embodiments, the cytokine is GITRL and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against GITRL. In some embodiments, the cytokine is GITRL and the masking moiety is GITR or a fragment thereof.

[0363] In some embodiments, the cytokine is LIGHT, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against LIGHT. In some embodiments, the cytokine is LIGHT, and the masking moiety is LTβR, HVEM, or a fragment thereof.

[0364] In some embodiments, the cytokine is OX40, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against OX40. In some embodiments, the cytokine is OX40, and the masking moiety is OX40 or a fragment thereof.

[0365] In some embodiments, the cytokine is TALL-1, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TALL-1. In some embodiments, the cytokine is TALL-1, and the masking moiety is BCMA, TACI, or a fragment thereof.

[0366] In some embodiments, the cytokine is TRAIL, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TRAIL. In some embodiments, the cytokine is TRAIL, and the masking moiety is TRAILR1-4 or a fragment thereof.

[0367] In some embodiments, the cytokine is TWEAK, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TWEAK. In some embodiments, the cytokine is TWEAK, and the masking moiety is Apo3 or a fragment thereof.

[0368] In some embodiments, the cytokine is TRANCE, and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TRANCE. In some embodiments, the cytokine is TRANCE, and the masking moiety is RANK, OPG, or a fragment thereof.

[0369] In some embodiments, the cytokine is TGF-β1 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TGF-β1. In some embodiments, the cytokine is TGF-β1 and the masking moiety is TGF-βR1 or a fragment thereof.

[0370] In some embodiments, the cytokine is TGF-β2 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TGF-β2. In some embodiments, the cytokine is TGF-β2 and the masking moiety is TGF-βR2 or a fragment thereof.

[0371] In some embodiments, the cytokine is TGF-β3 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TGF-β3. In some embodiments, the cytokine is TGF-β3 and the masking moiety is TGF-βR3 or a fragment thereof.

[0372] In some embodiments, the cytokine is Epo and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against Epo. In some embodiments, the cytokine is Epo and the masking moiety is EpoR or a fragment thereof.

[0373] In some embodiments, the cytokine is Tpo and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against Tpo. In some embodiments, the cytokine is Tpo and the masking moiety is TpoR or a fragment thereof.

[0374] In some embodiments, the cytokine is Flt-3L and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against Flt-3L. In some embodiments, the cytokine is Flt-3L and the masking moiety is Flt-3 or a fragment thereof.

[0375] In some embodiments, the cytokine is SCF and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against SCF. In some embodiments, the cytokine is SCF and the masking moiety is CD117 or a fragment thereof.

[0376] In some embodiments, the cytokine is M-CSF and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against M-CSF. In some embodiments, the cytokine is M-CSF and the masking moiety is CD115 or a fragment thereof.

[0377] In some embodiments, the cytokine is MSP and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against MSP. In some embodiments, the cytokine is MSP and the masking moiety is CDw136 or a fragment thereof.

[0378] In some embodiments, the cytokine is an IL-15 agonist polypeptide and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-15. In some embodiments, the cytokine portion comprises an IL-15 agonist polypeptide and the chimeric molecule further comprises the sushi domain of IL-15 receptor a (IL-15Ra sushi domain).

[0379] In some embodiments, the cytokine is an IL-2 agonist polypeptide and the masking moiety is the extracellular domain of the IL-21 receptor a (IL-21Ra ECD) or a functional analog thereof. In some embodiments, the cytokine is an IL-2 agonist polypeptide or an IL-15 agonist polypeptide and the masking moiety is the extracellular domain of the IL-2 receptor b (IL-2RβECD). In some embodiments, the cytokine is an IL-21 agonist polypeptide and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-21.

[0380] In some embodiments, the cytokine is an IL-2 agonist polypeptide and the masking moiety is the extracellular domain of the IL-21 receptor a (IL-21Ra ECD) or a functional analog thereof. In some embodiments, the cytokine is an IL-2 agonist polypeptide or an IL-15 agonist polypeptide and the masking moiety is the extracellular domain of the IL-2 receptor b (IL-2RβECD). In some embodiments, the cytokine is an IL-21 agonist polypeptide and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-21.

[0381] Antibody or antigen-binding fragment The masking moiety masks the cytokine or therapeutic active domain in the masked cytokine or masked therapeutic active molecule, thereby reducing or preventing the binding of the cytokine or therapeutic active domain to its cognate receptor or ligand.

[0382] In some embodiments, the masking moiety is an antibody or an antigen-binding fragment thereof that prevents the therapeutic active domain from binding to its cognate ligand or receptor. In some embodiments, the masking moiety is an antibody or an antigen-binding fragment thereof that specifically binds to the therapeutic active domain. In some embodiments, the masking moiety is an antibody or an antigen-binding fragment thereof that prevents the cytokine from binding to its cognate ligand or receptor. In some embodiments, the masking moiety is an antibody or an antigen-binding fragment thereof that specifically binds to the cytokine.

[0383] In some embodiments, the masking moiety comprises an antibody or an antigen-binding fragment thereof that specifically binds to a therapeutic active domain. In some embodiments, the masking moiety comprises an antibody or an antigen-binding fragment thereof that specifically binds to a cytokine. In some embodiments, the masking moiety comprises an antibody or an antigen-binding fragment thereof that specifically binds to the IL-2 cytokine. In some embodiments, the masking moiety comprises an antibody or an antigen-binding fragment thereof that specifically binds to the IL-15 cytokine. In some embodiments, the masking moiety comprises an antibody or an antigen-binding fragment thereof that specifically binds to the IL-12 cytokine. In some embodiments, the masking moiety comprises an antibody or an antigen-binding fragment thereof that specifically binds to the IL-18 cytokine.

[0384] In some embodiments, the masking moiety comprises an scFv that specifically binds to the IL-2 cytokine. In some embodiments, the masking moiety comprises an scFv that specifically binds to the IL-15 cytokine. In some embodiments, the masking moiety comprises an scFv that specifically binds to the IL-12 cytokine. In some embodiments, the masking moiety comprises an scFv that specifically binds to the IL-18 cytokine.

[0385] In some embodiments, the masking moiety comprises a VHH that specifically binds to the IL-2 cytokine. In some embodiments, the masking moiety comprises a VHH that specifically binds to the IL-15 cytokine. In some embodiments, the masking moiety comprises a VHH that specifically binds to the IL-12 cytokine. In some embodiments, the masking moiety comprises a VHH that specifically binds to the IL-18 cytokine.

[0386] Anti-IL-2 scFv In some embodiments, the masking moiety comprises an anti-IL-2 scFv having a variable heavy chain (VH) of SEQ ID NO: 124. In some embodiments, the masking moiety comprises an anti-IL-2 scFv having a variable light chain (VL) of SEQ ID NO: 125. In some embodiments, the masking moiety comprises an anti-IL-2 scFv having the VH of SEQ ID NO: 124 and the VL of SEQ ID NO: 125.

[0387] In some embodiments, the masking moiety comprises an anti-IL-2 scFv having hCDR1 of SEQ ID NO: 126, hCDR2 of SEQ ID NO: 127, and hCDR3 of SEQ ID NO: 128. In some embodiments, the masking moiety comprises an anti-IL-2 scFv having lCDR1 of SEQ ID NO: 129, lCDR2 of SEQ ID NO: 130, and lCDR3 of SEQ ID NO: 131. In some embodiments, the masking moiety comprises an anti-IL-2 scFv having hCDR1 of SEQ ID NO: 126, hCDR2 of SEQ ID NO: 127, hCDR3 of SEQ ID NO: 128, lCDR1 of SEQ ID NO: 129, lCDR2 of SEQ ID NO: 130, and lCDR3 of SEQ ID NO: 131.

[0388] Exemplary anti-IL-2 scFv sequences [Table 5]

[0389] In some embodiments, the masking portion comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 124. In some embodiments, the masking portion comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 124. In some embodiments, the masking portion comprises an amino acid sequence having at least 87% identity to SEQ ID NO: 124. In some embodiments, the masking portion comprises an amino acid sequence having at least 88% identity to SEQ ID NO: 124. In some embodiments, the masking portion comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 124. In some embodiments, the masking portion comprises an amino acid sequence having at least 92% identity to SEQ ID NO: 124. In some embodiments, the masking portion comprises an amino acid sequence having at least 93% identity to SEQ ID NO: 124. In some embodiments, the masking portion comprises an amino acid sequence having at least 94% identity to SEQ ID NO: 124. In some embodiments, the masking portion comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 124. In some embodiments, the masking portion comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 124. In some embodiments, the masking portion comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 124. In some embodiments, the masking portion comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 124. In some embodiments, the masking portion comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 124.

[0390] In some embodiments, the masking portion comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 125. In some embodiments, the masking portion comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 125. In some embodiments, the masking portion comprises an amino acid sequence having at least 87% identity to SEQ ID NO: 125. In some embodiments, the masking portion comprises an amino acid sequence having at least 88% identity to SEQ ID NO: 125. In some embodiments, the masking portion comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 125. In some embodiments, the masking portion comprises an amino acid sequence having at least 92% identity to SEQ ID NO: 125. In some embodiments, the masking portion comprises an amino acid sequence having at least 93% identity to SEQ ID NO: 125. In some embodiments, the masking portion comprises an amino acid sequence having at least 94% identity to SEQ ID NO: 125. In some embodiments, the masking portion comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 125. In some embodiments, the masking portion comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 125. In some embodiments, the masking portion comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 125. In some embodiments, the masking portion comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 125. In some embodiments, the masking portion comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 125.

[0391] In some embodiments, the masking portion comprises a VL linked to a VH via a non-cleavable linker. In some embodiments, the masking portion comprises a VH linked to a VL via a non-cleavable linker. In some embodiments, the non-cleavable linker is GGGGG SGGGGSGGGGSGGGGS (SEQ ID NO: 141).

[0392] In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 142. NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQTYDSIDVYFGGGTKLTVLGGGGGSGGGGSGGGGSGGGGSQLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYKSGSAYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTPTVTGDWFDPWGRGTLVTVSS (SEQ ID NO: 142)

[0393] In some embodiments, the masking portion comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence having at least 87% identity to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence having at least 88% identity to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence having at least 92% identity to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence having at least 93% identity to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence having at least 94% identity to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 142.

[0394] In some embodiments, the masking moiety is linked to the engineered Fc domain via a non-cleavable linker of SEQ ID NO: 140 (GGSSGSGGSGGGSGSGGG). In some embodiments, the masking moiety is linked to the engineered Fc domain via a non-cleavable linker of SEQ ID NO: 141 (GGGGGSGGGGSGGGGSGGGGS). In some embodiments, the masking moiety is linked to the engineered Fc domain via a non-cleavable linker of SEQ ID NO: 210 (GGSSGSGGSGGGSGSGGGSGGSGG).

[0395] Anti-IL-2 VHH In some embodiments, the masking moiety comprises an anti-IL-2 VHH having the amino acid sequence of SEQ ID NO: 121. In some embodiments, the masking moiety comprises an anti-IL-2 VHH having hCDR1 of SEQ ID NO: 132, hCDR2 of SEQ ID NO: 133, and hCDR3 of SEQ ID NO: 134.

[0396] Exemplary anti-IL-2 scFv sequences [Table 6]

[0397] In some embodiments, the masking moiety comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence having at least 87% identity to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence having at least 88% identity to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence having at least 92% identity to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence having at least 93% identity to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence having at least 94% identity to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 121.

[0398] In some embodiments, the masking moiety is linked to the engineered Fc domain via a non-cleavable linker of SEQ ID NO: 140. In some embodiments, the masking moiety is linked to the engineered Fc domain via a non-cleavable linker of SEQ ID NO: 141. In some embodiments, the masking moiety is linked to the engineered Fc domain via a non-cleavable linker of SEQ ID NO: 210.

[0399] CD122 (masking portion of IL-2 and IL-15) The masking portion masks the cytokine or a functional fragment thereof in the masked cytokine, thereby reducing or preventing the binding of the cytokine or a functional fragment thereof to its cognate receptor. In some embodiments, the masking portion reduces or prevents the binding of the IL-2 cytokine or a functional fragment thereof to IL-2Rα (CD25). In some embodiments, the masking portion provided herein refers to a portion that can bind to an IL-2 cytokine or a functional fragment thereof, such as an anti-IL-2 antibody or an IL-2 cognate receptor protein, or can exhibit other affinity. In some embodiments, the masking portion reduces or prevents the binding of the IL-15 cytokine or a functional fragment thereof to IL-15Rα. In some embodiments, the masking portion provided herein refers to a portion that can bind to an IL-15 cytokine or a functional fragment thereof, such as an anti-IL-15 antibody or an IL-15 cognate receptor protein, or can exhibit other affinity. Methods for determining the degree of binding of a protein (e.g., a cytokine) to its cognate protein (e.g., a cytokine receptor) are well known in the art.

[0400] In some embodiments, the masking portion comprises an IL-2 cytokine receptor, or a subunit or a functional fragment thereof.

[0401] In some embodiments, the masking portion comprises CD122 (also referred to as IL-2Rβ) or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-2 and IL-15.

[0402] In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 50. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 50)

[0403] In some embodiments, the masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the masking portion comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 50 with 1 to 4 amino acid substitutions. In some embodiments, the masking portion comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 50 with 1 or 2 amino acid substitutions.

[0404] In some embodiments, CD122 or a fragment, portion, or variant thereof has a mutation at amino acid position C122 as compared to CD122 of SEQ ID NO: 50.

[0405] In some embodiments, CD122 or a fragment, portion, or variant thereof has the mutation C122S at amino acid position 122 as compared to CD122 of SEQ ID NO: 50.

[0406] In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 50 having a C122 mutation.

[0407] In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 50 having a C122S mutation.

[0408] In some embodiments, CD122 or a fragment, portion, or variant thereof has a mutation at amino acid position C168 as compared to CD122 of SEQ ID NO: 50.

[0409] In some embodiments, CD122 or a fragment, portion, or variant thereof has the mutation C168S at amino acid position 168 as compared to CD122 of SEQ ID NO: 50.

[0410] In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 50 having a C168 mutation.

[0411] In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 50 having a C168S mutation.

[0412] In some embodiments, CD122 or a fragment, portion, or variant thereof has mutations at amino acid positions C122 and C168 as compared to CD122 of SEQ ID NO: 50.

[0413] In some embodiments, CD122 or a fragment, portion, or variant thereof has the mutations C122S and C168S as compared to CD122 of SEQ ID NO: 50.

[0414] In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 51. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRSNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWISLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 51)

[0415] In some embodiments, the masking portion comprises an amino acid sequence having at least about 80% identity to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence having at least about 91% identity to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence having at least about 92% identity to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence having at least about 93% identity to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence having at least about 94% identity to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 51.

[0416] In some embodiments, the masking portion has a mutation at amino acid position F8 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation F8C compared to CD122 of SEQ ID NO: 50.

[0417] In some embodiments, the masking portion has a mutation at amino acid position A94 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation A94C compared to CD122 of SEQ ID NO: 50.

[0418] In some embodiments, the masking portion has a mutation at amino acid position L106 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation L106C compared to CD122 of SEQ ID NO: 50.

[0419] In some embodiments, the masking portion has a mutation at amino acid position V117 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation V117C compared to CD122 of SEQ ID NO: 50.

[0420] In some embodiments, the masking portion has a mutation at amino acid position C122 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation C122S compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation C122V compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation C122A compared to CD122 of SEQ ID NO: 50.

[0421] In some embodiments, the masking portion has a mutation at amino acid position N123 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation N123C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation N123Q compared to CD122 of SEQ ID NO: 50.

[0422] In some embodiments, the masking portion has a mutation at amino acid position C168 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation C168S compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation C168V compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation C168A compared to CD122 of SEQ ID NO: 50.

[0423] In some embodiments, the masking portion has a mutation at amino acid position L169 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation L169C compared to CD122 of SEQ ID NO: 50.

[0424] In some embodiments, the masking portion has a mutation at amino acid position Q177 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation Q177C compared to CD122 of SEQ ID NO: 50.

[0425] In some embodiments, the masking portion has a mutation at amino acid position V184 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation V184C compared to CD122 of SEQ ID NO: 50.

[0426] In some embodiments, the masking portion has a mutation at amino acid position S195 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation S195C compared to CD122 of SEQ ID NO: 50.

[0427] In some embodiments, the masking portion has a mutation at amino acid position R204 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion has the mutation R204C compared to CD122 of SEQ ID NO: 50.

[0428] In some embodiments, the masking portion has the mutation C122V / C168V compared to CD122 of SEQ ID NO: 50.

[0429] In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 52. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 52)

[0430] In some embodiments, the masking portion has mutations C122A / C168V compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 53. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 53)

[0431] In some embodiments, the masking portion has the mutation C168V compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 54. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 54)

[0432] In some embodiments, the masking portion has mutations C122V / C168A compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 55. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 55)

[0433] In some embodiments, the masking portion has mutations C122A / N123C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 56. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRACISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 56)

[0434] In some embodiments, the masking portion comprises an amino acid sequence having at least about 80% identity to SEQ ID NO: 56. In some embodiments, the masking portion comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 56. In some embodiments, the masking portion comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 56. In some embodiments, the masking portion comprises an amino acid sequence having at least about 91% identity to SEQ ID NO: 56. In some embodiments, the masking portion comprises an amino acid sequence having at least about 92% identity to SEQ ID NO: 56. In some embodiments, the masking portion comprises an amino acid sequence having at least about 93% identity to SEQ ID NO: 56. In some embodiments, the masking portion comprises an amino acid sequence having at least about 94% identity to SEQ ID NO: 56. In some embodiments, the masking portion comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 56. In some embodiments, the masking portion comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 56. In some embodiments, the masking portion comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 56. In some embodiments, the masking portion comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 56. In some embodiments, the masking portion comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 56.

[0435] In some embodiments, the masking portion has the mutations C122V / N123C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 57. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRVCISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 57)

[0436] In some embodiments, the masking portion has mutations C122A / C168A compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 58. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ ID NO: 58)

[0437] In some embodiments, the masking portion has mutations V117C / N123Q / C168A compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 59. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHCETHRCQISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 59)

[0438] In some embodiments, the masking portion has mutations N123Q / C168A / L169C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 60. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCQISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIACETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 60)

[0439] In some embodiments, the masking portion has mutations L106C / C122A / C168A / S195C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 61. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWCPWSQPLAFRTKPAALGKD (SEQ ID NO: 61)

[0440] In some embodiments, the masking portion has mutations L106C / C122A / C168A / V184C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 62. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRCKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 62)

[0441] In some embodiments, the masking portion has mutations C122A / C168A / V184C / S195C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 63. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRCKPLQGEFTTWCPWSQPLAFRTKPAALGKD (SEQ ID NO: 63)

[0442] In some embodiments, the masking portion has mutations C122A / C168A / Q177C / R204C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 64. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTCYEFQVRVKPLQGEFTTWSPWSQPLAFCTKPAALGKD (SEQ ID NO: 64)

[0443] In some embodiments, the masking moiety has mutations L106C / C122V / C168V / S195C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 65. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMAPISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWCPWSQPLAFRTKPAALGKD (SEQ ID NO: 65)

[0444] In some embodiments, the masking moiety has mutations F8C / A94C / C122V / C168V compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 66. AVNGTSQCTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMCIQDFKPFENLRLMAPISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 66)

[0445] IL-12 receptor The masking moiety masks the IL-12 cytokine or a functional fragment thereof in the targeted cytokine, thereby reducing or preventing the binding of the IL-cytokine or a functional fragment thereof to its cognate receptor.

[0446] IL-12 receptor, beta 1, or IL-12Rβ1 is a subunit of the IL-12 receptor complex. IL-12Rβ1 is also known as CD212. This protein binds interleukin-12 (IL-12) with low affinity. This protein forms disulfide-bonded oligomers, which are required for IL-12 binding activity. IL-12 receptor, beta 2, or IL-12Rβ2 is a subunit of the IL-12 receptor complex. Co-expression of IL-12Rβ1 and IL-12Rβ2 proteins has been shown to lead to the formation of a high-affinity IL-12 binding site.

[0447] In some embodiments, the masking moiety comprises an extracellular domain of an IL-12 cytokine receptor, or a subunit or functional fragment thereof.

[0448] Interleukin-12 receptor subunit beta-1, also called CD212, has the following sequence. MEPLVTWVVPLLFLFLLSRQGAACRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRQLGSQGSSWSKWSSPVCVPPENPPQPQVRFSVEQLGQDGRRRLTLKEQPTQLELPEGCQGLAPGTEVTYRLQLHMLSCPCKAKATRTLHLGKMPYLSGAAYNVAVISSNQFGPGLNQTWHIPADTHTEPVALNISVGTNGTTMYWPARAQSMTYCIEWQPVGQDGGLATCSLTAPQDPDPAGMATYSWSRESGAMGQEKCYYITIFASAHPEKLTLWSTVLSTYHFGGNASAAGTPHHVSVKNHSLDSVSVDWAPSLLSTCPGVLKEYVVRCRDEDSKQVSEHPVQPTETQVTLSGLRAGVAYTVQVRADTAWLRGVWSQPQRFSIEVQVSDWLIFFASLGSFLSILLVGVLGYLGLNRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEGAPELALDTELSLEDGDRCKAKM (SEQ ID NO: 67)

[0449] Interleukin-12 receptor subunit beta-2 has the following sequence. MAHTFRGCSLAFMFIITWLLIKAKIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTGMLDVWYMKRHIDYSRQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGMDNILVTWQPPRKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSSILGNSKHKAPLSGPHINAITEEKGSILISWNSIPVQEQMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNSHPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKANWMAFVAPSICIAIIMVGIFSTHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML (SEQ ID NO: 68)

[0450] Bold indicates a propeptide, underlined italics indicate an extracellular domain, italics indicate a transmembrane domain, and underlined bold indicates a cytoplasmic domain.

[0451] In some embodiments, the masking moiety comprises the extracellular domain of human IL-12Rβ1, or a fragment, portion, or variant thereof, that retains or otherwise exhibits affinity for IL-12.

[0452] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of human IL-12Rβ1 (SEQ ID NO: 68) with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of human IL-12Rβ1 with 1 or 2 amino acid substitutions.

[0453] In some embodiments, the masking moiety comprises residues 24-237 of human IL-12Rβ1, i.e., the sequence having SEQ ID NO: 69, or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-12. CRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRQLGSQGSSWSKWSSPVCVPPENP (SEQ ID NO: 69)

[0454] In some embodiments, the masking moiety comprises IL-12Rβ1 having SEQ ID NO: 69. In some embodiments, the masking moiety comprises an amino acid sequence having a sequence identity of about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to any one of the amino acid sequences of SEQ ID NO: 69. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 69. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 69. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 69. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 69. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 69. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 69. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 69. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 69. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 69. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 69.

[0455] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 69 with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 69 with 1 or 2 amino acid substitutions.

[0456] In some embodiments, the masking moiety comprises residues 24 to 545 of human IL-12Rβ1, i.e., the sequence having SEQ ID NO: 70, or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-12. CRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRQLGSQGSSWSKWSSPVCVPPENPPQPQVRFSVEQLGQDGRRRLTLKEQPTQLELPEGCQGLAPGTEVTYRLQLHMLSCPCKAKATRTLHLGKMPYLSGAAYNVAVISSNQFGPGLNQTWHIPADTHTEPVALNISVGTNGTTMYWPARAQSMTYCIEWQPVGQDGGLATCSLTAPQDPDPAGMATYSWSRESGAMGQEKCYYITIFASAHPEKLTLWSTVLSTYHFGGNASAAGTPHHVSVKNHSLDSVSVDWAPSLLSTCPGVLKEYVVRCRDEDSKQVSEHPVQPTETQVTLSGLRAGVAYTVQVRADTAWLRGVWSQPQRFSIEVQVSD (SEQ ID NO: 70)

[0457] In some embodiments, the masking moiety comprises IL-12Rβ1 having SEQ ID NO: 70. In some embodiments, the masking moiety comprises an amino acid sequence having a sequence identity of about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to any one of the amino acid sequences of SEQ ID NO: 70. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 70. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 70. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 70. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 70. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 70. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 70. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 70. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 70. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 70. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 70.

[0458] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 70 with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 70 with 1 or 2 amino acid substitutions.

[0459] In some embodiments, the masking moiety comprises the extracellular domain of human IL-12Rβ2, or a fragment, portion, or variant thereof, that retains or otherwise exhibits affinity for IL-12. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of human IL-12Rβ2 (SEQ ID NO: 68) with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of human IL-12Rβ2 with 1 or 2 amino acid substitutions.

[0460] In some embodiments, the masking moiety comprises the sequence having residues 24-212 of human IL-12Rβ2, i.e., SEQ ID NO: 71. KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSL (SEQ ID NO: 71)

[0461] In some embodiments, the masking moiety comprises IL-12Rβ1 having SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having a sequence identity of about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to any one of the amino acid sequences of SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having a sequence identity of at least about 85% to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having a sequence identity of at least about 90% to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having a sequence identity of at least about 92% to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having a sequence identity of at least about 93% to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having a sequence identity of at least about 94% to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having a sequence identity of at least about 95% to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having a sequence identity of at least about 96% to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having a sequence identity of at least about 97% to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having a sequence identity of at least about 98% to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having a sequence identity of at least about 99% to SEQ ID NO: 71.

[0462] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 71 with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 71 with 1 or 2 amino acid substitutions.

[0463] In some embodiments, the masking portion comprises the residues 24-222 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 72. KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIV (SEQ ID NO: 72)

[0464] In some embodiments, the masking moiety comprises IL-12Rβ1 having SEQ ID NO: 72. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences of SEQ ID NO: 72. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 72. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 72. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 72. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 72. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 72. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 72. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 72. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 72. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 72. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 72.

[0465] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 72 with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 72 with 1 or 2 amino acid substitutions.

[0466] In some embodiments, the masking portion comprises the residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 73. KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEE (SEQ ID NO: 73)

[0467] In some embodiments, the masking moiety comprises IL-12Rβ2 having SEQ ID NO: 73. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences of SEQ ID NO: 73. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 73. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 130. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 73. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 73. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 73. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 73. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 73. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 73. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 73. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 73.

[0468] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 73 with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 73 with 1 or 2 amino acid substitutions.

[0469] In some embodiments, the masking moiety comprises the sequence of residues 24 to 319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 73, and has one or more cysteine substitutions. In some embodiments, the masking moiety comprises the sequence of residues 24 to 319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 73, and has an amino acid substitution at position C242. In some embodiments, the amino acid substitution at position C242 is C242S. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 74. KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRSTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEE (SEQ ID NO: 74)

[0470] 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: 74. In some embodiments, the masking moiety consists of the amino acid sequence of SEQ ID NO: 74.

[0471] In some embodiments, the masking moiety comprises the sequence of residues 24 to 622 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 75. KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTGMLDVWYMKRHIDYSRQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGMDNILVTWQPPRKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSSILGNSKHKAPLSGPHINAITEEKGSILISWNSIPVQEQMGCLLHYRIYWKER (SEQ ID NO: 75)

[0472] In some embodiments, the masking moiety comprises IL-12Rβ1 having SEQ ID NO: 75. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences of SEQ ID NO: 75. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 75. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 75. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 75. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 75. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 75. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 75. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 75. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 75. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 75. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 75.

[0473] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 75 with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 75 with 1 or 2 amino acid substitutions.

[0474] In some embodiments, the masking portion comprises the residues 24-227 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 76. KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPP (SEQ ID NO: 76)

[0475] In some embodiments, the masking portion comprises IL-12Rβ1 having SEQ ID NO: 76. In some embodiments, the masking portion comprises an amino acid sequence having a sequence identity of about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to any one of the amino acid sequences of SEQ ID NO: 76. In some embodiments, the masking portion comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 76. In some embodiments, the masking portion comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 76. In some embodiments, the masking portion comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 76. In some embodiments, the masking portion comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 76. In some embodiments, the masking portion comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 76. In some embodiments, the masking portion comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 76. In some embodiments, the masking portion comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 76. In some embodiments, the masking portion comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 76. In some embodiments, the masking portion comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 76. In some embodiments, the masking portion comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 76.

[0476] In some embodiments, the masking portion comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 76 with 1 to 4 amino acid substitutions. In some embodiments, the masking portion comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 76 with 1 or 2 amino acid substitutions.

[0477] Targeted cytokine format As contemplated, the cleavable Fc domain-binding cytokine may further include a targeting moiety for forming a targeted cytokine. In some embodiments, the targeted cytokine is in a bivalent targeting format, the format including: 1) a first chain including a variable heavy chain region and a heavy chain constant region of IgG1 or IgG4 having "hole mutations" (Y349C, T366S, L368A, and Y407V); 2) a second chain including a variable heavy chain region and a heavy chain constant region of IgG1 or IgG4 having "knob mutations" (S354C and T366W) fused to a cytokine or variant thereof, wherein the CH1 domain and the CH2 domain are from IgG1 or IgG4 and the CH3 domain is from IgG3; and 3) a third chain including a variable light chain region and a constant region of immunoglobulin kappa or lambda.

[0478] In some embodiments, the targeted cytokine is in a bivalent targeting format, the format including: 1) a first chain including a variable heavy chain region and a heavy chain constant region of IgG1 or IgG4 having "hole mutations" (Y349C, T366S, L368A, and Y407V); 2) a second chain including a variable heavy chain region and a heavy chain constant region of IgG1 or IgG4 having "knob mutations" (S354C and T366W) and "RF mutations" (H435R and Y436F) fused to a cytokine or variant thereof; and 3) a third chain including a variable light chain region and a constant region of immunoglobulin kappa or lambda.

[0479] In some embodiments, the targeted cytokine is in a bivalent targeting format, which format comprises: 1) a first chain comprising a variable heavy chain region and a heavy chain constant region of IgG1 or IgG4 having a "hole mutation" (Y349C, T366S, L368A, and Y407V) fused to a cytokine or a variant thereof; 2) a second chain comprising a variable heavy chain region and a heavy chain constant region having a "knob mutation" (S354C and T366W) fused to a masking moiety, wherein the CH1 domain and the CH2 domain are from IgG1 or IgG4 and the CH3 domain is from IgG3; and 3) a third chain comprising a variable light chain region and a constant region of immunoglobulin kappa or lambda.

[0480] In some embodiments, the targeted cytokine is in a bivalent targeting format, which format comprises: 1) a first chain comprising a variable heavy chain region and a heavy chain constant region of IgG1 or IgG4 having a "hole mutation" (Y349C, T366S, L368A, and Y407V) fused to a cytokine or a variant thereof; 2) a second chain comprising a variable heavy chain region and a heavy chain constant region from IgG1 or IgG4 having a "knob mutation" (S354C and T366W) and an "RF mutation" (H435R and Y436F) fused to a masking moiety; and 3) a third chain comprising a variable light chain region and a constant region of immunoglobulin kappa or lambda.

[0481] In some embodiments, the targeted cytokine is in a bivalent targeting format, which format comprises: 1) a first chain comprising a variable heavy chain region and a heavy chain constant region of IgG1 or IgG4 having a "hole mutation" (Y349C, T366S, L368A, and Y407V) fused to a masking moiety; 2) a second chain comprising a variable heavy chain region and a heavy chain constant region having a "knob mutation" (S354C and T366W) fused to a cytokine or a variant thereof, wherein the CH1 domain and the CH2 domain are from IgG1 or IgG4 and the CH3 domain is from IgG3; and 3) a third chain comprising a variable light chain region and a constant region of immunoglobulin kappa or lambda.

[0482] In some embodiments, the targeted cytokine is in a bivalent targeting format, which format comprises: 1) a first chain comprising a variable heavy chain region and a heavy chain constant region of IgG1 or IgG4 having a "hole mutation" (Y349C, T366S, L368A, and Y407V) fused to a masking moiety; 2) a second chain comprising a variable heavy chain region and a heavy chain constant region derived from IgG1 or IgG4 having a "knob mutation" (S354C and T366W) and an "RF mutation" (H435R and Y436F) fused to a cytokine or a variant thereof; and 3) a third chain comprising a variable light chain region and a constant region of immunoglobulin kappa or lambda.

[0483] In some embodiments, the targeted cytokine is in a monovalent targeting format, which format comprises: 1) a first chain comprising a Fab fused to a first Fc polypeptide chain derived from IgG1 or IgG4 having a "hole mutation" (Y349C, T366S, L368A, and Y407V); and 2) a second chain comprising a second Fc polypeptide chain derived from IgG1 or IgG4 having a "knob mutation" (S354C and T366W) and an "RF mutation" (H435R and Y436F).

[0484] In some embodiments, the targeted cytokine is in a monovalent targeting format, which format comprises: 1) a first chain comprising a Fab fused to a first Fc polypeptide chain derived from IgG1 or IgG4 having a "hole mutation" (Y349C, T366S, L368A, and Y407V); and 2) a second chain comprising a second Fc polypeptide chain having a "knob mutation" (S354C and T366W), wherein the CH1 domain and the CH2 domain are derived from IgG1 or IgG4 and the CH3 domain is derived from IgG3.

[0485] In some embodiments, the targeted cytokine is in a monovalent targeting format and comprises: 1) a first chain comprising a first Fc polypeptide chain derived from IgG1 or IgG4 having "hole mutations" (Y349C, T366S, L36...

Claims

1. An engineered Fc domain comprising one or more amino acid substitutions compared to a wild-type Fc domain, and comprising a protease cleavage site.

2. An engineered Fc domain, wherein the engineered Fc domain includes one or more amino acid substitutions in a hinge region, a CH2 domain, a CH3 domain, and / or a CH2-CH3 linker, such that the engineered Fc domain includes a protease cleavage site.

3. The manipulated Fc domain according to claim 2, wherein the manipulated Fc domain comprises one or more amino acid substitutions in the G chain, FG loop, or F chain of the CH3 domain.

4. An engineered Fc domain, wherein the engineered Fc domain includes a protease cleavage site, by EU numbering (a) 436th to 447th place, (b) 416th to 425th place, or (c) 426th to 437th An engineered Fc domain containing one or more amino acid substitutions.

5. The aforementioned protease cleavage site is Tumor-related protease cleavage sites, Tissue-selective protease cleavage sites, or Inflammation-related protease cleavage sites The manipulated Fc domain according to claim 4.

6. The manipulated Fc domain according to claim 1, comprising amino acid substitutions S442G, L443G, S444P, P445L, and G447L.

7. The therapeutic activity domain, The masked area and, A manipulated Fc domain containing tumor-associated protease cleavage sites, A mask-type therapeutic active molecule containing, A mask-type therapeutic molecule in which the manipulated Fc domain is fused to the therapeutic active domain or the masking portion such that the masking portion binds to the therapeutic active domain and the therapeutic active domain is released from the masking portion upon cleavage of the tumor-associated protease cleavage site on the manipulated Fc domain.

8. a) The manipulated Fc domain is fused to the therapeutically active domain or the masking portion via a non-cleavable linker, b) The manipulated Fc domain is directly fused to the therapeutically active domain or the masking portion, The mask-type therapeutic active molecule according to claim 7.

9. The mask-type therapeutic active molecule according to claim 7, wherein the therapeutic active domain is a cell engager or a co-stimulatory domain.

10. The cytokine portion, The masked area and, A mask-type cytokine comprising an engineered Fc domain containing a tumor-associated protease cleavage site, A masked cytokine, wherein the manipulated Fc domain is fused to the cytokine portion or the masking portion such that the masking portion binds to the cytokine portion and the cytokine portion is released from the masking portion upon cleavage of the tumor-associated protease cleavage site on the manipulated Fc domain.

11. a) The manipulated Fc domain is fused to the cytokine portion or the masking portion via a non-cleavable linker, b) The manipulated Fc domain is directly fused to the cytokine portion or the masking portion, The mask-type cytokine according to claim 10.

12. The cytokine portion, The masked area and, A mask-type cytokine comprising a carrier portion containing an engineered tumor-associated protease cleavage site, A mask-type cytokine in which the carrier portion is fused to the cytokine portion and / or the masking portion such that the masking portion binds to the cytokine portion and the cytokine portion is activated upon cleavage of the manipulated tumor-associated protease cleavage site.

13. a) The carrier portion comprises a first Fc domain and a second Fc domain, wherein one of the first or second Fc domains comprises the manipulated tumor-associated protease cleavage site, b) The carrier portion is a half-life extension domain selected from albumin, transferrin, or tissue factor, or c) The carrier portion is selected from immunoglobulin, Fab, scFv, VHH, nanobody, VH, VL, single-domain Ab immunoglobulin kappa constant region (CK), and immunoglobulin lambda constant region (CL), The mask-type cytokine according to claim 12.

14. a) The carrier portion is fused to the cytokine portion and / or the masking portion via a non-cleavable linker, b) The carrier portion is directly fused to the cytokine portion and / or the masking portion, The mask-type cytokine according to claim 12.

15. The mask-type cytokine according to claim 10, wherein the mask-type cytokine does not contain a cleavable linker.

16. The aforementioned proteases are MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP27, MMP28, cathepsin (cathepsin B, cathepsin D, cathepsin F, cathepsin K, cathepsin L, cathepsin V, cathepsin S, cathepsin W), ADAM, ADAMTS, An engineered Fc domain according to claim 1, selected from kallikrein 1-15, HTRA1-2-3, HGFAc, PRSS, TMPRSS, elastase, PR-3, granzyme (granzyme A, B, M, H, and K), fibroblast-activating protein (FAP), plasmin, urokinase plasminogen activator (uPA), tryptase, caspase, thrombin, regmine, chymase, collagenase, napsin A, matryptase 1-2, and matryptase.

17. The manipulated Fc domain, a) N434I, Y436V, and Q438L mutations; b) H435S, Q438I, and S440G mutations; c) T437S, Q438N, and K438E mutations; d) K439V and L441S mutations; e) Q438P, K439T, and L441T mutations; f) K439E, P445E and G446E mutations; or g) K439A, S444A, and G446V mutations The manipulated Fc domain according to claim 1, including the manipulated Fc domain according to claim 1.

18. a) The manipulated Fc domain includes one of the amino acid substitution sets listed in Table 5, Table 8a, Table 11, Table 11a, Table 11b, or Table 11c, b) The protease cleavage sites are VPLSLYSG, IHVTLKSL, NSYTIKGL, SNESLSLS, SQESLSLS, QVSSSLSSP, PTSTSLSP, ESLSLSEE, ASLSLAPV, PLGL, GLFG, SRLRSPQG, KPMQFLGD, FRLWS, SLLLRTGN, SLGRRPGG, SLFRAPGP, AGLRSPGGG, SGRRSPGG, SLSGRRGG, FRLWA, SLSLGRRG, SLFRSAGP, F Containing the amino acid sequence of RSGVPGG, ALFKSSFP, SLSLSGRR, GGPRNL, FLVGGGASL, GGPKLL, SLSSGRSG, GGPRML, GGPLRL, RGSRGG, KQLRVVNG, GGPRNL, KGSRAG, PLRLSRA, GGPRRL, RGSRAG, SLSGRSGG, GRSRGG, GRSRAG, GKSRAG, RQYRVVGG, LSGK, LSGR, KQLRHMRG, or SSGRSPGG, and / or c) The protease cleavage site contains one of the amino acid sequences among sequence numbers 95, 97, 99-100, 102, 104, 105, 107-110, 119, 122, 123, 135-139, 143-208, and 209. The manipulated Fc domain according to claim 1.

19. The cytokine is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-33, TNF-α, TNF-β, CXCL8 (IL-8), G-CSF, GM-CSF, LI The mask-type cytokine according to claim 10, which is F, OSM, IFN-α, IFN-β, IFN-γ, CD154, LT-β, ​​4-1BBL, April, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β, M-CSF, or MSP, or a fragment thereof.

20. a) The IL-2 is an IL-2 cytokine or a functional fragment thereof that has been modified compared to the sequence of mature IL-2 having sequence number 13. b) The IL-15 polypeptide contains the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least one amino acid modification compared to the amino acid sequence of SEQ ID NO: 16, c) An IL-12 polypeptide or a functional fragment thereof comprises an IL-12p40 polypeptide or a functional fragment thereof covalently bonded to an IL-12p35 polypeptide or a functional fragment thereof. The mask-type cytokine according to claim 19.

21. a) The modified IL-2 cytokine or its functional fragment comprises modified R38A, F42A, Y45A, and E62A relative to the sequence of mature IL-2 having SEQ ID NO: 13, b) The modified IL-2 cytokine or its functional fragment contains modified C125A relative to the sequence of mature IL-2 having SEQ ID NO: 13, c) The modified IL-2 cytokine or its functional fragment comprises R38A, F42A, Y45A, E62A, and C125A relative to the sequence of mature IL-2 having SEQ ID NO: 13, and / or d) The modified IL-2 cytokine or its functional fragment includes a mutation that reduces binding to CD25, The mask-type cytokine according to claim 20.

22. The masking portion is a mask-type cytokine according to claim 10, wherein the masking portion binds to the cytokine.

23. a) The masking portion is a receptor for the cytokine or the fragment thereof, b) The masking portion is a peptide, polypeptide, protein, ligand, or drug that specifically binds to the cytokine or the fragment thereof, or c) The masking portion includes a linear peptide, a cyclic peptide, a Fab, a single-chain Fv (scFv), a single-domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibody (bsFab), a single-domain antibody-conjugated Fab (s-Fab), an antibody, or a combination thereof. The mask-type cytokine according to claim 22.

24. a) The first Fc domain comprises a first IgG1, IgG2, or IgG4 Fc domain, or a fragment thereof. b) The second Fc domain includes a second IgG1, IgG2, or IgG4 Fc domain, or a fragment thereof. c) Each of the first and / or second Fc domains comprises one or more modifications that promote non-covalent association of the first and second Fc polypeptides, or d) The first Fc domain includes a CH3 domain that has been modified to reduce or eliminate binding to protein A, and the second Fc domain includes a CH3 domain that binds to protein A. The mask-type cytokine according to claim 13.

25. The mask-type cytokine according to claim 10, further comprising a targeting portion.

26. The targeted portion includes PD-1, PD-L1, PD-L2, CTLA-4, TIGIT, TIM-3, LAG-3, CD25, CD16a, CD16b, NKG2D, NKP44, NKP30, CD19, CD20, CD30, CD38, BCMA, human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epidermal growth factor receptor (EGFR), glypican-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGFR), and vascular endothelial growth factor receptor 2 (VEGFR). R2), Nectin-4, Liv-1, Glycoprotein NMB (GPNMB), Prostate-Specific Membrane Antigen (PSMA), Trop-2, Carbonic Anhydrase IX (CA9), Endothelin B Receptor (ETBR), Six-Transmembrane Epithelial Antigen of the Prostate (STEAPl), Folate Receptor Alpha (FR-a), SLIT and NTRK-like Protein 6 (SLITRK6), Carbonic Anhydrase VI (CA6), Ectonucleotide Pyrophosphatase / Phosphodiesterase Family Member 3 (ENPP3), Mesothelin, Trophoblastic Glycoprotein (TPBG) The mask-type cytokine according to claim 25, which specifically binds to CD19, CD20, CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD98, CD123, CD138, CD352, CD47, signal regulatory protein alpha (SIRPα), claudin 18.2, claudin 6, 5T4, fibroblast-activating protein alpha (FAPα), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epithelial cell adhesion molecule (EPCAM), or a combination thereof.

27. ​​A nucleic acid encoding an manipulated Fc domain according to any one of claims 1 to 6 and 16 to 18, a mask-type therapeutic active molecule according to any one of claims 7 to 9, or a mask-type cytokine according to any one of claims 10 to 15 and 19 to 26.

28. A method for producing a mask-type cytokine, comprising culturing a host cell containing the nucleic acid described in claim 27 under conditions for producing a mask-type cytokine.

29. A pharmaceutical composition comprising an manipulated Fc domain according to any one of claims 1 to 6 and 16 to 18, a mask-type therapeutically active molecule according to any one of claims 7 to 9, or a mask-type cytokine according to any one of claims 10 to 15 and 19 to 26, and a pharmaceutically acceptable carrier.

30. a) For use in medical treatment, b) For use in the treatment of neoplastic diseases, c) For use in the treatment or prevention of inflammatory diseases, d) For use in the treatment or prevention of autoimmune diseases, A pharmaceutical composition comprising an manipulated Fc domain according to any one of claims 1 to 6 and 16 to 18, a mask-type therapeutically active molecule according to any one of claims 7 to 9, or a mask-type cytokine according to any one of claims 10 to 15 and 19 to 26.

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