Engineered cleavable Fc domains as vectors and methods of use thereof

Abstract

The present disclosure relates to a cleavable vector and a cytokine prodrug linked to the cleavable vector, wherein the cleavable vector is an engineered Fc domain comprising at least one tumor-associated protease cleavage site. Upon cleavage at the cleavage site of the vector Fc domain, the cytokine is released from the masking moiety. The platform provides enzymatically induced prodrug activation. The present disclosure also provides pharmaceutical compositions comprising the cleavable vector-linked cytokine prodrugs, for use as medicaments and methods of treating diseases and administering.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 389,476, filed on July 15, 2022, and U.S. Provisional Application No. 63 / 448,943, filed on February 28, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Cross-reference to sequence listing

[0004] This application is submitted with a sequence listing submitted electronically in XML format. The sequence listing file named XTX_UNIVFC_WO1_SL.XML was created on July 12, 2023 and is 210,539 bytes in size; the information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Background Art

[0005] Cancer is the second leading cause of death in the United States, causing more deaths than the next five leading causes (chronic respiratory disease, stroke, accidents, Alzheimer's disease, and diabetes). Although great progress has been made, especially in targeted therapies, there is still a lot of work to be done in this field. Immunotherapy and a subfield of this field, immuno-oncology, are generating viable and exciting therapeutic options for treating malignancies. Specifically, it is now recognized that one of the hallmarks of cancer is immune evasion, and significant efforts have been made to identify targets and develop therapies directed against these targets to reactivate the immune system to recognize and treat cancer.

[0006] Cytokine therapy is an effective strategy to stimulate the immune system to induce anti-tumor cytotoxicity. Unfortunately, the cytokines administered to patients usually have a very short half-life, which requires frequent administration. For example, the product label of aldesleukin sold under the brand name Puliujing shows that the drug has a half-life of 85 minutes in patients receiving 5 minutes of intravenous (IV) infusion. In addition, the administration of high-dose cytokines can cause adverse health outcomes such as vascular leakage by systemic immune activation. In order to extend the half-life and reduce toxicity, carrier moieties (such as Fc domains, albumin and PEG) have been fused to cytokines by cleavable joints, which allows the release of active cytokines once the fusion molecule reaches the tumor microenvironment. Summary of the invention

[0007] The present invention provides, in particular, compositions and methods for precision cancer treatment based on cleavable carriers. In particular, the present invention is based on cleavable carriers (e.g., cleavable Fc domains), which can be connected to therapeutic agents (particularly masked therapeutic agents) to deliver and activate the connected therapeutic agents in specific targets (such as different tumor microenvironments). For example, the cleavable carrier can be an Fc domain that is engineered to contain a cleavage site that can be cleaved by a tumor-specific protease. Such engineered cleavable Fc domains are connected to cytokines or masked portions of cytokines so that cytokines are released from their masked portions only when the Fc domains are cleaved by tumor-specific proteases. Prior to the present invention, existing tumor-specific therapeutic agents generally comprise cleavable joints. The inventive method described herein allows for more flexible joint design without being constrained by cleavage sites for more effective masking and activation. Therefore, the present invention is a major improvement in the field of precision medicine, and is expected to provide cancer patients with safer and more effective treatment methods.

[0008] In one aspect of the invention, a masked therapeutic agent is provided, comprising a therapeutic molecule, a masking portion and a cleavable carrier portion, the 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 the therapeutic molecule and / or the masking portion, and when the engineered tumor-associated protease site in the cleavable carrier portion is cleaved, the therapeutic molecule is released from the masking portion. In some embodiments, the cleavable carrier portion is fused to the therapeutic portion and / or the masking portion via a non-cleavable joint. In other embodiments, the cleavable carrier portion is fused to the therapeutic portion and / or the masking portion via a cleavable joint.

[0009] In some embodiments, the cleavable carrier portion is a cleavable Fc domain. The cleavable Fc domain includes a first Fc domain and a second Fc domain, wherein one of the first Fc domain or the second Fc domain includes an engineered tumor-associated protease cleavage site. In some embodiments, the carrier portion can be a half-life extension domain selected from the group consisting of: albumin, transferrin, and tissue factor. In other embodiments, the carrier portion can be an antigen targeting domain selected from the group consisting of: immunoglobulin, Fab, F(ab)2, scFv, VHH, ScAb, nanobodies, VH, VL, single domain antibodies, CL, and CK; wherein the tumor-associated protease cleavage site is engineered within the antigen targeting domain.

[0010] In one aspect, the present invention provides, inter alia, an engineered Fc domain comprising one or more amino acid substitutions compared to a wild-type Fc domain, wherein the engineered Fc domain comprises a protease cleavage site.

[0011] 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.

[0012] In one embodiment, the cleavable carrier portion is an engineered cleavable Fc domain comprising at least one tumor-associated protease cleavage site. The Fc domain is a human Fc domain.

[0013] The therapeutic molecule can be any agent that has a therapeutic effect, such as inhibiting tumor growth, invasion and progression. In some embodiments, the therapeutic molecule is a cytokine, a variant thereof, or a functional fragment thereof.

[0014] In one embodiment, the present disclosure provides a kind of cytokine of masking.The cytokine of masking comprises cytokine molecule, masking part and carrier part including engineered tumor-associated protease cleavage site, wherein carrier part is fused with cytokine and / or masking part, and when engineered tumor-associated protease cleavage site is cut, the cytokine of masking is released from masking part.In a preferred embodiment, the present invention provides a kind of cytokine of masking, it comprises cytokine molecule, masking part and engineered Fc domain including tumor-associated cleavage site, wherein engineered Fc domain is fused with cytokine part or masking part, so that masking part is combined with cytokine part, and when tumor-associated protease cleavage site on engineered Fc domain is cut, cytokine molecule is released from masking part.The engineered Fc domain is fused with cytokine molecule and / or masking part via non-cleavable joint.In certain embodiments, engineered Fc domain is directly fused with cytokine molecule and / or masking part.

[0015] 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.

[0016] In some embodiments, the protease cleavage site is a tumor-associated protease cleavage site. Therefore, in some embodiments, the protease cleavage site in 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 so that the Fc domain is cleaved. As a non-limiting example, the tumor-associated protease is 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 another embodiment, the tumor-associated protease is MMP9. In another embodiment, the tumor-associated protease is MMP10. Other disease-associated and tissue-selective proteases include, but are not limited to, cathepsins (cathepsin B, cathepsin D, cathepsin F, cathepsin K, cathepsin L, cathepsin V, cathepsin S, cathepsin W), ADAM, ADAMTS, kallikrein 1 to 15, HTRA1-2-3, HGFAc, PRSS, TMPRSS, elastase, PR-3, granzymes (granzymes 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-associated and tissue-selective protease is cathepsin B. In another embodiment, the disease-associated and tissue-selective protease is matriptase.

[0017] According to the present disclosure, the cleavable Fc domain is engineered to include at least one protease cleavage site. As a non-limiting example, compared with the wild-type Fc domain, the engineered Fc domain includes one or more amino acid replacements, wherein the replacement produces a protease cleavage site. In certain embodiments, the engineered Fc domain includes one or more amino acid replacements in the hinge region, in the CH2 domain, in the CH3 domain and / or in the CH2-CH3 joint region, so that the engineered Fc domain includes a protease cleavage site in the hinge region, in the CH2 domain, in the CH3 domain or in the CH2-CH3 joint region. In certain embodiments, the engineered Fc domain is included in one or more amino acid replacements in the position 439 to 446 according to EU numbering; so that replacement produces a protease cleavage site in the position 439 to 446 of the Fc domain.

[0018] In one aspect, the present invention provides, inter alia, an engineered Fc domain comprising one or more amino acid replacements in the hinge region, in the CH2 domain, in the CH3 domain and / or in the CH2-CH3 linker, such that the engineered Fc domain comprises a protease cleavage site.

[0019] In some embodiments, one or more amino acid substitutions are located in the hinge region. In some embodiments, one or more amino acid substitutions are located in the hinge region. In some embodiments, one or more amino acid substitutions are located in the CH2 domain. In some embodiments, one or more amino acid substitutions are located in the CH3 domain. In some embodiments, one or more amino acid substitutions are located in the CH2-CH3 linker.

[0020] In one aspect, the invention provides, inter alia, an engineered Fc domain comprising one or more amino acid substitutions in positions 436 to 447 according to EU numbering, such that the engineered Fc domain comprises a protease cleavage site.

[0021] 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 EU numbering.

[0022] In some embodiments, the engineered Fc domain comprises at least one set of combined mutations comprising: N434I, Y346V and Q438L mutations; H435S, Q438I and S440G mutations; T437S, Q438N and K438E mutations; K439V and L441S mutations; Q438P, K439T and L441T mutations; K439E, P445E and G446E mutations; and K439A, S444A and G446V mutations.

[0023] 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.

[0024] In another aspect, the present invention provides, inter alia, an engineered Fc domain comprising one or more amino acid substitutions in positions 416 to 425 according to EU numbering, such that the engineered Fc domain comprises a protease cleavage site.

[0025] In another aspect, the present invention provides, inter alia, an engineered Fc domain comprising one or more amino acid substitutions in positions 426 to 437 according to EU numbering, such that the engineered Fc domain comprises a protease cleavage site. As a non-limiting example, the present disclosure provides an engineered cleavable Fc domain comprising a tumor-associated protease cleavage site having an amino acid sequence of VPLSLYSG (SEQ ID NO: 95). As a non-limiting example, the present disclosure provides an engineered cleavable Fc domain comprising a tumor-associated protease cleavage site having an amino acid sequence of VPLSLYSGP (SEQ ID NO: 209). In one embodiment, the engineered Fc domain of the present invention comprises a tumor-associated protease cleavage site having an amino acid sequence of IHVTLKSL (SEQ ID NO: 99). In one embodiment, the engineered Fc domain of the present invention comprises a tumor-associated protease cleavage site having an amino acid sequence of NSYTIKGL (SEQ ID NO: 100). In one embodiment, the engineered Fc domain of the present invention comprises a tumor-associated protease cleavage site having an amino acid sequence of SNESLSLS (SEQ ID NO: 102). In one embodiment, the engineered Fc domain of the present invention 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 of the present invention 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 of the present invention 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 of the present invention 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 of the present invention 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 of the present invention comprises a tumor-associated protease cleavage site having the amino acid sequence of PLGL (SEQID NO: 110).

[0026] In some embodiments, the protease site includes any one of SEQ ID NOs: 95, 97, 99-100, 102, 104, 105, 107-110, 119, 122, 123, 135-139, 143-208, and 209.

[0027] In some embodiments, the engineered Fc domain of any of the preceding claims, wherein the engineered Fc domain comprises any one of the amino acid substitution groups listed in Table 5, Table 8a, Table 11, Table 11a, Table 11b, or Table 11c.

[0028] In some embodiments, the engineered Fc domain comprises at least one set of combinatorial substitutions in Table 5.

[0029] In some embodiments, the engineered Fc domain comprises at least one set of combinatorial substitutions in Table 8a.

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

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

[0032] In some embodiments, the engineered Fc domain comprises at least one set of combinatorial substitutions in Table 11b.

[0033] In some embodiments, the engineered Fc domain comprises at least one set of combinatorial substitutions in Table 11c.

[0034] In some embodiments, the engineered cleavable Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, wherein one of the first Fc polypeptide or the second Fc polypeptide comprises a protease cleavage site. In some instances, one of the first Fc polypeptide or the second Fc polypeptide comprises a tumor-associated protease cleavage site as described herein.

[0035] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide each contains one or more modifications that promote non-covalent association of the first Fc polypeptide and the second Fc polypeptide.

[0036] 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.

[0037] In some embodiments, the cytokine molecule of the masked and / or targeted cytokine of the present invention 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 or fragment thereof. As a non-limiting example, the cytokine molecule of the masked or targeted cytokine of the present invention is IL-2, IL-15 or IL-12 or a variant or fragment thereof.

[0038] In some embodiments, the cytokine molecule is IL-2, wherein the IL-2 is a modified IL-2 cytokine or a functional fragment thereof compared to the sequence of a mature IL-2 having the amino acid sequence SEQ ID NO: 13. In some embodiments, the modified IL-2 cytokine or a functional fragment thereof comprises modifications R38A, F42A, Y45A and E62A relative to the sequence of a mature IL-2 having the amino acid sequence SEQ ID NO: 13. In some embodiments, the modified IL-2 cytokine or a functional fragment thereof comprises a modification C125A relative to the sequence of a mature IL-2 having SEQ ID NO: 13. In other embodiments, the modified IL-2 cytokine or a functional fragment thereof comprises modifications R38A, F42A, Y45A, E62A and C125A relative to the sequence of a mature IL-2 having SEQ ID NO: 13.

[0039] In another embodiment, the cytokine molecule is IL-15, wherein the IL-15 polypeptide comprises 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.

[0040] In another embodiment, the cytokine molecule is IL-12, wherein the IL-12 polypeptide or its functional fragment comprises an IL-12p40 polypeptide or its functional fragment covalently linked to an IL-12p35 polypeptide or its functional fragment. The length of the IL-12p40-IL-12p35 linker is between 5 amino acids and 20 amino acids. In some embodiments, the IL-12p40-IL-12p35 linker is rich in amino acid residues G and S.

[0041] According to the present disclosure, the masked cytokines of the present invention include a masking moiety that is associated with a cytokine molecule.

[0042] In some embodiments, the masking moiety is a receptor for a cytokine or a variant or fragment thereof. As a 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 or fragment thereof.

[0043] In one embodiment, the masking part is CD122. In certain embodiments, CD122 is an engineered CD122 polypeptide or a fragment thereof, and an engineered CD122 polypeptide or a fragment thereof includes one or more mutations relative to a wild-type CD122 amino acid sequence. Engineered CD122 includes 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.

[0044] In some embodiments, the masking moiety is a peptide, polypeptide, protein, ligand or agent that specifically binds to a cytokine or variant or fragment thereof.

[0045] In some embodiments, the masking portion comprises Fab, single-chain Fv (scFv), single domain antibody (VHH), one or more CDRs, variable heavy chain (VH), variable light chain (VL), Fab-like bispecific antibody (bsFab), single domain antibody-linked Fab (s-Fab), single heavy chain antibody (HcAb), antibody or a combination thereof.

[0046] 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 an scFv having SEQ ID NO: 124 and SEQ ID NO: 125. In some embodiments, the masking moiety comprises an scFv having SEQ ID NO: 124 connected to SEQ ID NO: 125 by a linker. In some embodiments, the masking moiety comprises an scFv having SEQ ID NO: 142.

[0047] 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.

[0048] In some embodiments, the masked cytokines of the present disclosure include an engineered cleavable Fc domain portion that is further modified. In some embodiments, the first Fc polypeptide and the second Fc polypeptide of the engineered cleavable Fc domain portion of the present invention further include the same amino acid replacement as described herein. In other embodiments, the first Fc polypeptide and the second Fc polypeptide of the engineered cleavable Fc domain portion of the present invention include different amino acid replacements as described herein. As a non-limiting example, the engineered Fc domain of the present invention includes a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide includes Y349C, T366S, L368A, Y407V and N297A mutations, and the second Fc polypeptide includes S354C, T366W and N297A mutations. Alternatively, the engineered Fc domain of the present invention comprises a first Fc polypeptide and a second Fc polynucleotide, the first Fc polypeptide comprising S354C, T366W and N297A mutations, and the second Fc polynucleotide comprising Y349C, T366S, L368A, Y407V and N297A mutations. In another example, the engineered Fc domain of the present invention comprises a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide comprising Y349C, T366S, L368A, Y407V, N297A and I253A mutations, and the second Fc polypeptide comprising S354C, T366W, N297A and I253A mutations. Alternatively, the engineered Fc domain of the invention comprises a first Fc polypeptide comprising S354C, T366W, N297A and I253A mutations and a second Fc polynucleotide comprising Y349C, T366S, L368A, Y407V, N297A and I253A mutations.

[0049] The cytokine of the masking of the present disclosure includes an engineered cleavable Fc domain portion that is further modified. In certain embodiments, the first Fc polypeptide of the engineered Fc domain includes a CH3 domain, and the CH3 domain includes a modification that reduces or eliminates the combination with protein A, and the second Fc domain includes a CH3 domain combined with protein A. In certain embodiments, the CH3 domain combined with protein A is a human IgG1, IgG2 or IgG4 sequence. The second CH3 domain can be a human IgG1, IgG2 or IgG4 sequence included in the modification at the position H435 and / or Y436 according to Kabat numbering. As a non-limiting example, the second CH3 domain includes the modification of H435R and Y436F according to Kabat numbering.

[0050] In some embodiments, the first CH3 domain comprises a human IgG3 sequence.

[0051] In some embodiments, the masked cytokines of the present invention further include a targeting moiety; the targeting moiety includes one or more antigen binding domains, peptides, polypeptides, proteins, ligands or agents that specifically bind to an antigen or a ligand. As a non-limiting example, the targeting moiety includes an antigen binding domain selected from the group consisting of: Fab, single-chain Fv (scFv), single domain antibody (VHH), one or more CDRs, variable heavy chain (VH), variable light chain (VL), Fab-like bispecific antibody (bsFab), single domain antibody-linked Fab (s-Fab), antibodies and combinations thereof.

[0052] In one embodiment, the targeting moiety includes a first antigen binding domain and a second antigen binding domain. In some embodiments, the first antigen binding domain and the second antigen binding domain specifically bind to the same target. The first antigen binding domain and the second antigen binding domain include the same amino acid sequence. In other examples, the first antigen binding domain and the second antigen binding domain specifically bind to different targets. The first antigen binding domain and the second antigen binding domain include different amino acid sequences.

[0053] The targeting moiety can bind to a molecule that is specific for a target, thereby targeting the masked cytokine to the target, such as a target cell, tissue, and organ. In some embodiments, the targeting moiety specifically binds to 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), glycine-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGF R1), vascular endothelial growth factor receptor 2 (VEGF R2), 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 (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, trophoblast glycoprotein (TPBG), CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD94, CD96, CD98, CD 123, 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.

[0054] In a preferred embodiment, the targeting moiety binds PD-1.

[0055] According to the present disclosure, the masked cytokine of the present invention is partially covalently linked. In some embodiments, the targeting moiety is connected to the engineered Fc domain through one or both of the first Fc polypeptide and the second Fc polypeptide. In one example, the targeting moiety is connected to the N-terminus of the first Fc polypeptide of the engineered Fc domain, and the C-terminus of the first Fc polypeptide is connected to the N-terminus of the cytokine or its fragment. In another example, the C-terminus of the targeting moiety is connected to the N-terminus of the second Fc polypeptide of the engineered Fc domain, and the C-terminus of the second Fc polypeptide is connected to the N-terminus of the masking moiety.

[0056] According to the present disclosure, the protease cleavage site has an in vitro cleavage efficiency that produces at least 10% active cytokine. In some embodiments, about 10% to 30% of the active cytokine is released after cleavage.

[0057] In some embodiments, the disclosure provides nucleic acid molecules encoding engineered Fc domains that include a protease cleavage site. The disclosure also provides nucleic acid molecules encoding masked cytokines.

[0058] In some embodiments, the disclosure provides vectors, host cells, the vectors, host cells comprising nucleic acid molecules, the nucleic acid molecules encoding engineered Fc domains and masked cytokines comprising protease cleavage sites as described herein. Provided herein is a method for producing masked cytokines of the present invention; the method comprising: culturing a host cell comprising a nucleic acid molecule encoding an engineered Fc domain or a masked cytokine under conditions that produce an engineered Fc domain or a masked cytokine, the engineered Fc domain comprising a protease cleavage site.

[0059] The present disclosure also provides compositions, pharmaceutical compositions and kits, which include engineered Fc domains or masked cytokines. Pharmaceutical compositions including engineered cleavable Fc domains and / or masked cytokines also include pharmaceutically acceptable excipients. In certain embodiments, pharmaceutical compositions including engineered cleavable Fc domains and / or masked cytokines are formulated for administration via limited routes, such as intravenous (IV) infusion.

[0060] In another aspect of the present disclosure, methods of using engineered cleavable Fc domains and masked cytokines are provided. In some embodiments, the present disclosure provides a method for treating or preventing a neoplastic disease in a subject; the method comprises administering to the subject an effective amount of a masked cytokine, a composition comprising a masked cytokine of the present invention, or a pharmaceutical composition.

[0061] In some embodiments, the masked cytokines, compositions comprising the masked cytokines, or pharmaceutical compositions of the invention are used in a method of treating or preventing an inflammatory or autoimmune disease in a subject; the method comprises administering to the subject an effective amount of the masked cytokines, compositions comprising the masked cytokines, or pharmaceutical compositions of the invention.

[0062] In some embodiments, the engineered cleavable Fc domains of the invention are used alone or in combination with other therapeutic agents to treat neoplastic diseases, inflammatory diseases, and / or autoimmune diseases.

[0063] In one aspect, the present invention provides, inter alia, a masked cytokine comprising a cytokine portion, a masking portion, and an engineered Fc domain, wherein the engineered Fc domain comprises 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 upon cleavage by the tumor-associated protease cleavage site on the engineered Fc domain, the cytokine portion is released from the masking portion.

[0064] In one aspect, the present invention provides, inter alia, a masked therapeutically active molecule comprising a therapeutically active domain, a masking moiety, and an engineered Fc domain, wherein the engineered Fc domain comprises a tumor-associated cleavage site, wherein the engineered Fc domain is fused to the therapeutically active domain or the masking moiety, such that the masking moiety binds to the therapeutically active domain, and upon cleavage by the tumor-associated protease cleavage site on the engineered Fc domain, the therapeutically active domain is released from the masking moiety.

[0065] In some embodiments, the engineered Fc domain is fused to the therapeutically active domain or masking moiety via a non-cleavable linker. In some embodiments, the engineered Fc domain is fused directly to the therapeutically active domain or masking moiety.

[0066] In some embodiments, the therapeutically active domain is a cell engager. In some embodiments, the therapeutically active domain is a co-stimulatory domain. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The drawings are for illustration purposes only and are not intended to be limiting.

[0068] Figure 1A is a schematic representation showing the design of a masked cytokine with a cleavable Fc domain including a protease cleavage site (protease substrate). Figure 1B is an exemplary schematic of a masked cytokine including a protease cleavage site located in a linker.

[0069] Figure 2 Two exemplary universal cleavable Fc platforms showing two different protease substrates (VPLSLYSG and an MMP7-specific substrate) are shown.

[0070] Figure 3A and Figure 3B The structure of the human Fc beta chain and loop regions are shown. Figure 3C The hFc sequence into which the protease cleavage site is incorporated is listed.

[0071] Figure 4 Fusion of a mutated IL-12 (muIL-12) to the C-terminus of the knob chain of Fc is shown.

[0072] Figure 5A and Figure 5B is an SDS-PAGE image showing the cleavage of several constructs (except #19) with VPLSLYSG substrate by MMP10: Figure 5A ) and #23( Figure 5B ), as well as complete cleavage of constructs #7, 9, 10, and 11 ( Figure 5A ) and #19, 20, and 21 ( Figure 5B Partial cutting of the ).

[0073] Fig. 6A and Figure 6B is an SDS-PAGE image showing cleavage of several constructs (except #19) with VPLSLYSG substrate by MMP2, MMP3, and MMP9: almost complete cleavage of constructs #13, 15, 16, and 17 ( Fig. 6A ), and partial cleavage of constructs #19 and 20 ( Figure 6B ).

[0074] Fig. 7A Is a series of exemplary EC50 graphs of masked IL-2 cytokines with cleavable Fc domains in the absence of matrix metalloproteinases (MMPs), as performed in a cell-based reporter assay (HEK Blue IL-2 assay), which determines the calculated % active cytokine. Recombinant human IL-2 (rhIL-2) and CM3 (unmasked, fused to the Fc domain of IL-2) were used as positive controls. Figure 7B ] is a series of exemplary EC50 graphs for IL-2 cytokines masked with a cleavable Fc domain in the presence of MMPs as performed in a cell-based reporter assay (HEK Blue IL-2 assay) which determined the calculated % active cytokine.

[0075] Figure 8 is an exemplary schematic diagram of a targeted cytokine construct containing a cleavable Fc domain. The cross indicates the location of the cleavage site. One Fc polypeptide is fused to the cytokine via a non-cleavable linker, and another Fc polypeptide is fused to a masking portion (e.g., an anti-cytokine VHH) via a non-cleavable linker. Each Fc polypeptide also contains a targeting portion (e.g., Fab) that can specifically bind to a target of interest (e.g., PD-1).

[0076] Fig.9AThe mechanism of the PD-1 / PD-L1 blocking bioassay used in Example 8 is shown. This assay is a biologically relevant MOA-based assay for measuring the efficacy and stability of antibodies and other biologics designed to block PD-1 / PD-L1 interactions. When the two cell types are co-cultured, the PD-1 / PD-L1 interaction inhibits TCR signaling and NFAT-mediated luciferase activity. Adding antibodies that block PD-1 or PD-L1 releases the inhibitory signal and generates TCR signaling and NFAT-mediated luciferase activity. Fig. 9B is an exemplary schematic diagram of a targeted "unmasked" cytokine construct used as a positive control. Figure 8 Compared to the targeted cytokines shown, the "unmasked" versions do not contain the masking moiety.

[0077] Fig.10 is a series of exemplary graphs showing effective blockade of PD-1 / PD-L1 interaction by PD-1 targeted cytokines incorporating a cleavable Fc domain.

[0078] Fig.11A and Fig. 11B Figure 2 is a series of exemplary EC50 graphs for targeted IL-2 cytokines with a cleavable Fc domain in the absence and presence of matrix metalloproteinases (MMPs), as performed in a cell-based reporter gene assay (HEK Blue IL-2 assay), which determined the calculated % active cytokine.

[0079] Fig.12 is a series of exemplary graphs showing % body weight change, tumor growth inhibition, and overall survival of mice treated with the targeted cytokines of the invention, demonstrating in vivo efficacy.

[0080] FIG. 13A to FIG. 13E are exemplary graphs showing the cleavage kinetics of a masked cytokine comprising a cleavable Fc domain by MMP7, MMP8, MMP14, MMP9, and MMP10, respectively.

[0081] FIG. 14A to FIG. 14C are exemplary graphs showing the cleavage kinetics of UCM-75-UCM84 by MMP7, MMP9, and MMP14, respectively.

[0082] Fig.15A Shown is the % cleavage of the cleavable Fc molecules UCM75-UCM84 by ex vivo human tumors. Fig. 15B It was shown that the cleavable Fc molecules UCM75-UCM84 are not cleaved by human plasma.

[0083] definition

[0084] In order to make the present invention more easily understood, certain terms are first defined below. The following terms and other definitions of other terms are set forth throughout the specification. The publications and other reference materials that describe the background of the present invention and provide additional details about its practice mentioned herein are hereby incorporated by reference.

[0085] It is to be understood that the present invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0086] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an IL-2 polypeptide" optionally includes a combination of two or more such polypeptides, etc.

[0087] As used herein, the term "about" refers to the common error range of the corresponding value that is easily known to those skilled in the art. References herein to "about" values ​​or parameters include (and describe) embodiments for the value or parameter itself.

[0088] It should be understood that the aspects and embodiments of the present invention described herein include "comprising the aspects and embodiments," "consisting of the aspects and embodiments," and "consisting essentially of the aspects and embodiments."

[0089] As used herein, the term "and / or" refers to any one, any combination, or all of the terms associated with the term. For example, the phrase "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A and B or C; B and A or C; C and A or B; A (alone); B (alone); and C (alone).

[0090] Antibody: As used herein, the term "antibody" is used in the broadest sense, including polyclonal antibodies, monoclonal antibodies (including full-length antibodies with immunoglobulin Fc regions), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), bifunctional antibodies and single-chain molecules (e.g., scFv and heavy chain antibodies only (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 naturally occurring or artificial.

[0091] The basic 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of 5 basic heterotetramer units together with another polypeptide referred to as J chain, and contain 10 antigen binding sites, while IgA antibodies include 2-5 basic 4-chain units, which can be polymerized to form a multivalent aggregate in combination with J chain. In the case of IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is connected to the H chain by a covalent disulfide bond, and according to the H chain isotype, two H chains are connected to each other by one or more disulfide bonds. Each H and L chain also has a regularly spaced intrachain disulfide bridge. Each H chain has a variable domain (VH) at the N-terminal, followed by three constant domains (CH) of each chain in a chain and y chain, and four CH domains of p isotype and s isotype. Each L chain has a variable domain (VL) at the N-terminal, followed by a constant domain at its other end. VL is aligned with VH, and CL is aligned with the first constant domain (CHI) of the heavy chain. It is believed that specific amino acid residues form the interface between the light chain variable domain and the heavy chain variable domain. The pairing of VH and VL forms a single antigen binding site together. For the structure and characteristics 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, page 71 and chapter 6.

[0092] The L chain from any vertebrate species can be designated as one of two distinct types called κ and λ based on the amino acid sequence of its constant domain. Depending on the amino acid sequence of the constant domain (CH) of its heavy chain, immunoglobulins can be designated as different species or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, which have heavy chains named a, 8, e, y and p, respectively. Based on the relatively minor differences in CH sequence and function, y and a classes are further divided into subclasses, for example, people express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. IgG1 antibodies can exist in a variety of polymorphic variants called allotypes (reviewed in Jefferis and Lefranc 2009, "Monoclonal Antibodies (mAbs)" Vol. 1, No. 4: 1-7), which are all applicable to the present invention. Common allotype variants in the human population are variants indicated by the letters a, f, n and z.

[0093] An "isolated" antibody is an antibody that has been identified, separated and / or recovered (e.g., naturally or recombinantly) from a component of its production environment. In some embodiments, an isolated polypeptide is independent of all other components of its production environment. Contaminant components in its production environment (e.g., produced by recombinant transfected cells) are materials that will generally interfere with the research, diagnostic, or therapeutic use of the antibody, and may include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the polypeptide is purified: (1) to greater than 95% by weight of the antibody, as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (1) by using a spinning cup sequencer to a degree sufficient to obtain at least 15 N-terminal residues or internal amino acid sequence, or (3) by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver staining. An isolated antibody contains the antibody in situ within a recombinant cell, since at least one component of the antibody's natural environment will not be present. However, an isolated polypeptide is typically prepared by at least one purification step.

[0094] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for mutations and / or post-translational modifications (e.g., isomerization, amidation) that may occur naturally in a small amount, the individual antibodies comprising the population are identical. In certain embodiments, the monoclonal antibody has a C-terminal cut at the heavy chain and / or light chain. For example, at the C-terminal of the heavy chain and / or light chain, 1, 2, 3, 4 or 5 amino acid residues are cut. In certain embodiments, the C-terminal cut removes the C-terminal lysine from the heavy chain. In certain embodiments, the monoclonal antibody has an N-terminal cut at the heavy chain and / or light chain. For example, at the N-terminal of the heavy chain and / or light chain, 1, 2, 3, 4 or 5 amino acid residues are cut. In certain embodiments, a truncated form of the monoclonal antibody can be prepared by recombinant technology. In certain embodiments, the monoclonal antibody has high specificity for a single antigenic site. In certain embodiments, the monoclonal antibody has high specificity (such as a bispecific antibody or a multispecific antibody) for multiple antigenic sites. The modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including, for example, the hybridoma method, recombinant DNA methods, phage display technology, and techniques for producing human or human-like antibodies in animals having a portion or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0095] The terms "full-length antibody", "intact antibody" or "complete antibody" are used interchangeably to refer to an antibody in substantially complete form, relative to an antibody fragment. Specifically, a complete antibody comprises an antibody having a heavy chain and a light chain comprising an Fc region. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, a complete antibody may have one or more effector functions.

[0096] "Antibody fragments" include a portion of an intact antibody, such as an antigen binding region and / or 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 (dAb), Fab, Fab', F(ab')2, and Fv fragments; bifunctional antibodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062

[1995] ); single-chain antibody molecules, and multispecific antibodies formed by antibody fragments, 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), Fabs (s-Fab) linked to single domain antibodies, and combinations thereof. Single heavy chain antibodies or single light chain antibodies can be engineered, or in the case of the heavy chain, isolated from camelids, sharks, libraries or mice engineered to produce single heavy chain molecules.

[0097] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a name reflecting the ability to crystallize easily. The Fab fragment consists of the entire L chain together with the variable region domain (VH) of the H chain and the first constant domain (CHI) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, that is, it has a single antigen binding site. Pepsin treatment of antibodies produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen binding activities and is still able to cross-link antigen. The Fab' fragment differs from the Fab fragment in that it has several additional residues at the carboxyl terminus of the CHI domain containing one or more cysteines from the hinge region of the antibody. Fab'-SH is the name for Fab' herein, in which the cysteine ​​residues of the constant domains carry free thiol groups. F(ab')2 antibody fragments were originally produced in pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxyl-terminal portions of two H chains held together by disulfide bonds. The effector functions of antibodies are determined by the sequences and glycans in the Fc region, which is also recognized by Fc receptors (FcR) found on certain types of cells.

[0098] The term "diabody" refers to small antibody fragments with two antigen-binding sites comprising a heavy-chain variable (VH) domain connected to a light-chain variable (VL) domain in the same polypeptide chain (VH-VL).

[0099] "Amino acid sequence identity percentage (%)" about 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 a reference polypeptide sequence after the sequences are aligned and, if necessary, spaces are introduced to achieve the maximum sequence identity percentage and any conservative substitutions are not considered as part of the sequence identity. Alignment for the purpose of determining the amino acid sequence identity percentage can be achieved in various ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequence. For example, the amino acid sequence identity percentage of a given amino acid sequence A with, and or for a given amino acid sequence B (which can be alternatively described as a given amino acid sequence A with, and or for a given amino acid sequence B having or including a certain amino acid sequence identity percentage) is calculated as follows:

[0100] Multiply 100 by the fraction X / Y

[0101] Where X is the number of amino acid residues scored as identical matches in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A.

[0102] Antibody "effector function" refers to the biological activity attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region) and varies with 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.

[0103] Amino Acid: As used herein, the term "amino acid" refers in the 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 naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a d-amino acid; in some embodiments, the amino acid is an l-amino acid. "Standard amino acid" refers to any of the twenty standard l-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether it is synthetically prepared or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids, including the carboxyl and / or amino terminal amino acids in a peptide, can be modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups that can alter the circulating half-life of the peptide without adversely affecting its activity. Amino acids can participate in disulfide bonds. An amino acid may include one or more post-translational modifications, such as association with one or more chemical entities (e.g., a methyl group, an acetate group, an acetyl group, a phosphate group, a formyl moiety, an isoprenoid group, a sulfate group, a polyethylene glycol moiety, a lipid moiety, a carbohydrate moiety, a biotin moiety, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to a free amino acid and / or an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

[0104] 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, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals may be transgenic animals, genetically engineered animals, and / or clones.

[0105] At risk: As used herein, an individual who is "at risk for a disorder" may or may not have had a detectable disease or disease symptom prior to treatment methods described herein, and may or may not display a detectable disease or disease symptom. "At risk" means that the individual has one or more risk factors, which are measurable parameters associated with the development of a disease as known in the art. An individual who has one or more of these risk factors has a higher likelihood of developing the disorder than an individual who does not have one or more of these risk factors.

[0106] Biological activity: As used herein, the phrase "biological activity" refers to the property of any agent that is active in a biological system, particularly in an organism. For example, an agent that has a biological effect on an organism when administered to the organism is considered to be biologically active.

[0107] 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 a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0108] Pharmaceutical carrier: As used herein, the term "carrier" includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers are typically aqueous pH buffered solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citric acid 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 dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN TM , polyethylene glycol (PEG) and PLURONICS TM .

[0109] Chronic: As used herein, "chronic" administration refers to administration of a drug in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is a treatment that is performed non-continuously without interruption, but is cyclic in nature.

[0110] Disease: As used herein, the term "disease" is a pathological condition, e.g., one that can be identified by symptoms or other identifying factors as emanating from a healthy or normal state. 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.

[0111] Delivery: As used herein, the term "delivery" encompasses both local delivery and systemic delivery. For example, delivery of mRNA encompasses situations where the mRNA is delivered to the target tissue and the encoded protein is expressed and retained in the target tissue (also referred to as "local distribution" or "local delivery"), and situations where the mRNA is delivered to the target tissue and the encoded protein is expressed and secreted into the patient's circulatory system (e.g., serum) and is distributed systemically and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[0112] Dosing interval: As used herein, in the context of a method for treating a disease, the term "dosing interval" is the frequency of administering a therapeutic composition (e.g., an mRNA composition) at an effective dose of mRNA in a subject (mammal) in need thereof, such that one or more symptoms associated with the disease are alleviated; or one or more biomarkers associated with the disease are reduced at least within the dosing interval period. In the present disclosure, dosing frequency and dosing interval can be used interchangeably.

[0113] Effective amount: As used herein, "effective amount" refers to an amount that effectively achieves the desired or indicated effect (including therapeutic or preventive results) at least at the required dosage and time period. The effective amount can be provided in one or more administrations. A "therapeutically effective amount" is at least the minimum concentration required to achieve a measurable improvement in a specific condition. In this article, the therapeutically effective amount can vary according to factors such as the disease state, the patient's age, sex and weight, and the ability of the antibody to elicit a desired response in an individual. The therapeutically effective amount can also be an amount in which the therapeutically beneficial effect exceeds any toxic or deleterious effect of the targeted cytokine. A "preventive effective amount" refers to an amount that effectively achieves the desired preventive result at the required dosage and time period. Usually, but not necessarily, because the preventive dose is used for subjects before the disease or at an earlier stage of the disease, the preventive effective amount is less than the therapeutically effective amount.

[0114] Effective dose: As used herein, an effective dose is a dose of mRNA in a pharmaceutical composition that, when administered to a subject in need thereof, according to the methods of the present invention, and thus a mammalian subject, is effective in producing a desired result in the subject, such as alleviation of symptoms associated with a disease.

[0115] Host cell: As used herein, the term "host cell" is a single cell or cell culture that can be or has been a recipient of any recombinant vector(s) or isolated polynucleotide(s), such as a vector or polynucleotide for engineering a cleavable Fc domain, or a masked cytokine of the present disclosure. 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.

[0116] Improvement, increase or decrease: As used herein, the terms "improvement," "increase," or "decrease," or grammatical equivalents, refer to values ​​relative to a baseline measurement, such as a measurement in the same individual prior to starting a treatment described herein, or a measurement in a control subject (or multiple control subjects) in the absence of a treatment described herein. A "control subject" is a subject having the same form of disease as the subject being treated, who is approximately the same age as the subject being treated.

[0117] In conjunction with: As used herein, "in conjunction with" or "in combination with" refers to the administration of a therapeutic modality in addition to another therapeutic modality. Thus, "in conjunction with" or "in combination with" refers to the administration of another therapeutic modality before, during, or after the administration of one therapeutic modality to an individual.

[0118] Individual: As used herein, an "individual" or "subject" is a mammal. For the purposes of treatment, "mammals" include humans, domestic and farm animals, and zoo, sport or pet animals, such as dogs, horses, rabbits, cows, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual or subject is a human.

[0119] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than in a multicellular organism.

[0120] In vivo: As used herein, the term "in vivo" refers to events that occur in multicellular organisms such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0121] 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 contaminant nucleic acid molecule that is normally associated with the environment in which it is produced. In some embodiments, the isolated nucleic acid is free of all components associated with the environment in which it is produced. The isolated nucleic acid molecules encoding the polypeptides and cytokine polypeptides herein are in a form that is different from the form or environment in which they are found in nature. Thus, isolated nucleic acid molecules are distinguished from nucleic acids encoding the polypeptides and cytokine polypeptides herein that are naturally present in cells.

[0122] Pharmaceutical formulation: As used herein, the term "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

[0123] Moiety: As used herein, the term "moiety" refers to a substructure that is a part of a molecule.

[0124] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to substances that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0125] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed of an amino group with an inorganic acid (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid) or an organic acid (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid), or by using other methods used in the art (such as ion exchange) to form salts. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, sulfonate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, dodecylsulfate, 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, sodium p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N+(C1-4 alkyl)4 salts. Representative alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Where appropriate, additional pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates and arylsulfonates. Additional pharmaceutically acceptable salts include salts formed by quaternization of amines, which are carried out using appropriate electrophilic reagents (e.g., alkyl halides) to form quaternized alkylated amino salts.

[0126] Polypeptide: As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, such as a polymer of at least 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. Throughout this disclosure, the standard three-letter or one-letter names for amino acids are used. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0127] Polynucleotide: As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of at least 5, 6, 7, 8, 9 or 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is intended to include single-stranded and double-stranded forms of DNA and / or RNA. Throughout this disclosure, the term is used interchangeably with "nucleic acid molecule."

[0128] Prevention: As used herein, the term "prevention" includes providing prevention of the occurrence or recurrence of a disease in an individual. An individual may be susceptible to a disease, be susceptible to a disease, or be at risk of developing a disease, but has not yet been diagnosed with the disease. In some embodiments, the targeted cytokines described herein are used to delay the development of a disease.

[0129] 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). Humans include prenatal and postnatal forms. In many embodiments, the subject is a human. The subject can be a patient, which refers to a person who presents to a medical provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient". A subject may have a disease or condition or be susceptible to a disease or condition, but may or may not show symptoms of the disease or condition.

[0130] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the total or nearly total range or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, reach completion and / or proceed to completion or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0131] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by a disease to be treated. In some embodiments, target tissues include those tissues that display disease-associated pathology, symptoms, or characteristics.

[0132] 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 a disease, disorder and / or condition. One of ordinary skill in the art will appreciate that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0133] Treatment: As used herein, the terms "treat," "treatment," or "treating" refer to any method for partially or completely alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset, reducing the severity of, and / or reducing the incidence of, one or more symptoms or features of a particular disease, disorder, and / or condition. The term may refer to a clinical intervention designed to alter the natural processes of the treated individual or cell during the course of clinical pathology. Treatment may be applied to subjects who do not show signs of the disease and / or only show early signs of the disease in order to reduce the risk of developing pathology associated with the disease. For example, if one or more symptoms associated with a disorder (e.g., a neoplastic disease) are alleviated or eliminated, the individual is successfully "treated." For example, if the treatment improves the quality of life of an individual with the disease, reduces the dose of other drugs required to treat the disease, reduces the frequency of recurrence of the disease, reduces the severity of the disease, slows the development or progression of the disease, and / or prolongs the survival of the individual, the individual is successfully "treated."

[0134] Vector: As used herein, the term "vector" refers to a macromolecule or complex of molecules, typically a virus or plasmid, comprising a polynucleotide that is delivered to a host cell or organism in vitro or in vivo.

[0135] Various aspects of the present invention are described in detail in the following sections. The use of sections is not intended to limit the present invention. Each section may apply to any aspect of the present invention. In this application, the use of "or" means "and / or" unless otherwise stated. DETAILED DESCRIPTION

[0136] I. Introduction

[0137] It is desirable to specifically deliver and activate therapeutic agents such as cytokines to tumor cells to increase the safety of such treatments (e.g., to avoid targeting normal cells). The present invention provides methods and compositions for precision treatment of such cancers. In particular, the present invention relates to a universal cleavable carrier platform, together with the masking portion of the present invention, for precision tumor treatment, etc. The cleavable carrier of the present invention can be connected to a therapeutic agent (e.g., a cytokine) for tumor-specific delivery and activation.

[0138] In general, 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 the therapeutic molecule and / or the masking portion via a non-cleavable linker. The masked therapeutic agent comprising such a cleavable carrier portion is capable of releasing an active therapeutic molecule from the masking portion when the engineered tumor-associated protease site within the cleavable carrier portion is cleaved.

[0139] As used herein, the term "cleavable carrier" refers to any agent that can be enzymatically or non-enzymatically cleaved from the composition of the invention. For example, the cleavable carrier can be a protein, a polypeptide, and a domain thereof. In the context of the present disclosure, the terms "cleavable carrier" and "cleavable domain" are used interchangeably. The cleavable carrier portion of the present invention provides enzymatically induced prodrug activation, characterized in that cleavage of the carrier portion controls the release of the active therapeutic agent.

[0140] The cleavable carrier portion can be a half-life extension domain derived from albumin, transferrin or tissue factor. The cleavable carrier portion can be an antigen targeting domain selected from the group consisting of: immunoglobulin, Fab, F(ab)2, scFv, VHH, ScAb and nanobodies. Tumor-associated protease cleavage sites are engineered within the antigen targeting domain. The carrier portion can be an Fc domain, wherein at least one tumor-associated protease cleavage site is engineered, for example, by amino acid replacement at a specific position of the Fc domain.

[0141] In addition to the aforementioned cytokines, therapeutic molecules may comprise any therapeutic agent that has a functional effect on the disease, such as therapeutic proteins, cell engagers, and co-stimulatory domains.

[0142] The masking moiety in the masked therapeutic agent of the present invention binds to the therapeutic molecule, shielding the therapeutic molecule 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.

[0143] The masked therapeutic agent may further include a targeting moiety; the targeting moiety may 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 case, the cytokine is also referred to as a targeted cytokine.

[0144] According to the present disclosure, the masked therapeutic agent can be interpreted as a fusion polypeptide. After the engineered tumor-associated protease site in the carrier portion is cleaved, the polypeptide is cleaved, and the masked therapeutic molecule is then released and activated to perform its function in the target cell, tissue and / or organ (e.g., in tumor tissue). The fusion polypeptide can be produced by recombinant techniques known in the art.

[0145] In particular, the present disclosure provides an engineered Fc domain, which includes a tumor-associated protease cleavage site. The engineered Fc domain with a tumor-associated protease cleavage site provides a universal cleavable platform that can be connected to a therapeutic agent (such as a masked cytokine) to control its activation. The engineered cleavable Fc domain of the present invention can be fused with a cytokine, a masking moiety and / or a targeting moiety to produce a prodrug. The prodrug comprising the engineered Vc domain of the present invention becomes active at the disease site and can specifically target the cells of interest to effectively treat cancer without causing undesirable side effects.

[0146] The present disclosure also provides, inter alia, therapeutic agents and pharmaceutical compositions comprising engineered Fc domains and / or targeted cytokines and formulations thereof. Methods of using such agents and compositions and formulations for treating diseases (eg, cancer) are also provided.

[0147] III. Composition

[0148] In the present invention, a cleavable carrier and a composition comprising the cleavable carrier are particularly provided. As a non-limiting example, the cleavable carrier is a cleavable Fc domain. The composition of the present invention includes a cleavable Fc domain. In the composition described herein, a masked cytokine is provided. The masked cytokine includes a cytokine portion, a masking portion, and an engineered cleavable Fc domain including a tumor-associated cleavage site, wherein the engineered cleavable Fc domain is fused to the cytokine portion or the masking portion so that the masking portion binds the cytokine portion, and when the tumor-associated protease cleavage site of the engineered Fc domain is cleaved, the cytokine portion is released from the masking portion. The engineered cleavable Fc domain is fused to the cytokine molecule and / or the masking portion via a non-cleavable linker. The cytokine prodrug connected to the cleavable Fc domain increases the therapeutic efficacy of the cytokine in vivo.

[0149] Cleavable vector

[0150] As used herein, the term "cleavable carrier" refers to any agent that can be enzymatically or non-enzymatically cleaved from the composition of the invention. For example, the cleavable carrier can be a protein, a polypeptide, and a domain thereof. In the context of the present disclosure, the terms "cleavable carrier" and "cleavable domain" are used interchangeably. The cleavable carrier portion of the present invention provides enzymatically induced prodrug activation, characterized in that cleavage of the carrier portion controls the release of the active therapeutic agent.

[0151] The cleavable carrier portion can be a half-life extension domain derived from albumin, transferrin or tissue factor. The cleavable carrier portion can be an antigen targeting domain selected from the group consisting of: immunoglobulin, Fab, F(ab)2, scFv, VHH, ScAb and nanobodies. Tumor-associated protease cleavage sites are engineered within the antigen targeting domain. The carrier portion can be an Fc domain, wherein at least one tumor-associated protease cleavage site is engineered, for example, by amino acid substitution at a specific position of the Fc domain.

[0152] Engineering cleavable Fc domain

[0153] According to the present disclosure, the carrier portion is an Fc domain derived from an immunoglobulin such as IgM, IgG1, IgG2, IgG3, IgG4, IgD, IgE and IgA, or a variant or fragment thereof. Thus, the Fc domain is genetically engineered to be enzymatically cleavable. The engineered Fc domain is referred to as a "cleavable Fc domain".

[0154] As used herein, the term "Fc domain" refers to a polypeptide or fragment from an immunoglobulin. The Fc domain or its fragment can be any antibody or fragment thereof. The Fc domain is derived from an antibody or fragment thereof that is naturally present in an animal (e.g., a mammal) or prepared by any technique known in the art. In some embodiments, the Fc domain is a human Fc domain.

[0155] In some embodiments, the Fc domain includes a first Fc polypeptide and a second Fc polypeptide. In some embodiments, the Fc domain is from any antibody or fragment thereof including a heavy chain Fc polypeptide or a light chain Fc polypeptide. In some embodiments, the Fc domain includes a portion of a heavy chain polypeptide or a light chain polypeptide. In some embodiments, an antibody or a fragment thereof includes an Fc domain or a fragment thereof. In some embodiments, the Fc domain is derived from an antibody or a fragment thereof including a hinge region, a CH2 domain, and a CH3 domain or a fragment thereof. In some embodiments, the Fc domain includes only the constant domain of a heavy chain polypeptide. In some embodiments, the Fc domain includes only the constant domain of a light chain polypeptide. In some embodiments, the Fc domain includes a first Fc polypeptide having a CH2 domain and a CH3 domain, and a second Fc polypeptide having a CH2 domain and a CH3 domain. In some embodiments, the Fc domain includes a first Fc polypeptide having a CH3 domain and a second Fc polypeptide having a CH3 domain. In some embodiments, the Fc domain includes a first Fc polypeptide having a CH2 domain and a CH3 domain, and a second Fc polypeptide having a CH3 domain. In some embodiments, the Fc domain includes a first Fc polypeptide having a CH2 domain and a CH3 domain, 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 a CH2 domain and a CH3 domain.

[0156] In some embodiments, the first Fc polypeptide and the second Fc polypeptide are connected via a linker (such as a short peptide linker). In some embodiments, the linker is non-cleavable.

[0157] The engineered cleavable Fc domain includes one or more cleavable sites. As used herein, "cleavage site" refers to a recognizable site for cutting a portion of a cleavable peptide found in any Fc domain."cleavage site" can be an amino acid sequence recognized and cut by a cleavage agent, such as a short peptide motif. The cleavage site can be an amino acid sequence naturally occurring in the Fc domain. Additionally and / or alternatively, the cleavage site can be introduced into the cleavable portion of the Fc domain by mutation (e.g., amino acid insertion, substitution and deletion). Mutation does not change other activities of the Fc domain. In certain embodiments, one or more cleavage peptide motifs can be engineered in a cleavable Fc domain as described herein. The cleavage agent can be an enzyme, such as a protease. In certain embodiments, the cleavage site is a protease cleavage site, so that the cleavable Fc domain is proteolytically cleavable. Protease is an enzyme that cuts and hydrolyzes the peptide bond between two specific amino acid residues of the target substrate protein. Protease generally recognizes a specific peptide motif and cuts the peptide bond between two specific amino acid residues in the short peptide motif.

[0158] The Fc domain can be engineered to contain 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 non-limiting examples, the cleavable Fc domains described herein can be cleaved by disease-associated or tissue-selective proteases selected from the group consisting of matrix metalloproteinases (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, tissue cathepsin F, cathepsin K, cathepsin L, cathepsin V, cathepsin S, cathepsin W), ADAM, ADAMTS, kallikreins 1 to 15, HTRA1-2-3, HGFAc, PRSS, TMPRSS, elastase, PR-3, granzymes (granzymes A, B, M, H and K), fibroblast activation protein (FAP), plasmin, urokinase plasminogen activator (uPA), tryptase, caspase, thrombin, asparagine endopeptidase, chymotrypsin, collagenase, aspartic proteinase A and matriptase1-2.

[0159] In some embodiments, the cleavable Fc domain as described herein includes at least one engineered tumor-associated protease cleavage site. As provided herein, a "tumor-associated protease cleavage site" is an amino acid sequence recognized by a protease, and the expression of the protease is specific or upregulated for tumor cells or their tumor cell environment. In some embodiments, the tumor-associated protease is a matrix metalloproteinase (MMP), which is 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 another embodiment, the tumor-associated protease is MMP9. In another embodiment, the tumor-associated protease is MMP10. In some embodiments, the protease is cathepsin B. In other embodiments, the protease is matriptase.

[0160] The advantage of such engineered cleavable Fc domains is that a general design framework is provided; such that fusion of the engineered cleavable Fc domain to a therapeutic agent and subsequent cleavage at the cleavage site allows release of the agent in a specific environment (e.g., a tumor microenvironment).

[0161] The tumor cell environment is complex and can include a variety of different proteases. Therefore, the precise site at which a given cleavable peptide will be cleaved in a tumor cell environment can vary between tumor types, between patients with the same tumor type, and even between cleavage products formed in the same tumor depending on a specific tumor cell environment. In addition, even after cleavage, additional modification of the initial cleavage product, such as by removing one or two terminal amino acids, can also occur through further action of the protease in the tumor cell environment. Therefore, after administration of a single structure of a targeted cytokine as described herein, a distribution of cleavage products can be expected to form in the patient's tumor cell environment.

[0162] It should be understood that the cleavage site as mentioned herein refers to the site between two specific amino acid residues in the cleavable peptide, which is the target of a protease known to be associated with the tumor cell environment. In this sense, there is more than one cleavage site in the cleavable peptide as described herein, wherein different proteases cut the cleavable peptide at different cleavage sites. It is also possible that more than one protease can act on the same cleavage site in the cleavable peptide. Discussion of protease cleavage sites can be found in the art.

[0163] Therefore, the cleavable Fc domain disclosed herein can be cleaved by one or more proteases. In some embodiments, the cleavage site of the cleavable Fc domain is a cleavage site of one or more tumor-associated proteases. In some embodiments, the cleavage site of the cleavable Fc domain is a cleavage site of one or more tissue-selective proteases. In some embodiments, the cleavage site of the cleavable Fc domain is a cleavage site of one or more inflammation-associated proteases. In other embodiments, the cleavable Fc domain includes one or more cleavage sites of other disease-associated proteases.

[0164] In some embodiments, the cleavable Fc domain is a substrate for a protease that is co-localized in a region or tissue or organ expressing a cytokine receptor.

[0165] The cleavable peptide motif in the Fc domain comprises at least 3 amino acid residues. In some embodiments, the cleavable peptide motif is a 3-mer (i.e., a peptide of 3 amino acids in length), a 4-mer (i.e., a peptide of 4 amino acids in length), a 5-mer (i.e., a peptide of 5 amino acids in length), a 6-mer (i.e., a peptide of 6 amino acids in length), a 7-mer (i.e., a peptide of 7 amino acids in length), an 8-mer (i.e., a peptide of 8 amino acids in length), a 9-mer (i.e., a peptide of 9 amino acids in length), a 10-mer (i.e., a peptide of 10 amino acids in length) , 11-mer (i.e., a peptide of 11 amino acids in length), 12-mer (i.e., a peptide of 12 amino acids in length), 13-mer (i.e., a peptide of 13 amino acids in length), 14-mer (i.e., a peptide of 14 amino acids in length), 15-mer (i.e., a peptide of 15 amino acids in length), 16-mer (i.e., a peptide of 16 amino acids in length), 17-mer (i.e., a peptide of 17 amino acids in length) or 18-mer (i.e., a peptide of 18 amino acids in length).

[0166] In some embodiments, the length of the cleavable peptide motif in the Fc domain is 3 to 18 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 5 to 10 amino acids, or 5 to 8 amino acids, or 6 to 10 amino acids, or 7 to 10 amino acids, or 6 to 12 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 3 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 4 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 5 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 6 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 7 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 8 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 9 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 10 amino acids.

[0167] In some embodiments, the protease cleavage site in the engineered cleavable Fc domain is located in the hinge region, in the CH2 domain, in the CH3 domain and / or in the CH2-CH3 domain joint region. In some embodiments, the protease cleavage site in the engineered cleavable Fc domain is located in the F chain region. In some embodiments, the protease cleavage site in the engineered cleavable Fc domain is located in the FG loop region. In some embodiments, the protease cleavage site in the engineered cleavable Fc domain is located in the G chain region.

[0168] In some embodiments, a cleavable site within an engineered cleavable Fc domain can be generated based on a short amino acid motif that is close to the cleavage site of a protease.

[0169] Based on the short peptide sequence of the Fc domain, one or more mutations (e.g., amino acid substitutions) can be introduced into the peptide sequence to produce a protease cleavage site. As a non-limiting example, one or more protease cleavage sites can be produced based on the short peptide sequence of SEQ ID NO:98, 101, 103 and 106.

[0170] In some embodiments, a protease cleavage site is generated that can be recognized by MMP2. In some embodiments, a protease cleavage site is generated that can be recognized by MMP3. In some embodiments, a protease cleavage site is generated that can be recognized by MMP9. In some embodiments, a protease cleavage site is generated that can be recognized by MMP10. In some embodiments, a protease cleavage site is generated that can be recognized by cathepsin B. In some embodiments, a protease cleavage site is generated that can be recognized by matriptase.

[0171] 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 NO: 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 includes AGRRAAK (SEQ ID NO: 147). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes FRLWA (SEQ ID NO: 148). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes FRLWS (SEQ ID NO: 149). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain includes 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 comprises GIVRAKGV (SEQ ID NO: 155). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises ALFKSSFP (SEQ ID NO: 156). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SGRRSPGG (SEQ ID NO: 157).In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SLGRRPGG (SEQ ID NO: 158). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SLSGRRGG (SEQ ID NO: 159). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SLSLGRRG (SEQ ID NO: 160). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises SLSLSGRR (SEQ ID NO: 161). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises GGPRRL (SEQ ID NO: 162). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises GGPLRL (SEQ ID NO: 163). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises GGPKLL (SEQ ID NO: 164). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises 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 PLKLSRA (SEQ ID NO: 195).In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises PLGLSGRS (SEQ ID NO: 196). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises PLGLRSRA (SEQ ID NO: 197). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises PLGLKSRA (SEQ ID NO: 198). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises RGSRAG (SEQ ID NO: 199). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises RLSRGK (SEQ ID NO: 200). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises RGSRGG (SEQ ID NO: 201). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises KGSRAG (SEQ ID NO: 202). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises KLSRGK (SEQ ID NO: 203). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises GRSRAG (SEQ ID NO: 204). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises LRSRGK (SEQ ID NO: 205). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises GRSRGG (SEQ ID NO: 206). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises GKSRAG (SEQ ID NO: 207). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises LKSRGK (SEQ ID NO: 208). In some embodiments, the cleavable peptide motif within the engineered cleavable Fc domain comprises VPLSLYSGP (SEQ ID NO: 209).

[0172] Cleavable substrate incorporated into G chain of Fc domain

[0173] In some embodiments, the engineered cleavable Fc domain comprises one or more replacements in the CH3 domain. In some embodiments, the engineered cleavable Fc domain comprises one or more replacements in the C-terminal region within the Fc domain. In some embodiments, the engineered cleavable Fc domain comprises one or more replacements in the G chain within the Fc domain. In some embodiments, the engineered cleavable Fc domain comprises one or more replacements in a position between 436 and 447 according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises one or more replacements in a position between 438 and 447 according to EU numbering.

[0174] In some embodiments, the cleavable peptide motif is located between positions 438 and 445 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 439 and 446 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 440 and 447 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 438 and 447 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 437 and 444 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 440 and 443 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 441 and 444 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 442 and 445 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 444 and 447 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 441 and 447 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 443 and 447 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 436 and 443 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 442 and 447 according to EU numbering.

[0175] In some embodiments, the engineered cleavable Fc domain comprises Q438V, K439P, S440L, L441S, S442L, L443Y, P445G substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises K439V, S440P, S444Y, P445S substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440V, L441P, S442L, L443S, S444L, P445Y, G446S substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises Q438M, K439P, S440Y, L441D, S442L, L443Y, S444H substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises K439M, S440P, L441Y, S442D, S444Y, P445H, G446P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440M, L441P, S442Y, L443D, S444L, P445Y, G446H, G447P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises Q438R, K439A, S440A, L441A, S442V, L443K substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises K439R, S440A, L441A, S442A, L443V, S444K, P445S, G446P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440R, L441A, S442A, L443A, S444V, P445K, G446S, G447P replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises K439R, S440P, S442A, S444W, P445R, G446S replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises Q438A, K439P, S440A, L441G, S442L, L443I, S444V, G446Y, G447N replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises K439P, S440V, L441S, S442L, L443R, P445G, G446S replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises Q438P, K439A, S440N, S442V, L443A, S444P, P445D, G446P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440P, S442G substitutions according to EU numbering.In some embodiments, the engineered cleavable Fc domain comprises K439G, S440P, S442G replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L441P, S442L, L443G, S444L replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440G, L441P, S442L, L443G, S444L replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442P, S444G, P445L replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S444P, P445L, G447L replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L443G, S444P, P445L, G447L replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L443G, S444P, P445L, G447L substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440M, L441P, S442Y, L443D, S444L, P445Y, G446H, G447P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442G, L443G, S444P, P445L, G447L substitutions according to EU numbering.

[0176] In some embodiments, the engineered cleavable Fc domain comprises S440R, L441S, S442K, L443Y, S444L, P445A, G446T, G447A substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440G, L441R, S442P, L443R, S444H, P445Q, G447V substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S444G, P445L, G446F substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S444G, P445F, G446L substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L441A, S442G, L443R, S444R, P445A, G446A, G447K substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L443F, S444R, P445L, G446W, G447A substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L443F, S444R, P445L, G446W, G447S substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440N, L441F, S442F, L443G, S444V, P445G, G447E substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440P, L441M, S442K, L443R, S444L, P445T, G446L, G447A substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises K439P, S440M, L441K, S442R, S444T, P445L, G446A substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises Q438P, K439M, S440K, L441R, S442L, L443T, S444L, P445A substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440F, L441P, S442L, L443A, S444T, P445Y, G446A, G447P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440F, S442V, L443G, S444G, P445A, G446S, G447L substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440K, L441P, S442M, L443Q, S444F, P445L, G447D substitutions according to EU numbering.In some embodiments, the engineered cleavable Fc domain comprises S440G, L441I, S442V, L443R, S444A, P445K, G447V replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440A, S442F, L443K, P445S, G446F, G447P replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L441G, S442R, L443R replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442G, L443R, S444R replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L443G, S444R, P445R replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S444G, P445R, G446R replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises P445G, G446R, G447R replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442G, L443G, S444P, P445R, G446R, G447L replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442G, L443G, S444P, P445L, G446R, G447L replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442G, L443G, S444P, P445K, G446L, G447L replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442G, L443G, S444P, P445R, G446N, G447L replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442G, L443G, S444P, P445R, G446M, G447L replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440E, L441H, S442L, L443R replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440F, L441R, L443G, S444V replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442L, S444R, P445T, G447N replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440A, L441G, S442L, L443R replacements 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 includes S442F, L443R, S444A, G447P replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain includes S440W, S442F, L443R, G446L replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain includes L441R, S442L, L443R, G446Q replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain includes S442V, P445G, G446R, G447R replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain includes S440K, L441Q, S442L, L443R, S444H, P445M, G446R replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440L, L441S, S442G, L443R, P445D, G446N, G447H replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S444L, P445S, G447K replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S444L, P445S, G447R replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440R, L441Q, S442A, L443R, S444V, P445V replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440R, L441Q, S442R, L443R, S444V, P445V replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises K439R, S440Q, L441R, S442R, L443V, S444V, P445G substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises Q438R, K439Q, S440R, L441R, S442V, L443V, S444G, P445G substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440R, L441Q, S442Y, L443R, S444V, P445V substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L441K, S442G, L443R, P445L, G446I substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440P, L441R, S442F, L443K, S444I, P445I substitutions according to EU numbering.In some embodiments, the engineered cleavable Fc domain comprises S440K, L441Q, S442L, L443R, S444V, P445V, G446N substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440I, L441Q, S442P, L443R, S444I, P445T substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440K, L441Q, L443R, S444K, P445F, G446V, G447P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440G, L441R, S442Q, L443S, S444R, P445A substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L441G, S442R, L443S replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L441S, S442G, L443R replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L443G, S444R, P445S replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L443S, S444G, P445R, G446S replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises P445G, G446R, G447S replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L441P, S442L, L443R, S444L, P445S, G446R, G447A replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L441P, S442L, L443K, S444L, P445S, G446R, G447A substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440P, S442G, P445G, G446R, G447S substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440P, S442G, S444R, P445S, G446R, G447A substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S440P, S442G, S444K, P445S, G446R, G447A substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442R, L443G, P445R, G446A substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442R, P445R, G447K substitutions according to EU numbering.In some embodiments, the engineered cleavable Fc domain comprises S442R, L443G, P445R replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442K, L443G, P445R, G446A replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442K, P445R, G447K replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442G, L443R, P445R, G446A replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442L, L443R, P445R, G447K replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442G, L443R, P445R, G447K replacements according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442G, L443K, P445R, G446A substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises S442L, L443K, P445R, G447K substitutions according to EU numbering.

[0177] In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions shown in Table 5, Table 8a, or Table 11.

[0178] Table 11. Exemplary engineered Fc domains including cleavable substrates in the G chain

[0179]

[0180]

[0181]

[0182]

[0183]

[0184] In some embodiments, the engineered cleavable Fc domain also includes one or more replacements that allow two Fc polypeptides to heterodimerize. In some embodiments, the engineered cleavable Fc domain also includes Y349C, T366S, L368A and Y407V replacements. In some embodiments, the engineered cleavable Fc domain also includes S354C and T366W replacements. In some embodiments, the engineered cleavable Fc domain also includes Y349C, T366S, L368A, Y407V and N297A replacements. In some embodiments, the engineered cleavable Fc domain also includes S354C, T366W and N297A replacements. In some embodiments, the engineered cleavable Fc domain also includes Y349C, T366S, L368A, Y407V, N297A and I253A replacements. In some embodiments, the engineered cleavable Fc domain also includes S354C, T366W, N297A and I253A replacements.

[0185] In some embodiments, the engineered cleavable Fc domain is used as a carrier portion of a cytokine for treating cancer. The engineered cleavable Fc domain is fused to a masked cytokine molecule such that upon cleavage by 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 portion.

[0186] In some embodiments, in addition to incorporating one or more protease cleavage sites, the cleavable Fc domain may include additional mutations described herein.

[0187] Cleavable substrate incorporated into the F chain of the Fc domain

[0188] 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 in a position between 416 and 425 according to EU numbering.

[0189] In some embodiments, the cleavable peptide motif is located between positions 416 and 423 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 416 and 425 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 416 and 423 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 417 and 425 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 417 and 424 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 418 and 425 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 419 and 422 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 419 and 426 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 420 and 423 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 421 and 424 according to EU numbering.

[0190] In some embodiments, the engineered cleavable Fc domain comprises R416A, W417P, Q418A, Q419G, G420L, N421I, F423P, S424Y, C425N substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises R416I, W417S, Q418S, Q419G, G420L, N421L, V422S, F423G, S424R, C425S substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises R416V, W417P, Q418L, Q419S, G420L, N421Y, V422S, F423G, C425G substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises substitutions according to EU numbering of W417P, Q418A, Q419N, G420L, N421V, V422A, F423P, S424D, C425P. In some embodiments, the engineered cleavable Fc domain comprises substitutions according to EU numbering of W417V, Q418P, Q419L, G420S, N421L, V422Y, F423S, S424G, C425G. In some embodiments, the engineered cleavable Fc domain comprises substitutions according to EU numbering of Q418M, Q419P, G420Y, N421D, V422L, F423Y, S424H, C425P. In some embodiments, the engineered cleavable Fc domain comprises Q418P, Q419V, G420S, N421L, V422R, F423S, S424G, C425S substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises Q418R, Q419A, G420A, N421A, F423K, C425P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises Q418R, Q419P, G420L, N421A, V422L, F423W, S424R, C425S substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises Q418V, Q419P, G420L, N421S, V422L, F423Y, C425G substitutions according to EU numbering.

[0191] In some embodiments, the engineered cleavable Fc domain comprises Q419P, G420L, N421G, V422L, C425G substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises Q418V, Q419P, G420L, N421S, V422L, F423Y, C425G substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises G420P, N421L, V422G, F423L, C425G substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises N421P, V422L, F423G, S424L, C425G substitutions according to EU numbering.

[0192] In some embodiments, the engineered cleavable Fc domain comprises the substitutions shown in Table 11a.

[0193] Table 11a. Exemplary engineered Fc domains including cleavable substrates in the F chain

[0194]

[0195]

[0196] In some embodiments, the engineered cleavable Fc domain further comprises a substitution that allows heterodimerization of two Fc polypeptides. In some embodiments, the engineered cleavable Fc domain comprises the substitutions shown in Table 11a and comprises a serine at position 367 according to 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.

[0197] In some embodiments, the engineered cleavable Fc domain includes a combination of mutations listed in Table 11a and mutations that stabilize the Fc domain. In some embodiments, the engineered cleavable Fc domain includes a combination of mutations listed in Table 11a and C367S. In some embodiments, the engineered cleavable Fc domain includes a combination of mutations listed in Table 11a and C425G. In some embodiments, the engineered cleavable Fc domain includes a combination of mutations listed in Table 11a and S375C. In some embodiments, the engineered cleavable Fc domain includes a combination of mutations listed in Table 11a and P396C. In some embodiments, the engineered cleavable Fc domain includes a combination of mutations listed in Table 11a and 432C. In some embodiments, the engineered cleavable Fc domain includes a combination of mutations listed in Table 11a and T437C. In some embodiments, the engineered cleavable Fc domain includes a combination of mutations listed in Table 11a and S408C. In some embodiments, the engineered cleavable Fc domain includes a combination of mutations listed in Table 11a and 379C. In some embodiments, the engineered cleavable Fc domain comprises a combination of a mutation listed in Table 11a and W381C. In some embodiments, the engineered cleavable Fc domain comprises a combination of a mutation listed in Table 11a and L410C. In some embodiments, the engineered cleavable Fc domain comprises a combination of a mutation listed in Table 11a and K370C. In some embodiments, the engineered cleavable Fc domain comprises a combination of a mutation listed in Table 11a and F405C. In some embodiments, the engineered cleavable Fc domain comprises a combination of a mutation listed in Table 11a and 371C. In some embodiments, the engineered cleavable Fc domain comprises a combination of a mutation listed in Table 11a and S403C.

[0198] In some embodiments, the engineered cleavable Fc domain also includes Y349C, T366S, L368A and Y407V replacements. In some embodiments, the engineered cleavable Fc domain also includes S354C and T366W replacements. In some embodiments, the engineered cleavable Fc domain also includes Y349C, T366S, L368A, Y407V and N297A replacements. In some embodiments, the engineered cleavable Fc domain also includes S354C, T366W and N297A replacements. In some embodiments, the engineered cleavable Fc domain also includes Y349C, T366S, L368A, Y407V, N297A and I253A replacements. In some embodiments, the engineered cleavable Fc domain also includes S354C, T366W, N297A and I253A replacements.

[0199] Cleavable substrates are incorporated into the FG loop of the Fc domain

[0200] 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 in positions between 426 and 437 according to EU numbering.

[0201] In some embodiments, the cleavable peptide motif is located between positions 430 and 437 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 428 and 434 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 428 and 433 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 428 and 437 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 429 and 437 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 434 and 437 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 433 and 436 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 432 and 435 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 431 and 434 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 430 and 433 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 429 and 432 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 428 and 431 according to EU numbering.

[0202] In some embodiments, the engineered cleavable Fc domain comprises E430V, A431P, H433S, N434L, H435Y, Y436S, T437G substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises E430R, L432A, H433A, N434V, H435K, Y436S, T437P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises E430M, A431P, L432Y, H433D, N434L, H435Y, Y436H, T437P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises 428-ins-VP, H429L, E430S, A431L, L432Y, H433S, N434G substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises 428-ins-RA, H429A, E430A, A431V, L432K, H433S, N434P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises 428-ins-MP, H429Y, E430D, A431L, L432Y, N434P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises 428-ins-VPL, H429S, E430L, A431Y, L432S, H433G substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises 428-ins-RAA, H429A, E430V, A431K, L432S, H433P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises 428-ins-MPY, H429D, E430L, A431Y, L432H, H433P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises 428-ins-I, H429S, E430S, A431G, H433L, N434S, H435G, Y436R, T437S substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises E430R, A431P, H433A, N434L, H435W, Y436R, T437S substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises E430T, A431Q, L432K, H433P, N434L, H435G, Y436L, T437S substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises 428-ins-A, H429P, E430A, A431G, H433I, N434V, H435P, T437N substitutions according to EU numbering.In some embodiments, the engineered cleavable Fc domain comprises E430P, A431V, L432S, H433L, N434R, H435S, Y436G, T437S substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises H429P, E430A, A431N, H433V, N434A, H435P, Y436D, T437P substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises N434P, H435L, Y436G, T437L substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises H433P, N434L, H435G, Y436L substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises L432P, H433L, N434G, H435L substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises A431P, H433G, N434L substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises E430G, A431P, H433G, N434L substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises E430P, A431L, L432G, H433L substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises H429G, E430P, A431L, L432G, H433L substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises 428-ins-G, H429P, E430L, A431G, H433G substitutions according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises 428-ins-GGP, H429L, E430G, A431L, L432G substitutions according to EU numbering.

[0203] In some embodiments, the engineered cleavable Fc domain comprises the protease cleavage sites in Table 11b. The engineered cleavable Fc domain may comprise the substitutions shown in Table 11b.

[0204] Table 11b. Exemplary engineered Fc domains including cleavable substrates in the FG loop

[0205]

[0206]

[0207] In some embodiments, the engineered cleavable Fc domain further comprises a substitution that allows heterodimerization of two Fc polypeptides. In some embodiments, the engineered cleavable Fc domain further comprises a substitution that extends the half-life of the polypeptide comprising the engineered cleavable Fc domain.

[0208] In some embodiments, the engineered cleavable Fc domain comprises the substitutions shown in Table 11b and comprises a 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.

[0209] In some embodiments, the engineered cleavable Fc domain also includes Y349C, T366S, L368A and Y407V replacements. In some embodiments, the engineered cleavable Fc domain also includes S354C and T366W replacements. In some embodiments, the engineered cleavable Fc domain also includes Y349C, T366S, L368A, Y407V and N297A replacements. In some embodiments, the engineered cleavable Fc domain also includes S354C, T366W and N297A replacements. In some embodiments, the engineered cleavable Fc domain also includes Y349C, T366S, L368A, Y407V, N297A and I253A replacements. In some embodiments, the engineered cleavable Fc domain also includes S354C, T366W, N297A and I253A replacements.

[0210] In some embodiments, an engineered cleavable Fc domain comprising one or more substitutions between positions 416 and 416 or between positions 428 and 437 has reduced binding to Protein A.

[0211] Exemplary Engineered Cleavable Fc Domains with Cathepsin B or Matriptase Substrates

[0212] In some embodiments, the engineered cleavable Fc domain comprises a non-MMP protease cleavage site in 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 set of substitutions in Table 11c.

[0213] In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions in positions between 436 and 447 according to EU numbering to incorporate a cleavage site for Cathepsin B or Matriptase.

[0214] In some embodiments, the cleavable peptide motif is located between positions 440 and 447 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 444 and 447 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 441 and 447 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 443 and 447 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 438 and 445 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 436 and 443 according to EU numbering. In some embodiments, the cleavable peptide motif is located between positions 442 and 447 according to EU numbering.

[0215] Table 11c: Exemplary engineered Fc domains including non-MMP cleavage substrates

[0216]

[0217]

[0218]

[0219]

[0220] In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions in positions between 416 and 425 according to EU numbering to incorporate a cleavage site in Table 11c. In some embodiments, the engineered cleavable Fc domain comprises one or more substitutions in positions between 426 and 437 according to EU numbering to incorporate a cleavage site in Table 11c.

[0221] In some embodiments, the engineered cleavable Fc domain further comprises a substitution that allows heterodimerization of two Fc polypeptides. In some embodiments, the engineered cleavable Fc domain further comprises a substitution that can extend the half-life of the polypeptide comprising the engineered cleavable Fc domain.

[0222] In some embodiments, the engineered cleavable Fc domain comprises the substitutions shown in Table 11c and comprises a serine at position 367 according to EU numbering. In some embodiments, the engineered cleavable Fc domain comprises the substitutions shown in Table 11c and further comprises a knob mutation. In some embodiments, the engineered cleavable Fc domain comprises the substitutions shown in Table 11c and further comprises a hole mutation.

[0223] In some embodiments, the engineered cleavable Fc domain also includes Y349C, T366S, L368A and Y407V replacements. In some embodiments, the engineered cleavable Fc domain also includes S354C and T366W replacements. In some embodiments, the engineered cleavable Fc domain also includes Y349C, T366S, L368A, Y407V and N297A replacements. In some embodiments, the engineered cleavable Fc domain also includes S354C, T366W and N297A replacements. In some embodiments, the engineered cleavable Fc domain also includes Y349C, T366S, L368A, Y407V, N297A and I253A replacements. In some embodiments, the engineered cleavable Fc domain also includes S354C, T366W, N297A and I253A replacements.

[0224] Fc engineering to promote heterodimerization or increase half-life

[0225] An Fc domain or fragment thereof capable of FcRn-mediated recycling can reduce or otherwise delay the clearance of the targeted cytokine from the 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.

[0226] The cleavable Fc domain or its fragment can be derived from any antibody or its fragment. However, in some embodiments, the first Fc polypeptide or the second Fc polypeptide may not be combined with an 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, but the targeted cytokine has an extended half-life due to the inclusion of a second Fc polypeptide that can interact with the FcRn receptor, such as by including a heavy chain polypeptide. It is recognized in the art that the recycling mediated by FcRn requires the FcRn receptor to be combined with the Fc region of an antibody or its fragment. For example, studies have shown that residues I253, S254, H435 and Y436 (numbered according to the Kabat EU index numbering system) are important for the interaction between the human Fc region and the human FcRn complex. See, e.g., Firan, M. et al., Int. Immunol. 13 (2001) 993-1002; Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604). Various mutants of residues 248-259, 301-317, 376-382, and 424-437 (numbered according to the Kabat EU index numbering system) were also examined and reported. Yeung, YA et al., J. Immunol. 182 (2009) 7667-7671.

[0227] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide of the cleavable Fc domain each contain one or more modifications that promote non-covalent association of the first Fc polypeptide and the second Fc polypeptide. In some embodiments, the first Fc polypeptide comprises an IgG1 Fc domain or fragment thereof comprising mutations Y349C, T366S, L368A, and Y407V to form a 'hole' in the first half-life extension domain, and the second Fc polypeptide comprises an IgG1 Fc domain or fragment thereof comprising mutations S354C and T366W to form a 'knob' in the second half-life extension domain.

[0228] In some embodiments, the first Fc polypeptide and the second Fc polypeptide are each an IgG1, IgG2, or IgG4 Fc domain or a fragment thereof. In some embodiments, the first Fc polypeptide and the second Fc polypeptide are each an IgG1 Fc domain or a fragment thereof. Human IgG1 immunoglobulin heavy constant γ1 has the following sequence:

[0229]

[0230] In some embodiments, the first and second Fc polypeptides are derived from the sequence of human IgG1 immunoglobulin heavy constant gamma 1 having SEQ ID NO: 1 ('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.

[0231] In some embodiments, the first and Fc polypeptides each include the portion of SEQ ID NO: 1 shown above in bold, optionally with one or more amino acid modifications, namely:

[0232] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:2)

[0233] In some embodiments, the first Fc polypeptide and the second Fc polypeptide include SEQ ID NO: 2 with amino acid substitutions that promote association of the first Fc polypeptide and the second Fc polypeptide according to the 'knob into holes' approach. In some embodiments, the sequence SEQ ID NO: 2 contains mutations Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU numbering system) for forming a 'hole' in the first Fc polypeptide and mutations S354C and T366W (numbered according to the Kabat EU numbering system) for forming a 'knob' in the second Fc polypeptide. These modified sequences have SEQ ID NOs: 3 and 4 shown below:

[0234] First Fc polypeptide (Y349C, T366S, L368A and Y407V) SEQ ID NO: 3:

[0235] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0236] Second Fc polypeptide (S354C and T366W) SEQ ID NO:4:

[0237] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0238] In some embodiments, the first half-life prolonging domain and the second half-life prolonging domain each further comprises an amino substitution N297A numbered according to the Kabat EU numbering system:

[0239] First Fc polypeptide (Y349C, T366S, L368A, Y407V and N297A) SEQ ID NO: 5:

[0240] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0241] Second Fc polypeptide (S354C, T366W and N297A) SEQ ID NO:6:

[0242] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0243] In some embodiments, the first Fc polypeptide and the second Fc polypeptide each further comprise an amino substitution I253A numbered according to the Kabat EU numbering system.

[0244] In some embodiments, the first Fc polypeptide and the second Fc polypeptide each further comprise both amino substitutions N297A and 1253A, numbered according to the Kabat EU numbering system.

[0245] First Fc polypeptide (Y349C, T366S, L368A, Y407V, N297A and I253A) SEQ ID NO:7:

[0246] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0247] Second Fc polypeptide (S354C, T366W, N297A and I253A) SEQ ID NO): 8:

[0248] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0249] 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 of the amino acid sequences of any of SEQ ID NOs: 2-8.

[0250] 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 of the amino acid sequences of any of SEQ ID NOs: 2-8.

[0251] 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) compared to the amino acid sequence 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) compared to the amino acid sequence of any one of SEQ ID NOs: 2-8. The one or more modifications may be any modification or change described herein, including any modification or change that promotes heterodimerization of polypeptide chains and / or inhibits homodimerization of polypeptide chains, alters effector function, or enhances effector function in some embodiments.

[0252] In some embodiments, a protease cleavage site as described herein can be introduced into any of SEQ ID NOs: 2-8.

[0253] In some embodiments, the cleavable Fc domain can further include one or more amino acid replacements that alter effector function. In some embodiments, the half-life extension domain is an IgG1 Fc domain or a fragment thereof and includes one or more amino acid replacements 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 amino acid substitutions: V234A and G237A numbered according to the Kabat EU numbering system; H268Q, V309L, A330S and A331S; and / or V234A, G237A, P238S, H268A, V309L and A330S. 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 amino acid substitutions: L235A, G237A, and E318A numbered according to the Kabat EU numbering system; S228P, L234A, and L235A; H268Q, V309L, A330S, and P331S; and / or S228P and L235A. 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.

[0254] In some embodiments, the cleavable Fc domain further comprises one or more amino acid substitutions to enhance effector function. In some embodiments, the half-life extension domain is an IgG1 Fc domain or a fragment thereof and comprises an amino acid substitution according to Kabat EU numbering system numbers S298A, E333A and K334A; S239D and I332E; S239D, A330L and I332E; P247I and A339D or A339Q; D280H and K290S; D280H, K290S and S298D or S298V; F243L, R292P and Y300L; F243L, R292P, Y300L and P396L; F243L, R292P, Y300L, V305I and P396L; G236A, S239D and I332E; K326A and E333A; K326W and E333S; K290E, S298G, and T299A; K290E, S298G, T299A, and K326E; K290N, S298G, and T299A; K290N, S298G, T299A, and K326E; K334V; L235S, S239D, and K334V; K334V and Q331M, S239D, F243V, E294L, or S298T; E233L, Q311M, and K334V; L234I, Q311M, and K334V; K334V and S298T, A330M, or A330F; K334V, Q311M, and A330M or A3 30F; K334V, S298T and A330M or A330F; K334V, S239D and A330M or S298T; L234Y, Y296W and K290Y, F243V, or E294L; 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 I332 E; 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 S239E or S239Q; S239E, V264I, A330Y and I332E; A330Y, I332E and S 239D or S239N; A330L, I332E and S239D or S239N; V264I, S298A and I332E; S298A, I332E and S239D or S239N; S239D, V264I and I332E; S239D, V264I, S298A and I332E; S239D, V264I, A330L and I332E; S239 D, I332E, and A330I; P230A; P230A, E233D, and I332E; E272Y; K274T, K274E, K274R, K274L, or K274Y; F275W; N276L; Y278T; V302I; E318R; S324D, S324I, or S324V; K326I or K326T; T335D, T335R, or T335Y; V240I and V266I; S239D, A330Y, I332E and L234I; S239D, A330Y, I332E and L235D; S239D, A330Y, I332E and V240I; S239D, A330Y, I332E and V264T; and / or S239D, A330Y, I332E and K326E or K326T. In some embodiments, the cleavable Fc domain is IgG1 Fc domain or fragment thereof, and comprising one or more amino acid replacements 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. ;

[0255] In some embodiments, the cleavable Fc domain further comprises one or more amino acid substitutions that enhance the binding of the half-life extension domain to FcRn. In some embodiments, the one or more amino acid substitutions increase the binding affinity of the Fc-containing polypeptide (e.g., heavy chain polypeptide or Fc domain or fragment thereof) to FcRn at acidic pH. In some embodiments, the half-life extension domain comprises one or more amino acid replacements 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.

[0256] The "Pestle and Mortar" Method

[0257] One strategy used to promote heterodimerization of two Fc polypeptides is the so-called "knob-in-hole" approach.

[0258] 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 a 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 a "knob-in-hole" technique, which is described in several examples in, for example, WO 1996 / 027011; Ridgway, JB et al., Protein Engineering (1996) 9(7):617-621; Merchant, AM et al., Nat. Biotechnol. (1998) 16(7):677-681. See also Klein et al. (2012), Monoclonal Antibodies, 4(6):653-663. Using the knob-in-hole approach, the interacting surfaces of the two CH3 domains are altered to increase heterodimerization of the two half-life extension domains containing the two altered CH3 domains. This occurs by introducing a large number of residues into the CH3 domain of one of the half-life extension domains that acts as a "knob". Then, in order to accommodate the large number of residues, a "hole" is formed in the other half-life extension domain that can accommodate the knob. Either of the altered CH3 domains can be the "knob" and the other can be the "hole". The introduction of disulfide bonds further stabilizes the heterodimer (Merchant, AM et al., Nature Biotechnology (1998) 16 (7); Atwell, S. et al., J. Mol. Biol. (1997) 270 (1): 26-35) and increases the yield.

[0259] It has been reported that heterodimerization yields above 97% can be achieved by introducing S354C and T366W mutations in the heavy chain to create a "knob" and Y349C, T366S, L368A and Y407V mutations in the heavy chain to create a "hole" (residues are numbered according to the Kabat EU numbering system). Carter et al. (2001), J. Immunol. Methods, 248:7-15; Klein et al. (2012), Monoclonal Antibodies, 4(6):653-663.

[0260] 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 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 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 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 amino acid mutations S354C and T366W (numbered according to the Kabat EU numbering system).

[0261] Additional examples of substitutions that can be made to form a knob and a mortise include those described in US20140302037A1, the contents of which are incorporated herein by reference. For example, in some embodiments, any of the following amino acid substitutions can be made to a first Fc polypeptide ("first domain") and a paired second Fc polypeptide ("second domain"), each containing an Fc domain: (a) Y407T in the first domain and T366Y in the second domain, numbered according to the Kabat EU numbering system; (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) Y407T in the first domain and T366Y in the second domain, numbered according to the Kabat EU numbering system. (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.

[0262] In some embodiments, any of the following amino acid replacements may be made to a first Fc polypeptide ("first domain") and a paired second Fc polypeptide ("second domain"), each containing an Fc domain: (a) Y407T in the second domain and T366Y in the first domain; (b) Y407A in the second domain and T366W in the first domain; (c) F405A in the second domain and T394W in the first domain; (d) F405W in the second domain and T394S in the first domain; (e) Y407T in the second domain and T366Y in the first domain; (f) Y407T in the second domain and T366Y in the first domain, numbered according to the Kabat EU numbering system. (g) T366W and F405W in the second domain and T394S and Y407A in the first domain; (h) F405W and Y407A in the second domain and T366W and T394S in the first domain; or (i) T366W in the second domain and T366S, L368A and Y407V in the first domain.

[0263] In embodiments comprising a first Fc polypeptide and a second Fc polypeptide that each comprises an Fc domain, any of the heterodimerization alterations described herein can be used in the Fc domain to promote heterodimerization of any of the targeted cytokines described herein.

[0264] RF mutation or CH3 domain swapping for heterodimeric protein purification

[0265] Two immunoglobulin heavy chains that differ by at least one amino acid allow for the separation of antigen binding proteins based on the differential affinities of the immunoglobulin heavy chains and modified or mutated immunoglobulin heavy chains for affinity reagents. Antigen binding proteins having IgG CH2 and CH3 regions with different affinities relative to Protein A allow for rapid separation by differential binding of the IgG regions to Protein A.

[0266] In one embodiment, the second Fc polypeptide comprises a 95R modification in the CH3 region (by IMGT exon numbering; 435R by EU numbering). In another embodiment, the second Fc polypeptide further comprises a 96F modification (IMGT; 436F by 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 95R / 96F modifications by 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 435R / 436F modifications by EU numbering.

[0267] 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.

[0268] 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" having a CH3 domain from IgG3.

[0269] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCREELTKNQVSLWCLVKGFYPSDIAVEWESSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGGSPG(SEQ ID NO:9)

[0270] 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" having an "RF mutation" (435R / 436F).

[0271] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGSPG(SEQ ID NO:10)

[0272] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO:11.

[0273] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG(SEQ ID NO:11)

[0274] In some embodiments, a protease cleavage site as described herein can be introduced into any of SEQ ID NOs: 9-11.

[0275] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 91% identical to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 92% identical to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 93% identical to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 94% identical to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 96% identical to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 97% identical to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 98% identical to SEQ ID NO: 11. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 11.

[0276] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 91% identical to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 92% identical to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 93% identical to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 94% identical to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 96% identical to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 97% identical to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 98% identical to SEQ ID NO: 5. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 5.

[0277] Masked or targeted cytokines

[0278] According to the present disclosure, an engineered cleavable Fc domain comprising a tumor-associated protease cleavage site can be fused to a masked cytokine, the masked cytokine comprising a cytokine molecule and a masking portion. Alternatively, an engineered cleavable Fc domain comprising a tumor-associated protease cleavage site can be fused to a targeted cytokine, the targeted cytokine comprising a cytokine molecule, a masking portion and a targeting portion. After, for example, the Fc domain is cleaved at the protease cleavage site, the masked and targeted therapeutically active molecule is released and becomes active at the disease site, and can specifically target the cells of interest to effectively treat various diseases without causing undesirable side effects.

[0279] In some embodiments, the cytokine is connected 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 connected to the engineered cleavable Fc domain via a non-cleavable linker. In some embodiments, the targeting moiety is connected to the engineered Fc domain with or without a non-cleavable linker.

[0280] In other embodiments, the engineered cleavable Fc domain of the invention is not directly linked to a cytokine, masking moiety, or targeting moiety through a linker.

[0281] Cytokines

[0282] The immune system is adept at communication and is designed to respond rapidly, specifically, and globally to protect the organism from foreign invaders and disease. The cytokine superfamily of proteins is an integral part of the signaling network between cells and is essential in generating and regulating the immune system. These interacting biological signals have remarkable abilities, such as influencing growth and development, hematopoiesis, lymphocyte recruitment, T cell subset differentiation, and inflammation.

[0283] Cytokines can be a part of a larger immune program (e.g., T cell subset differentiation). Mature CD4 and CD8 T cells leave the thymus with an original phenotype and produce various cytokines. In the periphery, these T cells encounter antigen presenting cells (APCs) showing major histocompatibility complex (MHC) class I molecules (presenting peptides produced in cytosol to CD8 T cells) or MHC class II molecules (presenting peptides degraded in intracellular vesicles to CD4 T cells). After activation, characteristic cytokines and chemokine secretion profiles allow CD4 T helper (Th) cells to be classified into two main subgroups in mice and people. 3-7Th1 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 therefore promote inflammation, cytotoxicity and delayed hypersensitivity (DTH). Th2 cells support humoral immunity and are used to downregulate the inflammatory effects of Th1 cells. This paradigm is a good example of integrating biological networks and is very useful in simplifying the understanding of typical immune responses and immune responses that become pathogenic. For example, the inability to "self" communicate may lead to the loss of tolerance to self-antigens and promote destructive immune responses to self-tissues and autoimmune diseases. Autoimmunity (the main focus of this book) is the underlying mechanism of a group of pathologies (such as type 1 diabetes, multiple sclerosis, and rheumatoid arthritis). Autoimmune diseases can be caused in part by cytokine- and chemokine-mediated Th cell subset differentiation disorders. In addition to the context of presenting antigens and co-stimulatory signals, the main factors affecting the development of Th subsets are cytokines and chemokines in the stimulating environment. A better understanding of the properties and interactions of individual cytokines and chemokines that play a role in Th cell activation may lead to more advanced treatments for autoimmune diseases.

[0284] The targeted cytokines of the present invention may include any cytokine or variant thereof known in the art. See, e.g., Cameron MJ, Kelvin DJ. Cytokines, chemokines and their receptors. In: Madame Curie Bioscience Database. Austin (Texas): Landes Bioscience; 2000-2013, the contents of which are incorporated in their entirety. For example, the cytokine incorporated into the target cytokine can 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.

[0285] 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 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.

[0286] Interleukin-2 (IL-2)

[0287] Provided herein is a kind of IL-2 cytokine or its functional fragment, and the IL-2 cytokine or its functional fragment is used for targeting cytokine or its cleavage product.Cytokine plays a role in cell signaling, particularly in cells of the immune system.IL-2 is an interleukin, which is a type of cytokine signaling molecule that regulates the activity of white blood cells in the immune system.The IL-2 cytokine suitable for the present invention can be any IL-2 or its functional fragment.In certain embodiments, IL-2 is naturally occurring IL-2, IL-2 (for example, IL-2 mutant protein or IL-2 variant) or truncated IL-2 including one or more replacements.In certain embodiments, IL-2 is a polypeptide retaining at least one characteristic of IL-2 biological activity.

[0288] In some embodiments, the IL-2 is a naturally occurring IL-2. In some embodiments, the IL-2 comprises a C125A substitution of mature IL-2 (SEQ ID NO: 13).

[0289] In some embodiments, the amino acid substitution reduces the affinity of the IL-2 polypeptide or a functional fragment thereof for CD25 (IL-2Rα).

[0290] In some embodiments, the IL-2 polypeptide or its functional fragment comprises an amino acid sequence generated by introducing one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or its functional fragment, and the amino acid substitutions increase the affinity of the IL-2 polypeptide or its functional fragment for IL-2Rb or IL-2Rγ. In some embodiments, the IL-2 polypeptide or its functional fragment comprises an amino acid sequence having one or more amino acid substitutions that enhance the affinity of the IL-2 polypeptide or its functional fragment for IL-2Rb (CD 122) compared to the amino acid sequence of wild-type IL-2. In some embodiments, the IL-2 polypeptide or its functional fragment comprises an amino acid sequence having one or more amino acid substitutions that reduce the affinity of the IL-2 polypeptide or its functional fragment for IL-2Ra (CD25) compared to the amino acid sequence of wild-type IL-2 and one or more amino acid substitutions that enhance the affinity of the IL-2 polypeptide or its functional fragment for IL-2R (CD122) compared to the amino acid sequence of wild-type IL-2.

[0291] In some embodiments, IL-2 binds to IL-2Ra with an affinity similar to or greater than wild-type IL-2. In some embodiments, IL-2 preferentially binds to CD25 (e.g., α-biased). In some embodiments, IL-2 has reduced affinity for CD122 and / or CD132. In some embodiments, IL-2 comprises N88D and C125A relative to SEQ ID NO: 13.

[0292] In eukaryotic cells, naturally occurring IL-2 is synthesized as a precursor polypeptide having 153 amino acids, which has SEQ ID NO:12.

[0293] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:12)

[0294] It is then processed into mature IL-2 by removing amino acid residues 1 to 20. This produces a mature form of IL-2 consisting of 133 amino acids (amino acid residues 21 to 153) having the sequence shown in SEQ ID NO: 13.

[0295] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF AQSIISTLT(SEQ IDNO: 13)

[0296] A "functional fragment" of an IL-2 cytokine includes a portion of a full-length cytokine protein that retains or has a modified 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 shown, for example, by the ability of the cytokine to bind to a cognate receptor of the cytokine or a component thereof (e.g., one or more chains of a heterotrimeric receptor complex).

[0297] In some embodiments, the IL-2 cytokine or its functional fragment is any naturally occurring interleukin-2 (IL-2) protein or a modified variant thereof that can bind to an interleukin-2 receptor, particularly an IL-2R α chain. In the case of IL-2 cytokine binding, the target protein may be IL-2R (including IL-2R α, IL-2R β and IL-2R γ chain), IL-2R α chain, IL-2R β chain or IL-2R α / β dimer complex. In some embodiments, the IL-2 cytokine or its functional fragment comprises an amino acid sequence of amino acid residues 21-153 of SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide or its functional fragment comprises the amino acid sequence of mature IL-2, SEQ ID NO: 13.

[0298] In some embodiments, the IL-2 cytokine or its functional fragment comprises an amino acid sequence having at least one amino acid modification compared to the amino acid sequence of SEQ ID NO: 13. Each amino acid modification in the at least one amino acid modification can be any amino acid modification, such as substitution, insertion or deletion. In some embodiments, the IL-2 cytokine or its functional fragment 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: 13. In some embodiments, the IL-2 cytokine or its functional fragment comprises an amino acid sequence having at least 5 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 13.

[0299] 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 to 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 to 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 to 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 to SEQ ID NO: 13.

[0300] 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 reduce the affinity of the IL-2 peptide or its functional fragment to IL-2Rα (CD25) 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 compared to the amino acid sequence of SEQ ID NO: 13, such that one or more of 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 compared to the amino acid sequence of SEQ ID NO: 13, such that amino acid residues 38, 42, 45 and 62 are alanine (A).

[0301] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence substitution of C125A compared to the amino acid sequence of SEQ ID NO:13.

[0302] In some embodiments, the IL-2 cytokine or its functional fragment comprises an amino acid sequence having one or more amino acid substitutions 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 its functional fragment 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 its functional fragment 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.

[0303] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:14.

[0304] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLE EALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF AQSIISTLT(SEQ IDNO: 14)

[0305] 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 with 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 about 80% identity with SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 85% identity with SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 92% identity with SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 95% identity with SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 97% identity with SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 99% identity with SEQ ID NO: 14.

[0306] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions 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 in which amino acid residue N88 is substituted for aspartic acid in the amino acid sequence of SEQ ID NO: 13.

[0307] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:118.

[0308] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITF AQSIISTLT(SEQ IDNO: 118)

[0309] 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 about 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 about 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 about 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 about 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 about 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 about 97% identity with SEQ ID NO: 118. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 99% identity with SEQ ID NO: 118.

[0310] In some embodiments, the IL-2 cytokine or its functional fragment has one or more amino acid residues removed, for example, 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 its functional fragment has one or more substituted amino acid residues 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 its functional fragment has one or more inserted amino acid residues in the region of, for example, 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 its functional fragment does not have an O-glycosylation site within residues 1-3.

[0311] Interleukin 15 (IL-15)

[0312] The present invention provides an IL-15 cytokine or its functional fragment, which is used for targeting cytokines or their cleavage products. Cytokines play a role in cell signaling, particularly in cells of the immune system. IL-15 is an interleukin, which is a type of cytokine signaling molecule that regulates the activity of white blood cells in the immune system.

[0313] In eukaryotic cells, IL-15 is synthesized as a precursor polypeptide having 162 amino acids (SEQ ID NO: 15), which is then processed into mature IL-15 by removing amino acid residues 1 to 48. This produces a mature form of IL-15 consisting of 114 amino acids (amino acid residues 49 to 162), which is secreted in a mature, active form (see SEQ ID NO: 16).

[0314] IL-15 precursor polypeptide (SEQ ID NO: 15):

[0315] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0316] IL-15 mature polypeptide (SEQ ID NO: 16):

[0317] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0318] 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. The term encompasses any natural IL-15 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., rats and mice). The term encompasses unprocessed IL-15 (e.g., a full-length precursor form of IL-15 consisting of amino acid residues 1-162) and any form of IL-15 produced by processing in cells (e.g., a mature form of IL-15 consisting of amino acid residues 49-162). Therefore, the term encompasses proteins encoded by the amino acid sequence of SEQ ID NO: 16 and sequence variants thereof. The term also encompasses naturally occurring variants of IL-15. The term also encompasses non-naturally occurring variants of IL-15, such as truncations, deletions, forms in which IL-15 is linked to another molecule, and variants caused by at least one amino acid change (e.g., by substitution, addition, or deletion) of the amino acid sequence. In some aspects, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the entire sequence or a portion of the sequence (e.g., a 50, 100 or 114 contiguous amino acid portion) compared to a naturally occurring IL-15 polypeptide (e.g., an 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, including variants thereof, such as variants generated by making one or more amino acid substitutions to the amino acid sequence of SEQ ID NO: 15 or 16.

[0319] A "functional fragment" of an IL-15 cytokine includes a portion of a full-length cytokine protein that retains or has a modified 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 shown, for example, by the ability of a cytokine to bind to a cognate receptor of a cytokine or a component thereof (e.g., one or more chains of a heterotrimeric receptor complex).

[0320] In some embodiments, the IL-15 cytokine or a functional fragment thereof is any naturally occurring interleukin-2 (IL-15) protein or a modified variant thereof that is capable of binding to the interleukin-2 receptor, particularly the IL-15Rα chain.

[0321] In some embodiments, the IL-15 cytokine or a fragment thereof comprises SEQ ID NO: 16 or a functional fragment thereof.

[0322] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:16.

[0323] In some embodiments, the IL-15 cytokine or its functional fragment comprises an amino acid sequence having at least one amino acid modification compared to the amino acid sequence of SEQ ID NO: 16. Each amino acid modification in the at least one amino acid modification can be any amino acid modification, such as substitution, insertion or deletion. In some embodiments, the IL-15 cytokine or its functional fragment 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: 16. In some embodiments, the IL-15 cytokine or its functional fragment comprises an amino acid sequence having at least 5 amino acid substitutions 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.

[0324] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 92% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 93% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 94% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 96% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 97% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO: 16.

[0325] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO:16.

[0326] 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 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 compared to the amino acid sequence of SEQ ID NO: 16.

[0327] 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 compared to the amino acid sequence of SEQ ID NO:16.

[0328] 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 compared to the amino acid sequence of SEQ ID NO:16.

[0329] 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 compared to the amino acid sequence of SEQ ID NO:16.

[0330] 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 compared to the amino acid sequence of SEQ ID NO:16.

[0331] 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 compared to the amino acid sequence of SEQ ID NO:16.

[0332] 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 compared to the amino acid sequence of SEQ ID NO:16.

[0333] 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 compared to the amino acid sequence of SEQ ID NO:16.

[0334] 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 compared to the amino acid sequence of SEQ ID NO:16.

[0335] 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 compared to the amino acid sequence of SEQ ID NO:16.

[0336] 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 compared to the amino acid sequence of SEQ ID NO:16.

[0337] 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 compared to the amino acid sequence of SEQ ID NO:16.

[0338] In some embodiments, the amino acid replacement at position D22 is D22A. In some embodiments, the amino acid replacement at position E46 is E46A. In some embodiments, the amino acid replacement at position E46 is E46R. In some embodiments, the amino acid replacement at position E46 is E46S. In some embodiments, the amino acid replacement at position E53 is E53A, E53R or E53S. In some embodiments, the amino acid replacement at position N71 is N71Q. In some embodiments, the amino acid replacement at position N79 is N79Q. In some embodiments, the amino acid replacement at position N112 is N112Q.

[0339] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution D22A compared to the amino acid sequence of SEQ ID NO:16.

[0340] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:17.

[0341] NWVNVISDLKKIEDLIQSMHIAATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:17)

[0342] 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.

[0343] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an 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 an amino acid sequence of SEQ ID NO:18.

[0344] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLALQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:18)

[0345] 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.

[0346] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46A and E53A 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 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.

[0347] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLALQVISLASGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:19)

[0348] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46R and E53R 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 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.

[0349] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLRLQVISLRSGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:20)

[0350] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46S and E53S 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 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.

[0351] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLSLQVISLSSGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:21)

[0352] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution E53A 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 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.

[0353] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLASGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:22)

[0354] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 23 and an 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.

[0355] NWVNVISDLKKIEDLIQS(SEQ ID NO:223)

[0356] KVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:24)

[0357] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution N71Q 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 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.

[0358] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILAQNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:25)

[0359] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution N79Q 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 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.

[0360] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:26)

[0361] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution N112Q 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 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.

[0362] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS(SEQ ID NO:27)

[0363] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions N71Q and N79Q 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 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.

[0364] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILAQNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS((SEQ ID NO:28)

[0365] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions N71Q and N112Q 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 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.

[0366] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILAQNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS((SEQ ID NO:29)

[0367] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions N79Q and N112Q 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 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.

[0368] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS(SEQ ID NO:30)

[0369] 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 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 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.

[0370] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILAQNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS(SEQ ID NO:31)

[0371] 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.

[0372] 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.

[0373] In some embodiments, additional mutations may be included in any of the above sequences at one or more of 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.

[0374] In some embodiments, additional mutations may be included in any of the above sequences at one or more of 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.

[0375] In some embodiments, for the purpose of removing O-glycosylation sites, the IL-15 cytokine or its functional fragment has one or more amino acid residues removed, for example, residues 1-3s, compared to the amino acid sequence of mature IL-15 of SEQ ID NO: 16. In some embodiments, for the purpose of removing O-glycosylation sites, the IL-15 cytokine or its functional fragment has one or more substituted amino acid residues compared to the amino acid sequence of mature IL-15 of SEQ ID NO: 16. In some embodiments, for the purpose of removing O-glycosylation sites, the IL-15 cytokine or its functional fragment has one or more amino acid residues inserted in the region of, for example, residues 1-3, compared to the amino acid sequence of mature IL-15 of SEQ ID NO: 16. In some embodiments, the IL-15 cytokine or its functional fragment does not have an O-glycosylation site within residues 1-3.

[0376] Interleukin 12 (IL-12)

[0377] The present invention provides an IL-12 cytokine or a functional fragment thereof, wherein the IL-12 cytokine or its functional fragment is used for targeting cytokines or their cleavage products. Cytokines play a role in cell signaling, particularly in cells of the immune system. IL-12 is an interleukin, which is a type of cytokine signaling molecule that regulates the activity of white blood cells in the immune system.

[0378] Endogenous IL-12 exists as two distinct molecules, IL-12p40 and IL-12p35, which dimerize in the cell during biosynthesis.

[0379] The full sequences of IL-12p40 and IL-12p35 are in bold (the propeptide that is cleaved during biosynthesis is shown):

[0380] IL-12p40 subunit:

[0381]

[0382] IL-12p35 subunit:

[0383]

[0384] The mature form is as follows:

[0385] IL-12p40 subunit:

[0386] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSA ERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS(SEQ ID NO:34)

[0387] IL-12p35 subunit:

[0388] RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ ID NO:35)

[0389] They are expressed as two chains that covalently dimerize during biosynthesis via a disulfide bond between the two subunits: cysteine ​​C199 of the p40 subunit associates with cysteine ​​C96 of the p35 subunit.

[0390] A "functional fragment" of an IL-12 cytokine includes a portion of a full-length cytokine protein that retains or has a modified 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 shown, for example, by the ability of a cytokine to bind to a cognate receptor of a cytokine or a component thereof.

[0391] In some embodiments, the IL-12 cytokine or a functional fragment thereof is any naturally occurring interleukin-2 (IL-12) protein or a modified variant thereof that is capable of binding to an interleukin-12 receptor.

[0392] In some embodiments, the IL-12 polypeptide or a functional fragment thereof comprises an IL-12p40 polypeptide or a functional fragment thereof covalently linked to an IL-12p35 polypeptide or a functional fragment thereof.

[0393] The IL-12p40 polypeptide or a functional fragment thereof can be linked to the first half-life extension domain such that the first polypeptide chain comprises:

[0394] N'HL1-L1-MM C'

[0395] And the second polypeptide chain comprises:

[0396] N'HL2-L2-[IL-12p40-Linker-IL-12p35]C'

[0397] 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.

[0398] 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 amino acid modification in 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.

[0399] The IL-12p40 polypeptide includes a glycosaminoglycan (GAG) binding domain. It has been shown that GAGs (such as heparin and heparan sulfate) bind to many growth factors and cytokines, including IL-12. The physiological significance of this binding is twofold. First, GAGs can act as co-receptors on the cell surface to maintain high local concentrations of cytokines. Second, GAGs can regulate the biological activity of growth factors and cytokines through a variety of mechanisms, including dimerization and protection from proteolytic degradation.

[0400] The GAG ​​binding domain in the mature form of the IL-12p40 subunit is shown in bold as follows:

[0401]

[0402] Modifications to the GAG ​​binding domain KSKREKKDRV (SEQ ID NO: 37) have been shown herein to increase the PK profile of constructs comprising IL-12 cytokines having a mutated GAG binding domain without any reduction in cytokine activity. Thus, in some embodiments, the IL-12p40 polypeptide comprises 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.

[0403] 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 SEQ ID NO:38.

[0404] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATL SAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTERVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS(SEQ ID NO: 38)

[0405] 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 SEQ ID NO:39.

[0406] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLS AERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS(SEQ ID NO:39)

[0407] 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 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 SEQ ID NO:39.

[0408] 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. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having an amino acid substitution at position C252 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.

[0409] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSA ERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS(SEQ ID NO:40)

[0410] In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having one or more cysteine ​​substitutions and at least one amino acid modification to the GAG ​​binding domain compared to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the IL-12p40 polypeptide comprises an amino acid substitution at position C252S 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 position C252S 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.

[0411] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLS AERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS(SEQ ID NO:42)

[0412] 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 amino acid modification in 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: 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.

[0413] In some embodiments, the IL-12p40-IL-12p35 linker is between 5 and 20 amino acids in length.

[0414] In some embodiments, the IL-12p40-IL-12p35 linker is rich in amino acid residues G and S.

[0415] In some embodiments, the IL-12p40-IL-12p35 linker comprises only amino acid residue types selected from the group consisting of G and S.

[0416] In some embodiments, the IL-12p40-IL-12p35 linker comprises [(G)nS], wherein n=4 or 5.

[0417] In some embodiments, the IL-12p40-IL-12p35 linker comprises a (GGGGS) (SEQ ID NO: 43) repeat sequence.

[0418] In some embodiments, the IL-12p40-IL-12p35 linker comprises SEQ ID NO:44 (GGGGSGGGGSGGGGS).

[0419] In some embodiments, the IL-12 cytokine or its functional fragment comprises SEQ ID NO: 45. In some embodiments, the IL-12 cytokine or its functional fragment comprises an amino acid sequence having at least one amino acid modification compared to the amino acid sequences of SEQ ID NOs: 34 and 35. Each amino acid modification in 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 its functional fragment 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 sequences of SEQ ID NOs: 34 and 35. In some embodiments, the IL-12 cytokine or its functional fragment comprises an amino acid sequence having at least 5 amino acid substitutions compared to the amino acid sequences of SEQ ID NOs: 34 and 35.

[0420] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTF LRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIK PDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGG GGSRLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMA(SEQ ID NO:45)

[0421] 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.

[0422] 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.

[0423] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTD LTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKD NTERVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKN ESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ IDNO:46)

[0424] 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.

[0425] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDL TFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDN TEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKN ESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ IDNO:47)

[0426] 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.

[0427] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDL TFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKR EKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTK NESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ IDNO:48)

[0428] 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.

[0429] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTF LRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIK PDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGG SRLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALC(SEQ ID NO: 49)

[0430] Masking part

[0431] Provided herein are masking moieties for use in masked or targeted cytokines or any other masked therapeutically active molecules. It should be understood that the masking moiety is cleaved from the targeted cytokine to form its cleavage product. The masking moiety is combined with the cytokine moiety and inhibits the biological activity of the cytokine. After cleavage, the masking moiety is released from the cytokine, thereby activating the function of the cytokine in the target of interest.

[0432] In some embodiments, the masking moiety comprises an agent, peptide or polypeptide that binds to a cytokine. In some embodiments, the masking moiety comprises a cyclic peptide that binds to a cytokine. In some embodiments, the masking moiety comprises a linear peptide that binds to a cytokine.

[0433] In some embodiments, the masking portion comprises Fab, single-chain Fv (scFv), single domain antibody (VHH), one or more CDRs, variable heavy chain (VH), variable light chain (VL), Fab-like bispecific antibody (bsFab), Fab (s-Fab) connected to a single domain antibody, antibody or a combination thereof. In some embodiments, the masking portion comprises a Fab that binds to a cytokine. In some embodiments, the masking portion comprises a single-chain Fv (scFv) that binds to a cytokine. In some embodiments, the masking portion comprises a single domain antibody (VHH) that binds to a cytokine. In some embodiments, the masking portion comprises one or more CDRs that bind to a cytokine. In some embodiments, the masking portion comprises a variable heavy chain (VH) that binds to a cytokine. In some embodiments, the masking portion comprises a variable light chain (VL) that binds to a cytokine. In some embodiments, the masking portion comprises a Fab-like bispecific antibody (bsFab) that binds to a cytokine. In some embodiments, the masking portion comprises a Fab (s-Fab) connected to a single domain antibody that binds to a cytokine. In some embodiments, the masking portion comprises an antibody or fragment thereof that binds to a cytokine. In some embodiments, the masking moiety comprises an antibody or a binding fragment of an antibody directed against a cytokine.

[0434] In some embodiments, the masking moiety is a receptor for a cytokine. In some embodiments, the masking moiety is a fragment of a receptor for a cytokine. In some embodiments, the masking moiety is an extracellular domain (ECD) of a receptor for a cytokine.

[0435] In some embodiments, the cytokine is IL-1α or IL-1β; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL-1α or IL-1β. In some embodiments, the cytokine is IL-1α or IL-1β; and wherein the masking moiety is CD121a, CDw121b, or a fragment thereof.

[0436] In some embodiments, the cytokine is IL-2; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL-2. In some embodiments, the cytokine is IL-2; and wherein the masking moiety is IL-2Rα, IL-2Rβ, CD25, CD122, CD132, or a fragment thereof.

[0437] In some embodiments, the cytokine is IL-18; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL-18. In some embodiments, the cytokine is IL-18; and wherein the masking moiety is IL-18Rα, IL-18Rβ, or a fragment thereof.

[0438] In some embodiments, the cytokine is IL-4; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL-4. In some embodiments, the cytokine is IL-4; and wherein the masking moiety is CD124, CD213a13, CD132, or a fragment thereof.

[0439] In some embodiments, the cytokine is IL-7; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL-7. In some embodiments, the cytokine is IL-7; and wherein the masking moiety is CD127, CD132, or a fragment thereof.

[0440] In some embodiments, the cytokine is IL-9; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL-9. In some embodiments, the cytokine is IL-9, and wherein the masking moiety is IL-9R, CD132, or a fragment thereof.

[0441] In some embodiments, the cytokine is IL-13; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL- 13. In some embodiments, the cytokine is IL-13; and wherein the masking moiety is CD213a1, CD213a2, or a fragment thereof.

[0442] In some embodiments, the cytokine is IL-15; and wherein the masking moiety is a Fab, a single chain Fv (scFv), or a single domain antibody to IL- 15. In some embodiments, the cytokine is IL-15; and wherein the masking moiety is IL-15Ra, CD122, CD132, or a fragment thereof.

[0443] In some embodiments, the cytokine is IL-3; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL-3. In some embodiments, the cytokine is IL-3; and wherein the masking moiety is CD123, CDwl31, or a fragment thereof.

[0444] In some embodiments, the cytokine is IL-5; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL-5. In some embodiments, the cytokine is IL-5; and wherein the masking moiety is CDwl25, CD131, or a fragment thereof.

[0445] In some embodiments, the cytokine is GM-CSF; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to GM-CSF. In some embodiments, the cytokine is GM-CSF; and wherein the masking moiety is CD116, CDwl31, or a fragment thereof.

[0446] In some embodiments, the cytokine is IL-6; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL-6. In some embodiments, the cytokine is IL-6; and wherein the masking moiety is CD126, CD130, or a fragment thereof.

[0447] In some embodiments, the cytokine is IL-11; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL-11. In some embodiments, the cytokine is IL-11; and wherein the masking moiety is IL-11Ra, CD130, or a fragment thereof.

[0448] In some embodiments, the cytokine is G-CSF; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to G-CSF. In some embodiments, the cytokine is G-CSF; and wherein the masking moiety is CD114 or a fragment thereof.

[0449] In some embodiments, the cytokine is IL-12; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL-12. In some embodiments, the cytokine is IL-12; and wherein the masking moiety is CD212 or a fragment thereof.

[0450] In some embodiments, the cytokine is LIF; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to LIF. In some embodiments, the cytokine is LIF; and wherein the masking moiety is LIFR, CD130, or a fragment thereof.

[0451] In some embodiments, the cytokine is OSM; and wherein the masking moiety is a Fab, a single chain Fv (scFv), or a single domain antibody to OSM. In some embodiments, the cytokine is OSM; and wherein the masking moiety is OSMR, CD130, or a fragment thereof.

[0452] In some embodiments, the cytokine is IL-10; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL-10. In some embodiments, the cytokine is IL-10; and wherein the masking moiety is CDw210 or a fragment thereof.

[0453] In some embodiments, the cytokine is IL-20; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL-20. In some embodiments, the cytokine is IL-20; and wherein the masking moiety is IL-20Rα, IL-20Rβ, or a fragment thereof.

[0454] In some embodiments, the cytokine is IL-14; and wherein the masking moiety is a Fab, a single chain Fv (scFv), or a single domain antibody to IL- 14. In some embodiments, the cytokine is IL-14; and wherein the masking moiety is IL-14R or a fragment thereof.

[0455] In some embodiments, the cytokine is IL-16; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL- 16. In some embodiments, the cytokine is IL-16; and wherein the masking moiety is CD4 or a fragment thereof.

[0456] In some embodiments, the cytokine is IL-17; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IL-17. In some embodiments, the cytokine is IL-17; and wherein the masking moiety is CDw217 or a fragment thereof.

[0457] In some embodiments, the cytokine is IFN-α; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IFN-α. In some embodiments, the cytokine is IFN-α; and wherein the masking moiety is CD118 or a fragment thereof.

[0458] In some embodiments, the cytokine is IFN-β; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IFN-β. In some embodiments, the cytokine is IFN-β; and wherein the masking moiety is CD118 or a fragment thereof.

[0459] In some embodiments, the cytokine is IFN-γ; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to IFN-γ. In some embodiments, the cytokine is IFN-γ; and wherein the masking moiety is CDwl 19 or a fragment thereof.

[0460] In some embodiments, the cytokine is CD154; and wherein the masking moiety is a Fab, a single chain Fv (scFv), or a single domain antibody to CD154. In some embodiments, the cytokine is CD154; and wherein the masking moiety is CD40 or a fragment thereof.

[0461] In some embodiments, the cytokine is LT-β; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to LT-β. In some embodiments, the cytokine is LT-β; and wherein the masking moiety is LT-βR or a fragment thereof.

[0462] In some embodiments, the cytokine is TNF-α; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to TNF-α. In some embodiments, the cytokine is TNF-α; and wherein the masking moiety is CD120a, CD120b, or a fragment thereof.

[0463] In some embodiments, the cytokine is TNF-β; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to TMF-β. In some embodiments, the cytokine is TNF-β; and wherein the masking moiety is CD120a, CD120b, or a fragment thereof.

[0464] In some embodiments, the cytokine is 4-1BBL; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to 4-1BBL. In some embodiments, the cytokine is 4-1BBL; and wherein the masking moiety is CDwl37, 4-1BB, or a fragment thereof.

[0465] In some embodiments, the cytokine is APRIL; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to APRIL. In some embodiments, the cytokine is APRIL; and wherein the masking moiety is BCMA, TACI, or a fragment thereof.

[0466] In some embodiments, the cytokine is CD70; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to CD70. In some embodiments, the cytokine is CD70; and wherein the masking moiety is CD27 or a fragment thereof.

[0467] In some embodiments, the cytokine is CD153; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to CD153. In some embodiments, the cytokine is CD153; and wherein the masking moiety is CD30 or a fragment thereof.

[0468] In some embodiments, the cytokine is CD178; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to CD178. In some embodiments, the cytokine is CD178; and wherein the masking moiety is CD95 or a fragment thereof.

[0469] In some embodiments, the cytokine is GITRL; and wherein the masking moiety is a Fab, a single chain Fv (scFv), or a single domain antibody to GITRL. In some embodiments, the cytokine is GITRL; and wherein the masking moiety is GITR or a fragment thereof.

[0470] In some embodiments, the cytokine is LIGHT; and wherein the masking moiety is a Fab, a single chain Fv (scFv), or a single domain antibody to LIGHT. In some embodiments, the cytokine is LIGHT; and wherein the masking moiety is LTβR, HVEM, or a fragment thereof.

[0471] In some embodiments, the cytokine is OX40; and wherein the masking moiety is a Fab, a single chain Fv (scFv), or a single domain antibody to OX40. In some embodiments, the cytokine is OX40; and wherein the masking moiety is OX40 or a fragment thereof.

[0472] In some embodiments, the cytokine is TALL-1; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to TALL-1. In some embodiments, the cytokine is TALL-1; and wherein the masking moiety is BCMA, TACI, or a fragment thereof.

[0473] In some embodiments, the cytokine is TRAIL; and wherein the masking moiety is a Fab, a single chain Fv (scFv), or a single domain antibody to TRAIL. In some embodiments, the cytokine is TRAIL; and wherein the masking moiety is TRAILR1-4 or a fragment thereof.

[0474] In some embodiments, the cytokine is TWEAK; and wherein the masking moiety is a Fab, a single chain Fv (scFv), or a single domain antibody to TWEAK. In some embodiments, the cytokine is TWEAK; and wherein the masking moiety is Apo3 or a fragment thereof.

[0475] In some embodiments, the cytokine is TRANCE; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to TRANCE. In some embodiments, the cytokine is TRANCE; and wherein the masking moiety is RANK, OPG, or a fragment thereof.

[0476] In some embodiments, the cytokine is TGF-β1; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to TGF-β1. In some embodiments, the cytokine is TGF-β1; and wherein the masking moiety is TGF-βR1 or a fragment thereof.

[0477] In some embodiments, the cytokine is TGF-β2; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to TGF-β2. In some embodiments, the cytokine is TGF-β2; and wherein the masking moiety is TGF-βR2 or a fragment thereof.

[0478] In some embodiments, the cytokine is TGF-β3; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to TGF-β3. In some embodiments, the cytokine is TGF-β3; and wherein the masking moiety is TGF-βR3 or a fragment thereof.

[0479] In some embodiments, the cytokine is Epo; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to Epo. In some embodiments, the cytokine is Epo; and wherein the masking moiety is EpoR or a fragment thereof.

[0480] In some embodiments, the cytokine is Tpo; and wherein the masking moiety is a Fab, a single chain Fv (scFv), or a single domain antibody to Tpo. In some embodiments, the cytokine is Tpo; and wherein the masking moiety is TpoR or a fragment thereof.

[0481] In some embodiments, the cytokine is Flt-3L; and wherein the masking moiety is a Fab, a single chain Fv (scFv), or a single domain antibody to Flt-3L. In some embodiments, the cytokine is Flt-3L; and wherein the masking moiety is Flt-3 or a fragment thereof.

[0482] In some embodiments, the cytokine is SCF; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to SCF. In some embodiments, the cytokine is SCF; and wherein the masking moiety is CD117 or a fragment thereof.

[0483] In some embodiments, the cytokine is M-CSF; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody to M-CSF. In some embodiments, the cytokine is M-CSF; and wherein the masking moiety is CD115 or a fragment thereof.

[0484] In some embodiments, the cytokine is MSP; and wherein the masking moiety is a Fab, a single chain Fv (scFv), or a single domain antibody to MSP. In some embodiments, the cytokine is MSP; and wherein the masking moiety is CDwl36 or a fragment thereof.

[0485] In some embodiments, the cytokine is an IL-15 agonist polypeptide; and wherein the masking moiety is a Fab, a single chain Fv (scFv), or a single domain antibody to IL- 15. In some embodiments, the cytokine portion comprises an IL-15 agonist polypeptide, wherein the chimeric molecule further comprises a sushi domain of IL-15 receptor alpha (IL-15Ra sushi domain).

[0486] In some embodiments, the cytokine is an IL-2 agonist polypeptide; and wherein the masking moiety is the extracellular domain of 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 wherein the masking moiety is the extracellular domain of IL-2 receptor b (IL-2RβECD). In some embodiments, the cytokine is an IL-21 agonist polypeptide; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody against IL-21.

[0487] In some embodiments, the cytokine is an IL-2 agonist polypeptide; and wherein the masking moiety is the extracellular domain of 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 wherein the masking moiety is the extracellular domain of IL-2 receptor b (IL-2RβECD). In some embodiments, the cytokine is an IL-21 agonist polypeptide; and wherein the masking moiety is a Fab, a single-chain Fv (scFv), or a single domain antibody against IL-21.

[0488] Antibodies or antigen-binding fragments

[0489] The masking moiety masks the cytokine or the therapeutically active domain in the masked cytokine or the masked therapeutically active molecule, thereby reducing or preventing the binding of the cytokine or the therapeutically active domain to its cognate receptor or ligand.

[0490] In some embodiments, the masking moiety is an antibody or antigen binding fragment thereof that prevents the therapeutically active domain from binding to its cognate ligand or receptor. In some embodiments, the masking moiety is an antibody or antigen binding fragment thereof that specifically binds to the therapeutically active domain. In some embodiments, the masking moiety is an antibody or antigen binding fragment thereof that prevents a cytokine from binding to its cognate ligand or receptor. In some embodiments, the masking moiety is an antibody or antigen binding fragment thereof that specifically binds to a cytokine.

[0491] In some embodiments, the masking portion comprises an antibody or antigen binding fragment thereof that specifically binds to the therapeutically active domain. In some embodiments, the masking portion comprises an antibody or antigen binding fragment thereof that specifically binds to a cytokine. In some embodiments, the masking portion comprises an antibody or antigen binding fragment thereof that specifically binds to an IL-2 cytokine. In some embodiments, the masking portion comprises an antibody or antigen binding fragment thereof that specifically binds to an IL-15 cytokine. In some embodiments, the masking portion comprises an antibody or antigen binding fragment thereof that specifically binds to an IL-12 cytokine. In some embodiments, the masking portion comprises an antibody or antigen binding fragment thereof that specifically binds to an IL-18 cytokine.

[0492] In some embodiments, the masking portion comprises a scFv that specifically binds to an IL-2 cytokine. In some embodiments, the masking portion comprises a scFv that specifically binds to an IL-15 cytokine. In some embodiments, the masking portion comprises a scFv that specifically binds to an IL-12 cytokine. In some embodiments, the masking portion comprises a scFv that specifically binds to an IL-18 cytokine.

[0493] In some embodiments, the masking moiety comprises a VHH that specifically binds to an IL-2 cytokine. In some embodiments, the masking moiety comprises a VHH that specifically binds to an IL-15 cytokine. In some embodiments, the masking moiety comprises a VHH that specifically binds to an IL-12 cytokine. In some embodiments, the masking moiety comprises a VHH that specifically binds to an IL-18 cytokine.

[0494] Anti-IL-2 scFv

[0495] 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 a VH of SEQ ID NO: 124 and a VL of SEQ ID NO: 125.

[0496] In some embodiments, the masking moiety comprises an anti-IL-2 scFv having an hCDR1 of SEQ ID NO: 126, an hCDR2 of SEQ ID NO: 127, and an hCDR3 of SEQ ID NO: 128. In some embodiments, the masking moiety comprises an anti-IL-2 scFv having an lCDR1 of SEQ ID NO: 129, an lCDR2 of SEQ ID NO: 130, and an lCDR3 of SEQ ID NO: 131. In some embodiments, the masking moiety comprises an anti-IL-2 scFv having an hCDR1 of SEQ ID NO: 126, an hCDR2 of SEQ ID NO: 127, an hCDR3 of SEQ ID NO: 128, an lCDR1 of SEQ ID NO: 129, an lCDR2 of SEQ ID NO: 130, and an lCDR3 of SEQ ID NO: 131.

[0497] Exemplary anti-IL-2 scFv sequences

[0498]

[0499]

[0500] In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO: 124. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 124. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 87% identical to SEQ ID NO: 124. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 88% identical to SEQ ID NO: 124. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 124. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 92% identical to SEQ ID NO: 124. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 93% identical to SEQ ID NO: 124. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 94% identical to SEQ ID NO: 124. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO: 124. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 96% identical to SEQ ID NO: 124. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 97% identical to SEQ ID NO: 124. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 98% identical to SEQ ID NO: 124. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 124.

[0501] In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO: 125. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 125. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 87% identical to SEQ ID NO: 125. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 88% identical to SEQ ID NO: 125. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 125. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 92% identical to SEQ ID NO: 125. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 93% identical to SEQ ID NO: 125. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 94% identical to SEQ ID NO: 125. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO: 125. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 96% identical to SEQ ID NO: 125. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 97% identical to SEQ ID NO: 125. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 98% identical to SEQ ID NO: 125. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 125.

[0502] In some embodiments, the masking moiety comprises a VL connected to a VH via a non-cleavable linker. In some embodiments, the masking moiety comprises a VH connected to a VL via a non-cleavable linker. In some embodiments, the non-cleavable linker is GGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 141).

[0503] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:142.

[0504] NFMLTQPHSVSESPGKTVTISCTRSSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQTYDSIDVYFGGGTKLTVLGGGGGSGGGGSGGGGSGG GGSQLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYKSGSAYYSPSLKSRVTISDTSKNQFSLKLSSVTAADTAVYYCARTPTVTGDWFDPWGRGTLVTVSS(SEQ ID NO:142)

[0505] In some embodiments, the masking portion comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 87% identical to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 88% identical to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 92% identical to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 93% identical to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 94% identical to SEQ ID NO: 142. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO: 142. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 96% identical to SEQ ID NO: 142. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 97% identical to SEQ ID NO: 142. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 98% identical to SEQ ID NO: 142. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 142.

[0506] In some embodiments, the masking moiety is connected to the engineered Fc domain via a non-cleavable linker of SEQ ID NO: 140 (GGSSGSGGSGGGSGSGGG). In some embodiments, the masking moiety is connected to the engineered Fc domain via a non-cleavable linker of SEQ ID NO: 141 (GGGGGSGGGGSGGGGSGGGGS). In some embodiments, the masking moiety is connected to the engineered Fc domain via a non-cleavable linker of SEQ ID NO: 210 (GGSSGSGGSGGGSGSGGGSGGSGG).

[0507] Anti-IL-2 VHH

[0508] 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.

[0509] Exemplary anti-IL-2 scFv sequences

[0510]

[0511] In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 87% identical to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 88% identical to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 92% identical to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 93% identical to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 94% identical to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 96% identical to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 97% identical to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 98% identical to SEQ ID NO: 121. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 121.

[0512] 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.

[0513] CD122 (masking portion of IL-2 and IL-15)

[0514] The masking portion masks the cytokine or its functional fragment in the masked targeted cytokine, thereby reducing or preventing the binding of the cytokine or its functional fragment to its cognate receptor. In some embodiments, the masking portion reduces or prevents the binding of IL-2 cytokine or its functional fragment to IL-2Rα (CD25). In some embodiments, the masking portion as provided herein refers to a portion that can bind to IL-2 cytokine or its functional fragment or otherwise show affinity for IL-2 cytokine or its functional fragment (such as anti-IL-2 antibody or IL-2 cognate receptor protein). In some embodiments, the masking portion reduces or prevents the binding of IL-15 cytokine or its functional fragment to IL-15Rα. In some embodiments, the masking portion as provided herein refers to a portion that can bind to IL-15 cytokine or its functional fragment or otherwise show affinity for IL-15 cytokine or its functional fragment (such as anti-IL-15 antibody or IL-15 cognate receptor protein). Methods for determining the degree of binding of a protein (e.g., cytokine) to a cognate protein (e.g., cytokine receptor) are well known in the art.

[0515] In some embodiments, the masking moiety comprises an IL-2 cytokine receptor or a subunit or functional fragment thereof.

[0516] In some embodiments, the masking moiety comprises CD122 (also known as IL-2Rβ) or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-2 and IL-15.

[0517] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:50.

[0518] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:50)

[0519] 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: 50. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 50 with one to four amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 50 with one or two amino acid substitutions.

[0520] In some embodiments, CD122 or a fragment, portion or variant thereof has a mutation at amino acid position C122 compared to CD122 of SEQ ID NO:50.

[0521] In some embodiments, CD122 or a fragment, portion or variant thereof has a mutation C122S at amino acid position 122 compared to CD122 of SEQ ID NO:50.

[0522] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:50 having a C122 mutation.

[0523] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:50 having a C122S mutation.

[0524] In some embodiments, CD122 or a fragment, portion or variant thereof has a mutation at amino acid position C168 compared to CD122 of SEQ ID NO:50.

[0525] In some embodiments, CD122 or a fragment, portion or variant thereof has a mutation C168S at amino acid position 168 compared to CD122 of SEQ ID NO:50.

[0526] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:50 having a C168 mutation.

[0527] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:50 having a C168S mutation.

[0528] In some embodiments, CD122 or a fragment, portion or variant thereof has mutations at amino acid positions C122 and C168 compared to CD122 of SEQ ID NO:50.

[0529] In some embodiments, CD122 or a fragment, portion or variant thereof has mutations C122S and C168S compared to CD122 of SEQ ID NO:50.

[0530] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:51.

[0531] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRSNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWISLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO: 51)

[0532] In some embodiments, the masking portion comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 91% identical to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 92% identical to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 93% identical to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 94% identical to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 96% identical to SEQ ID NO: 51. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 97% identical to SEQ ID NO: 51. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 98% identical to SEQ ID NO: 51. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO:51.

[0533] In some embodiments, the masking moiety has a mutation at amino acid position F8 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation F8C compared to CD122 of SEQ ID NO: 50.

[0534] In some embodiments, the masking moiety has a mutation at amino acid position A94 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation A94C compared to CD122 of SEQ ID NO: 50.

[0535] In some embodiments, the masking moiety has a mutation at amino acid position L106 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation L106C compared to CD122 of SEQ ID NO: 50.

[0536] In some embodiments, the masking moiety has a mutation at amino acid position V117 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation V117C compared to CD122 of SEQ ID NO: 50.

[0537] In some embodiments, the masking moiety has a mutation at amino acid position C122 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation C122S compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation C122V ​​compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation C122A compared to CD122 of SEQ ID NO: 50.

[0538] In some embodiments, the masking moiety has a mutation at amino acid position N123 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation N123C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation N123Q compared to CD122 of SEQ ID NO: 50.

[0539] In some embodiments, the masking moiety has a mutation at amino acid position C168 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation C168S compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation C168V compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation C168A compared to CD122 of SEQ ID NO: 50.

[0540] In some embodiments, the masking moiety has a mutation at amino acid position L169 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation L169C compared to CD122 of SEQ ID NO: 50.

[0541] In some embodiments, the masking moiety has a mutation at amino acid position Q177 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation Q177C compared to CD122 of SEQ ID NO: 50.

[0542] In some embodiments, the masking moiety has a mutation at amino acid position V184 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation V184C compared to CD122 of SEQ ID NO: 50.

[0543] In some embodiments, the masking moiety has a mutation at amino acid position S195 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation S195C compared to CD122 of SEQ ID NO: 50.

[0544] In some embodiments, the masking moiety has a mutation at amino acid position R204 compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety has a mutation R204C compared to CD122 of SEQ ID NO: 50.

[0545] In some embodiments, the masking moiety has the mutations C122V / C168V compared to CD122 of SEQ ID NO:50.

[0546] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:52.

[0547] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO: 52)

[0548] In some embodiments, the masking moiety has the mutation C122A / C168V compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 53.

[0549] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:553)

[0550] In some embodiments, the masking moiety has a mutation C168V compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 54.

[0551] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:54)

[0552] In some embodiments, the masking moiety has the mutation C122V / C168A compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 55.

[0553] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:55)

[0554] In some embodiments, the masking moiety has mutations C122A / N123C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 56.

[0555] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRACISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:56)

[0556] In some embodiments, the masking portion comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO:56. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO:56. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO:56. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 91% identical to SEQ ID NO:56. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 92% identical to SEQ ID NO:56. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 93% identical to SEQ ID NO:56. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 94% identical to SEQ ID NO:56. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO:56. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 96% identical to SEQ ID NO:56. In some embodiments, the masking portion comprises an amino acid sequence that is at least about 97% identical to SEQ ID NO:56. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 98% identical to SEQ ID NO: 56. In some embodiments, the masking moiety comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO:56.

[0557] In some embodiments, the masking moiety has a mutation C122V / N123C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety comprises an amino acid sequence of SEQ ID NO: 57.

[0558] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRVCISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:57)

[0559] In some embodiments, the masking moiety has the mutation C122A / C168A compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 58.

[0560] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:558)

[0561] In some embodiments, the masking moiety has mutations V117C / N123Q / C168A compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 59.

[0562] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHCETHRCQISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:59)

[0563] In some embodiments, the masking moiety has mutations N123Q / C168A / L169C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 60.

[0564] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRCQISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIACETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:60)

[0565] In some embodiments, the masking moiety has mutations L106C / C122A / C168A / S195C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 61.

[0566] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMA PISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWCPWSQPLAFRTKPAALGKD(SEQ IDNO:61)

[0567] In some embodiments, the masking moiety has mutations L106C / C122A / C168A / V184C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 62.

[0568] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMA PISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRCKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO: 62)

[0569] In some embodiments, the masking moiety has mutations C122A / C168A / V184C / S195C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 63.

[0570] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRCKPLQGEFTTWCPWSQPLAFRTKPAALGKD(SEQ IDNNO:63)

[0571] In some embodiments, the masking moiety has mutations C122A / C168A / Q177C / R204C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 64.

[0572] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTCYEFQVRVKPLQGEFTTWSPWSQPLAFCTKPAALGKD(SEQ IDNO:64)

[0573] In some embodiments, the masking portion has mutations L106C / C122V / C168V / S195C compared to CD122 of SEQ ID NO: 50. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 65.

[0574] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMA PISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWCPWSQPLAFRTKPAALGKD(SEQ IDNO:65)

[0575] 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.

[0576] AVNGTSQCTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMCIQDFKPFENLRLMA PISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:66)

[0577] IL-12 receptor

[0578] The masking moiety masks the IL-12 cytokine or its functional fragment among the targeted cytokines, thereby reducing or preventing the binding of the IL-12 cytokine or its functional fragment to its cognate receptor.

[0579] IL-12 receptor, β1 or IL-12Rβ1 is a subunit of the IL-12 receptor complex. IL-12Rβ1 is also known as CD212. This protein binds to interleukin-12 (IL-12) with low affinity. This protein forms disulfide-linked oligomers, which are required for its IL-12 binding activity. IL-12 receptor, β2 or IL-12Rβ2 is a subunit of the IL-12 receptor complex. It has been shown that co-expression of IL-12Rβ1 and IL-12Rβ2 proteins leads to the formation of a high affinity IL-12 binding site.

[0580] In some embodiments, the masking moiety comprises the extracellular domain of the IL-12 cytokine receptor, or a subunit or functional fragment thereof.

[0581] Interleukin-12 receptor subunit beta-1 (also known as CD212) has the following sequence:

[0582]

[0583] Interleukin-12 receptor subunit beta-2 has the following sequence:

[0584]

[0585] Bold indicates the propeptide, underlined italics indicate the extracellular domain, italics indicate the transmembrane domain and underlined bold indicates the cytoplasmic domain.

[0586] 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.

[0587] 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 one to four amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of human IL-12Rβ1 with one or two amino acid substitutions.

[0588] In some embodiments, the masking moiety includes residues 24 to 237 of human IL-12Rβ1, ie, having the sequence of SEQ ID NO: 69, or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-12.

[0589] CRTSECCFQDPPYPDADSGSASGPRDLLCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTL QLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRQLGSQGSSWSKWSSPVCVPPENP(SEQ IDNO:69)

[0590] In some embodiments, the masking moiety comprises an IL-12Rβ1 having SEQ ID NO: 69. 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 any 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.

[0591] In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 69, wherein the amino acid sequence has one to four amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 69, wherein the amino acid sequence has one or two amino acid substitutions.

[0592] In some embodiments, the masking moiety includes residues 24 to 545 of human IL-12Rβ1, ie, having the sequence of SEQ ID NO: 70, or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-12.

[0593] CRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRQLGSQGSSWSKWSSPVCVPPENPPQPQVRFSVEQLGQDGRRRLTLKEQPTQLELPEGCQGLAPGTEVTYRLQLHMLSCPCKAKATRTLHLGKMPYLSGAAYNVAVISSNQFGPGLNQTWHIPADTHTEPVALNISVGTNGTTMYWPARAQSMTYCIEWQPVGQDGGLATCSLTAPQDPDPAGMATYSWSRESGAMGQEKCYYITIFASAHPEKLTLWSTVLSTYHFGGNASAAGTPHHVSVKNHSLDSVSVDWAPSLLSTCPGVLKEYVVRCRDEDSKQVSEHPVQPTETQVTLSGLRAGVAYTVQVRADTAWLRGVWSQPQRFSIEVQVSD(SEQ ID NO:70)

[0594] In some embodiments, the masking moiety comprises an IL-12Rβ1 having SEQ ID NO: 70. 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 any 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.

[0595] In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 70, wherein the amino acid sequence has one to four amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 70, wherein the amino acid sequence has one or two amino acid substitutions.

[0596] In some embodiments, the masking moiety comprises an 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 NNO: 68), the amino acid sequence having one to four amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of human IL-12Rβ2, the amino acid sequence having one or two amino acid substitutions.

[0597] In some embodiments, the masking moiety includes residues 24 to 212 of human IL-12Rβ2, ie, having the sequence of SEQ ID NO:71.

[0598] KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSL(SEQ ID NO:71)

[0599] In some embodiments, the masking moiety comprises an IL-12Rβ1 having SEQ ID NO: 71. 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 any of the amino acid sequences of SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 71. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 71.

[0600] In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 71, wherein the amino acid sequence has one to four amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 71, wherein the amino acid sequence has one or two amino acid substitutions.

[0601] In some embodiments, the masking moiety includes residues 24 to 222 of human IL-12Rβ2, ie, having the sequence of SEQ ID NO:72.

[0602] KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAP EQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIV(SEQ ID NO:72)

[0603] In some embodiments, the masking moiety comprises an IL-12Rβ1 having SEQ ID NO: 72. 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 any 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.

[0604] In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 72, wherein the amino acid sequence has one to four amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 72, wherein the amino acid sequence has one or two amino acid substitutions.

[0605] In some embodiments, the masking moiety includes residues 24 to 319 of human IL-12Rβ2, ie, having the sequence of SEQ ID NO:73.

[0606] KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQC KDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEE(SEQ ID NO:73)

[0607] In some embodiments, the masking moiety comprises an IL-12Rβ2 having SEQ ID NO: 73. 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 any 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.

[0608] In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 73, wherein the amino acid sequence has one to four amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 73, wherein the amino acid sequence has one or two amino acid substitutions.

[0609] In some embodiments, the masking moiety comprises residues 24 to 319 of human IL-12Rβ2, i.e., a sequence having SEQ ID NO: 73, which has one or more cysteine ​​substitutions. In some embodiments, the masking moiety comprises residues 24 to 319 of human IL-12Rβ2, i.e., a sequence having SEQ ID NO: 73, which 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.

[0610] KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQC KDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFTFLDIVRPLPPWDIRIKFQKASVSRSTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEE(SEQ ID NO:74)

[0611] 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.

[0612] In some embodiments, the masking moiety includes residues 24 to 622 of human IL-12Rβ2, ie, having the sequence of SEQ ID NO:75.

[0613] KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTGMLDVWYMKRHIDYSRQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGMDNILVTWQPPRKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSSILGNSKHKAPLSGPHINAITEEKGSILISWNSIPVQEQMGCLLHYRIYWKER(SEQ ID NO:75)

[0614] In some embodiments, the masking moiety comprises an IL-12Rβ1 having SEQ ID NO: 75. 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 any 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.

[0615] In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 75, wherein the amino acid sequence has one to four amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 75, wherein the amino acid sequence has one or two amino acid substitutions.

[0616] In some embodiments, the masking moiety includes residues 24 to 227 of human IL-12Rβ2, ie, having the sequence of SEQ ID NO:76.

[0617] KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTTLFVCKLACINSDEIQICGAEIFVGVAPEQP QNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPP(SEQ ID NO:76)

[0618] In some embodiments, the masking moiety comprises an IL-12Rβ1 having SEQ ID NO: 76. 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 any of the amino acid sequences of SEQ ID NO: 76. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 76. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 76. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 76. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 76. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 76. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 76. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 76. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 76. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 76. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 76.

[0619] In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 76, wherein the amino acid sequence has one to four amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 76, wherein the amino acid sequence has one or two amino acid substitutions.

[0620] Targeted cytokine forms

[0621] As discussed, the cytokine linked to the cleavable Fc domain can also include a targeting moiety to form a targeted cytokine. In some embodiments, the targeted cytokine is a bivalent targeted form comprising: 1) a first chain comprising a variable heavy region with a "hole mutation" (Y349C; T366S; L368A; and Y407V) and an IgG1 or IgG4 heavy constant region; 2) a second chain comprising a variable heavy region with a "knob mutation" (S354C and T366W) fused to a cytokine or a variant thereof, a heavy constant region, wherein the CH1 and CH2 domains are from IgG1 or IgG4, and the CH3 domain is from IgG3; and 3) a third chain comprising a variable light region and an immunoglobulin kappa or lambda constant region.

[0622] In some embodiments, the targeted cytokine is a bivalent targeting format comprising: 1) a first chain comprising a variable heavy region having a "hole mutation" (Y349C; T366S; L368A; and Y407V) and an IgG1 or IgG4 heavy constant region; 2) a second chain comprising a variable heavy region, a heavy constant region from IgG1 or IgG4 fused to a cytokine or a variant thereof with a "knob mutation" (S354C and T366W) and an "RF mutation" (H435R and Y436F); and 3) a third chain comprising a variable light region and an immunoglobulin kappa or lambda constant region.

[0623] In some embodiments, the targeted cytokine is a bivalent targeted format comprising: 1) a first chain comprising a variable heavy region having a "hole mutation" (Y349C; T366S; L368A; and Y407V) and an IgG1 or IgG4 heavy constant region fused to a cytokine or a variant thereof; 2) a second chain comprising a variable heavy region having a "knob mutation" (S354C and T366W), a heavy constant region fused to a masking portion, wherein the CH1 and CH2 domains are from IgG1 or IgG4, and the CH3 domain is from IgG3; and 3) a third chain comprising a variable light region and an immunoglobulin kappa or lambda constant region.

[0624] In some embodiments, the targeted cytokine is a bivalent targeting format comprising: 1) a first chain comprising a variable heavy region with a "hole mutation" (Y349C; T366S; L368A; and Y407V) and an IgG1 or IgG4 heavy constant region fused to a cytokine or a variant thereof; 2) a second chain comprising a variable heavy region with a "knob mutation" (S354C and T366W) and an "RF mutation" (H435R and Y436F), a heavy constant region from IgG1 or IgG4 fused to a masking portion; and 3) a third chain comprising a variable light region and an immunoglobulin kappa or lambda constant region.

[0625] In some embodiments, the targeted cytokine is a bivalent targeted format comprising: 1) a first chain comprising a variable heavy region having a "hole mutation" (Y349C; T366S; L368A; and Y407V) and an IgG1 or IgG4 heavy constant region fused to a masking portion; 2) a second chain comprising a variable heavy region having a "knob mutation" (S354C and T366W), a heavy constant region fused to a cytokine or a variant thereof, wherein the CH1 and CH2 domains are from IgG1 or IgG4, and the CH3 domain is from IgG3; and 3) a third chain comprising a variable light region and an immunoglobulin kappa or lambda constant region.

[0626] In some embodiments, the targeted cytokine is a bivalent targeting format comprising: 1) a first chain comprising a variable heavy region having a "hole mutation" (Y349C; T366S; L368A; and Y407V) and an IgG1 or IgG4 heavy constant region fused to a masking portion; 2) a second chain comprising a variable heavy region, a heavy constant region 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 region and an immunoglobulin kappa or lambda constant region.

[0627] In some embodiments, the targeted cytokine is a monovalent targeted format comprising: 1) a first chain comprising a Fab fused to a first Fc polypeptide chain from IgG1 or IgG4 having a "hole mutation" (Y349C; T366S; L368A; and Y407V); and 2) a second chain comprising a second Fc polypeptide chain from IgG1 or IgG4 having a "knob mutation" (S354C and T366W) and an "RF mutation" (H435R and Y436F).

[0628] In some embodiments, the targeted cytokine is a monovalent targeted format comprising: 1) a first chain comprising a Fab fused to a first Fc polypeptide chain 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 and CH2 domains are from IgG1 or IgG4, and the CH3 domain is from IgG3.

[0629] In some embodiments, the targeted cytokine is a monovalent targeting format comprising: 1) a first chain comprising a first Fc polypeptide chain from an IgG1 or IgG4 with a "hole mutation" (Y349C; T366S; L368A; and Y407V); and 2) a second chain comprising a Fab fused to a second Fc polypeptide chain from an IgG1 or IgG4 with a "knob mutation" (S354C and T366W) and an "RF mutation" (H435R and Y436F). In some embodiments, the cytokine is fused to the first Fc polypeptide chain. In some embodiments, the cytokine is fused to the second Fc polypeptide chain. In some embodiments, the masking moiety is fused to the first Fc polypeptide chain. In some embodiments, the masking moiety is fused to the second Fc polypeptide chain.

[0630] In some embodiments, the targeted cytokine is a monovalent targeting format comprising: 1) a first chain comprising a first Fc polypeptide chain from IgG1 or IgG4 with a "hole mutation" (Y349C; T366S; L368A; and Y407V); and 2) a second chain comprising a Fab fused to a second Fc polypeptide chain with a "knob mutation" (S354C and T366W), wherein the CH1 and CH2 domains are from IgG1 or IgG4, and the CH3 domain is from IgG3. In some embodiments, the cytokine is fused to the second Fc polypeptide chain. In some embodiments, the masking moiety is fused to the first Fc polypeptide chain. In some embodiments, the masking moiety is fused to the second Fc polypeptide chain.

[0631] In some embodiments, the targeted cytokine is a monovalent targeting format comprising: 1) a first chain comprising ...

Claims

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

2. The engineered Fc domain of claim 1, wherein the protease cleavage site is a tissue-selective protease cleavage site.

3. The engineered Fc domain of claim 1, wherein the protease cleavage site is a tumor-associated protease cleavage site.

4. The engineered Fc domain of claim 1, wherein the protease cleavage site is an inflammation-associated protease cleavage site.

5. An engineered Fc domain comprising one or more amino acid replacements in the hinge region, in the CH2 domain, in the CH3 domain and / or in the CH2-CH3 linker, such that the engineered Fc domain comprises a protease cleavage site.

6. The engineered Fc domain of claim 5, wherein the engineered Fc domain comprises one or more amino acid substitutions in the G chain, FG loop, or F chain of the CH3 domain.

7. An engineered Fc domain comprising one or more amino acid substitutions in positions 436 to 447 according to EU numbering, such that the engineered Fc domain comprises a protease cleavage site.

8. An engineered Fc domain according to any of the preceding claims, wherein 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 EU numbering.

9. An engineered Fc domain comprising one or more amino acid substitutions in positions 416 to 425 according to EU numbering, such that the engineered Fc domain comprises a protease cleavage site.

10. The engineered Fc domain of claim 9, wherein the one or more substitutions reduce or eliminate binding of the engineered Fc domain to Protein A.

11. An engineered Fc domain according to claim 9 or 10, wherein the protease cleavage site is located between positions 416 and 423, between positions 416 and 425, between positions 416 and 423, between positions 417 and 425, between positions 417 and 424, between positions 418 and 425, between positions 419 and 422, between positions 419 and 426, between positions 420 and 423, or between positions 421 and 424 according to EU numbering.

12. An engineered Fc domain comprising one or more amino acid substitutions in positions 426 to 437 according to EU numbering, such that the engineered Fc domain comprises a protease cleavage site.

13. The engineered Fc domain of claim 12, wherein the one or more substitutions reduce or eliminate binding of the engineered Fc domain to Protein A.

14. The engineered Fc domain of claim 12 or 13, wherein the protease cleavage site is located between positions 430 and 437, between positions 428 and 434, between positions 428 and 433, between positions 428 and 437, between positions 429 and 437, between positions 434 and 437, between positions 433 and 436, between positions 432 and 435, between positions 431 and 434, between positions 430 and 433, between positions 429 and 432, or between positions 428 and 431 according to EU numbering.

15. The engineered Fc domain of any one of claims 7 to 14, wherein the protease cleavage site is a tumor-associated protease cleavage site.

16. The engineered Fc domain of any one of claims 7 to 15, wherein the protease cleavage site is a tissue selective protease cleavage site.

17. The engineered Fc domain of any one of claims 7 to 15, wherein the protease cleavage site is an inflammation-associated protease cleavage site.

18. The engineered Fc domain of any of the preceding claims, wherein the protease cleavage 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.

19. The engineered Fc domain of any of the preceding claims, wherein the engineered Fc domain comprises any one of the amino acid substitution groups listed in Table 8a.

20. The engineered Fc domain of any of the preceding claims, wherein the engineered Fc domain comprises any one of the amino acid substitution groups listed in Table 5.

21. The engineered Fc domain of any of the preceding claims, wherein the engineered Fc domain comprises any one of the amino acid substitution groups listed in Table 11.

22. The engineered Fc domain of any of the preceding claims, wherein the engineered Fc domain comprises any one of the amino acid substitution groups listed in Table 11a.

23. The engineered Fc domain of any of the preceding claims, wherein the engineered Fc domain comprises any one of the groups of amino acid substitutions listed in Table 11b.

24. The engineered Fc domain of any of the preceding claims, wherein the engineered Fc domain comprises any one of the amino acid substitution groups listed in Table 11c.

25. A masked cytokine comprising: Cytokine part, masking part; as well as An engineered Fc domain including 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 upon cleavage by the tumor-associated protease cleavage site on the engineered Fc domain, the cytokine portion is released from the masking portion.

26. The masked cytokine of claim 25, wherein the engineered Fc domain is fused to the cytokine moiety or the masking moiety via a non-cleavable linker.

27. The masked cytokine of claim 25, wherein the engineered Fc domain is fused directly to the cytokine moiety or the masking moiety.

28. A masked therapeutically active molecule comprising: Therapeutically active domain masking part; as well as An engineered Fc domain including a tumor-associated protease cleavage site; wherein the engineered Fc domain is fused to the therapeutically active domain or the masking moiety, such that the masking moiety binds to the therapeutically active domain and upon cleavage by the tumor-associated protease cleavage site on the engineered Fc domain, the therapeutically active domain is released from the masking moiety.

29. The masked therapeutically active molecule of claim 28, wherein the engineered Fc domain is fused to the therapeutically active domain or the masking moiety via a non-cleavable linker.

30. The masked therapeutically active molecule of claim 28, wherein the engineered Fc domain is directly fused to the therapeutically active domain or the masking moiety.

31. The masked therapeutically active molecule according to any one of claims 28 to 30, wherein the therapeutically active domain is a cell engager or a co-stimulatory domain.

32. A masked cytokine comprising: Cytokine part, masking part; as well as a vector portion comprising an engineered tumor-associated protease cleavage site; 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 at the engineered tumor-associated protease cleavage site.

33. The masked cytokine of claim 32, wherein the carrier portion comprises a first Fc domain and a second Fc domain, wherein one of the first Fc domain or the second Fc domain comprises the engineered tumor-associated protease cleavage site.

34. The masked cytokine of claim 32, wherein the carrier moiety is a half-life extending domain selected from albumin, transferrin or tissue factor.

35. The masked cytokine of claim 32, wherein the carrier moiety is selected from the group consisting of an immunoglobulin, Fab, scFv, VHH, nanobody, VH, VL, a single domain Ab immunoglobulin kappa constant region (CK) and an immunoglobulin lambda constant region (CL).

36. The masked cytokine according to any one of claims 32 to 35, wherein the carrier moiety is fused to the cytokine moiety and / or the masking moiety via a non-cleavable linker.

37. The masked cytokine according to any one of claims 32 to 35, wherein the carrier moiety is directly fused to the cytokine moiety and / or the masking moiety.

38. The masked cytokine according to any one of claims 25 to 28 and 32 to 35, wherein the masked cytokine does not contain a cleavable linker.

39. An engineered Fc domain or masked cytokine according to any of the preceding claims, wherein the protease is 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, MMP28, cathepsins (cathepsin B, cathepsin D, cathepsin F, cathepsin K , cathepsin L, cathepsin V, cathepsin S, cathepsin W), ADAM, ADAMTS, kallikreins 1 to 15, HTRA1-2-3, HGFAc, PRSS, TMPRSS, elastase, PR-3, granzymes (granzymes A, B, M, H, and K), fibroblast activation protein (FAP), plasmin, urokinase plasminogen activator (uPA), tryptase, caspase, thrombin, asparagine endopeptidase, chymotrypsin, collagenase, aspartic proteinase A, matriptase1-2, and matriptase.

40. The engineered Fc domain or masked cytokine of claim 39, wherein the tumor-associated protease is MMP2, MMP3, MMP9, cathepsin B, or matriptase.

41. The engineered Fc domain or masked cytokine of any one of the preceding claims, wherein the engineered Fc domain comprises N434I, Y436V and Q438L mutations.

42. The engineered Fc domain or masked cytokine of any one of the preceding claims, wherein the engineered Fc domain comprises H435S, Q438I and S440G mutations.

43. The engineered Fc domain or masked cytokine of any one of the preceding claims, wherein the engineered Fc domain comprises T437S, Q438N and K438E mutations.

44. The engineered Fc domain or masked cytokine of any one of the preceding claims, wherein the engineered Fc domain comprises K439V and L441S mutations.

45. The engineered Fc domain or masked cytokine of any one of the preceding claims, wherein the engineered Fc domain comprises Q438P, K439T and L441T mutations.

46. ​​The engineered Fc domain or masked cytokine of any one of the preceding claims, wherein the engineered Fc domain comprises K439E, P445E and G446E mutations.

47. The engineered Fc domain or masked cytokine of any one of the preceding claims, wherein the engineered Fc domain comprises K439A, S444A and G446V mutations.

48. The engineered Fc domain or masked cytokine of any of the preceding claims, wherein the engineered Fc domain comprises any one of the amino acid substitution groups listed in Table 5.

49. The engineered Fc domain or masked cytokine of any of the preceding claims, wherein the engineered Fc domain comprises any one of the amino acid substitution groups listed in Table 8a.

50. The engineered Fc domain or masked cytokine of any of the preceding claims, wherein the engineered Fc domain comprises any one of the amino acid substitution groups listed in Table 11.

51. The engineered Fc domain or masked cytokine of any of the preceding claims, wherein the engineered Fc domain comprises any one of the amino acid substitution groups listed in Table 11a.

52. The engineered Fc domain or masked cytokine of any one of the preceding claims, wherein the engineered Fc domain comprises any one of the amino acid substitution groups listed in Table 11b.

53. The engineered Fc domain or masked cytokine of any of the preceding claims, wherein the tumor-associated protease cleavage site comprises the amino acid sequence of VPLSLYSG.

54. The engineered Fc domain or masked cytokine of any one of the preceding claims, wherein the tumor-associated protease cleavage site comprises the amino acid sequence of IHVTLKSL.

55. The engineered Fc domain or masked cytokine of any of the preceding claims, wherein the tumor-associated protease cleavage site comprises the amino acid sequence of NSYTIKGL.

56. The engineered Fc domain or masked cytokine of any of the preceding claims, wherein the tumor-associated protease cleavage site comprises the amino acid sequence of SNESLSLS.

57. The engineered Fc domain or masked cytokine of any of the preceding claims, wherein the tumor-associated protease cleavage site comprises the amino acid sequence of SQESLSLS.

58. The engineered Fc domain or masked cytokine of any one of the preceding claims, wherein the tumor-associated protease cleavage site comprises the amino acid sequence of QVSSSLSP.

59. The engineered Fc domain or masked cytokine of any one of the preceding claims, wherein the tumor-associated protease cleavage site comprises the amino acid sequence of PTSTSLSP.

60. The engineered Fc domain or masked cytokine of any of the preceding claims, wherein the tumor-associated protease cleavage site comprises the amino acid sequence of ESLSLSEE.

61. An engineered Fc domain or masked cytokine according to any of the preceding claims, wherein the tumor-associated protease cleavage site comprises the amino acid sequence of ASLSLAPV.

62. The engineered Fc domain or masked cytokine of any of the preceding claims, wherein the tumor-associated protease cleavage site comprises the amino acid sequence of PLGL.

63. An engineered Fc domain or masked cytokine according to any of the preceding claims, wherein the tumor-associated protease cleavage site comprises the amino acid sequence of GLFG, SRLRSPQG, KPMQFLGD, FRLWS, SLLLRTGN, SLGRRPGG, SLFRAPGP, AGLRSPGG, SGRRSPGG, SLSGRRGG, FRLWA, SLSLGRRG, SLFRSAGP, FRSGVPGG, ALFKSSFP, SLSLSGRR, GGPRNL or FLVGGASL.

64. The engineered Fc domain or masked cytokine of any of the preceding claims, wherein the tumor-associated protease cleavage site comprises the amino acid sequence of GGPKLL, SLSSGRSG, GGPRML, GGPLRL, RGSRGG, KQLRVVNG, GGPRNL, KGSRAG, PLRLSRA, GGPRRL, RGSRAG, SLSGRSGG, GRSRGG, GRSRAG, GKSRAG, RQYRVVGG, LSGK, LSGR, KQLRHMRG, or SSGRSPGG.

65. The engineered Fc domain or masked cytokine of any of the preceding claims, wherein the tumor-associated protease cleavage site comprises the amino acid sequence of any one of SEQ ID NOs: 119, 122, 123, 135-139, 143-208 and 209.

66. The masked cytokine of any one of claims 25 to 65, wherein 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, 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.

67. The masked cytokine of claim 66, wherein the cytokine is IL-2, IL-15 or IL-12.

68. The masked cytokine according to claim 67, wherein the IL-2 is a modified IL-2 cytokine or a functional fragment thereof compared to the sequence of mature IL-2 having SEQ ID NO:

13.

69. The masked cytokine according to claim 68, wherein the modified IL-2 cytokine or a functional fragment thereof comprises modifications R38A, F42A, Y45A and E62A relative to the sequence of mature IL-2 having SEQ ID NO:

13.

70. The masked cytokine according to claim 68 or 69, wherein the modified IL-2 cytokine or a functional fragment thereof comprises a modification C125A relative to the sequence of mature IL-2 having SEQ ID NO:

13.

71. The masked cytokine according to any one of claims 68 to 70, wherein the modified IL-2 cytokine or a functional fragment thereof comprises R38A, F42A, Y45A, E62A and C125A relative to the sequence of mature IL-2 having SEQ ID NO:

13.

72. The masked cytokine according to claim 68, wherein the modified IL-2 cytokine or a functional fragment thereof comprises a mutation that reduces binding to CD25.

73. The masked cytokine of claim 67, wherein the IL-15 polypeptide comprises 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.

74. The masked cytokine according to claim 67, wherein the IL-12 polypeptide or the functional fragment thereof comprises an IL-12p40 polypeptide or the functional fragment thereof covalently linked to an IL-12p35 polypeptide or the functional fragment thereof.

75. The masked cytokine of claim 74, wherein the IL-12p40-IL-12p35 linker is between 5 and 20 amino acids in length.

76. The masked cytokine of claim 74 or 75, wherein the IL-12p40-IL-12p35 linker is rich in amino acid residues G and S.

77. The masked cytokine according to any one of claims 25 to 76, wherein the masking moiety binds to the cytokine.

78. The masked cytokine of claim 77, wherein said masking moiety is a receptor for said cytokine or said fragment thereof.

79. The masked cytokine of claim 78, wherein 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, CD21 2. LIFR, OSMR, IL-20Rα, IL-20Rβ, IL-14R, CD4, CDw127, CD118, CDw119, CD40, LTβR, CD120a, CD120b, CDw137, BCMA, TAC I, CD27, CD30, CD95, GITR, LTbR, HVEM, OX40, TRAILR1-4, Apo3, RANK, OPG, TGF-βR1, TGF-βR2, TGF-βR3, CD115 or CDw136.

80. The masked cytokine of claim 79, wherein the masking moiety is CD122.

81. The masked cytokine of claim 80, wherein the CD122 is an engineered CD122 polypeptide or a fragment thereof comprising one or more mutations relative to a wild-type CD122 amino acid sequence.

82. The masked cytokine of claim 81, wherein the one or more mutations are selected from the group consisting of F8C, A94C, L106C, C122S, C122V, C122A, N123C, N123Q, C168V, C168A, C168S, L169C, Q177C, V184C, S195C, and R204C.

83. The masked cytokine of claim 77, wherein said masking moiety is a peptide, polypeptide, protein, ligand or agent that specifically binds to said cytokine or said fragment thereof.

84. The masked cytokine of claim 83, wherein the masking moiety comprises 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-linked Fab (s-Fab), an antibody, or a combination thereof.

85. The masked cytokine of claim 84, wherein the masking moiety comprises a scFv having SEQ ID NO: 124 and / or SEQ ID NO:

125.

86. The masked cytokine of claim 84, wherein the masking moiety comprises a scFv having at least 80%, 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity to SEQ ID NO: SEQ ID NO:

142.

87. The masked cytokine of claim 86, wherein the masking moiety comprises a scFv having the amino acid sequence of SEQ ID NO:

142.

88. The masked cytokine of claim 84, wherein 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.

89. The masked cytokine of claim 88, wherein the VHH has at least 80%, 82%, 85%, 88%, 90%, 92%, 95%, 98% or 99% sequence identity to SEQ ID NO: SEQ ID NO:

121.

90. The masked cytokine of claim 89, wherein the VHH has the amino acid sequence of SEQ ID NO:

121.

91. The masked cytokine of any one of claims 33 to 90, wherein the first Fc domain comprises a first IgGl, IgG2 or IgG4 Fc domain or a fragment thereof.

92. The masked cytokine of any one of the preceding claims 33 to 91, wherein the second Fc domain comprises a second IgGl, IgG2 or IgG4 Fc domain or a fragment thereof.

93. The masked cytokine of any one of claims 33 to 92, wherein the first Fc domain and / or the second Fc domain each contains one or more modifications that promote non-covalent association of the first Fc polypeptide and the second Fc polypeptide.

94. The masked cytokine according to any one of claims 33 to 93, wherein a. the first Fc domain comprises Y349C, T366S, L368A, Y407V and N297A mutations, and the second Fc domain comprises S354C, T366W and N297A mutations; or b. The second Fc domain comprises Y349C, T366S, L368A, Y407V and N297A mutations, and the first Fc domain comprises S354C, T366W and N297A mutations.

95. The masked cytokine according to any one of claims 33 to 94, wherein a. the first Fc domain comprises Y349C, T366S, L368A, Y407V, N297A and I253A mutations, and the second Fc domain comprises S354C, T366W, N297A and I253A mutations; or b. The second Fc domain comprises Y349C, T366S, L368A, Y407V, N297A and I253A mutations, and the first Fc domain comprises S354C, T366W, N297A and I253A mutations.

96. The masked cytokine of any one of claims 33 to 95, wherein the first Fc domain comprises a CH3 domain comprising a modification that reduces or eliminates binding to Protein A, and the second Fc domain comprises a CH3 domain that binds to Protein A.

97. The masked cytokine of claim 96, wherein the CH3 domain that binds to Protein A is a human IgG1, IgG2 or IgG4 sequence.

98. The masked cytokine according to claim 96 or 97, wherein the second CH3 domain is a human IgGl, IgG2 or IgG4 sequence comprising modifications at positions H435 and / or Y436 according to Kabat numbering.

99. The masked cytokine of claim 98, wherein the modifications are H435R and Y436F according to Kabat numbering.

100. The masked cytokine according to any one of claims 98 or 99, wherein the first CH3 domain comprises a human IgG3 sequence.

101. The masked cytokine of any one of claims 25 to 100, further comprising a targeting moiety.

102. The masked cytokine of claim 101, wherein the targeting moiety comprises one or more antigen binding domains, peptides, polypeptides, proteins, ligands or agents that specifically bind to an antigen or ligand.

103. The masked cytokine of claim 102, wherein the targeting moiety comprises an antigen binding domain selected from the group consisting of Fab, single-chain Fv (scFv), single domain antibody (VHH), one or more CDRs, variable heavy chain (VH), variable light chain (VL), Fab-like bispecific antibody (bsFab), single domain antibody-linked Fab (s-Fab), antibody, and combinations thereof.

104. The masked cytokine of any one of claims 101 to 103, wherein the targeting moiety comprises a first antigen binding domain and a second antigen binding domain.

105. The masked cytokine of claim 104, wherein the first antigen binding domain and the second antigen binding domain specifically bind to the same target.

106. The masked cytokine of claim 105, wherein the first antigen binding domain and the second antigen binding domain comprise the same amino acid sequence.

107. The masked cytokine of claim 104, wherein the first antigen binding domain and the second antigen binding domain specifically bind to different targets.

108. The masked cytokine of claim 107, wherein the first antigen binding domain and the second antigen binding domain comprise different amino acid sequences.

109. The masked cytokine of any one of claims 101 to 108, wherein the targeting moiety specifically binds to 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 (VEGF R1), vascular endothelial growth factor receptor 2 (VEGF R2), adhesion protein-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-a), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), ectonucleotide pyrophosphatase / phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG), CD19, CD20, CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD98, CD 123, CD 138, CD352, CD47, signal regulatory protein alpha (SIRPα), Claudin18.2, Claudin 6. 5T4, fibroblast activation protein alpha (FAPα), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epithelial cell adhesion molecule (EPCAM), or a combination thereof.

110. The masked cytokine of claim 109, wherein the targeting moiety specifically binds to PD-1.

111. The masked cytokine of any one of claims 101 to 110, wherein the targeting moiety is linked to the Fc domain via one or both of the first Fc polypeptide and the second Fc polypeptide.

112. The masked cytokine of any one of claims 101 to 111, wherein the C-terminus of the targeting moiety is linked to the N-terminus of the first Fc polypeptide.

113. The masked cytokine according to any one of claims 103 to 112, wherein the C-terminus of the first Fc polypeptide is linked to the N-terminus of the cytokine or fragment thereof.

114. The masked cytokine of any one of claims 101 to 111, wherein the C-terminus of the targeting moiety is linked to the N-terminus of the second Fc polypeptide.

115. The masked cytokine according to any one of claims 103 to 114, wherein the C-terminus of the second Fc polypeptide is linked to the N-terminus of the masking moiety.

116. The masked cytokine according to any one of claims 25 to 115, wherein the protease cleavage site has an in vitro cleavage efficiency that produces at least 10% active cytokine.

117. A nucleic acid encoding the engineered Fc domain or the masked cytokine according to any one of the preceding claims.

118. A vector comprising the nucleic acid according to claim 117.

119. A host cell comprising the nucleic acid of claim 117, or the vector of claim 118.

120. A method of producing a masked cytokine, the method comprising culturing the host cell of claim 119 under conditions whereby the targeted cytokine is produced.

121. A composition comprising the engineered Fc domain or the masked cytokine according to any one of claims 1 to 116.

122. A pharmaceutical composition comprising the engineered Fc domain or the masked cytokine according to any one of claims 1 to 116 and a pharmaceutically acceptable carrier.

123. A kit comprising the engineered Fc domain or the masked cytokine according to any one of claims 1 to 116, or the composition according to claim 121 or the pharmaceutical composition according to claim 122.

124. A method of treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of the engineered Fc domain or the masked cytokine according to any one of claims 1 to 116, or the composition of claim 121 or the pharmaceutical composition of claim 122.

125. A method of treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of an engineered Fc domain or a masked cytokine according to any one of claims 1 to 116, or a composition according to claim 121, or a pharmaceutical composition according to claim 122.

126. An engineered Fc domain or masked cytokine according to any one of claims 1 to 116 for use in medicine.

127. The engineered Fc domain or masked cytokine according to any one of claims 1 to 116 for use in treating a neoplastic disease.

128. The engineered Fc domain or masked cytokine according to any one of claims 1 to 116 for use in treating or preventing an inflammatory disease.

129. The engineered Fc domain or masked cytokine according to any one of claims 1 to 116 for use in treating or preventing an autoimmune disease.

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