Targeting cytokines and methods of use thereof

Abstract

The present invention relates to a targeted masked cytokine comprising a cytokine or a functional fragment thereof, a masking moiety, a targeting moiety, and a proteolytically cleavable linker. The targeting moiety includes an antigen-binding moiety that specifically binds to an antigen expressed on the surface of a target cell. The masking portion masks the cytokine or functional fragment thereof, thereby reducing or preventing binding of the cytokine or functional fragment thereof to its homologous receptor, but after proteolytic cleavage of the cleavable linker at a target site, the cytokine or functional fragment thereof is activated, thereby reducing or preventing binding of the cytokine or functional fragment thereof to its homologous receptor. This enables or more binding of the cytokine or functional fragment thereof to its homologous receptor.

Description

[0001] This application is a divisional application of the patent application with the application number 202380023984.2, the filing date of February 28, 2023, and the invention title of "Targeted Cytokines and Methods of Use Thereof".

[0002] Cross - reference to related applications

[0003] This application claims the priority and benefit of U.S. Provisional Application No. 63 / 314,928, filed on February 28, 2022, and U.S. Provisional Application No. 63 / 314,926, filed on February 28, 2022, and the content of each of the U.S. Provisional Applications is hereby incorporated by reference in its entirety. Background of the Invention

[0004] In the United States, cancer is the second leading cause of death, causing more deaths than the next five leading causes (chronic respiratory diseases, stroke, accidents, Alzheimer's disease, and diabetes). Despite significant progress, especially in targeted therapies, there is still much work to be done in this field. Immunotherapy and a branch of this field, immuno - oncology, are generating viable and exciting treatment options for treating malignancies. Specifically, it is now recognized that one hallmark of cancer is immune evasion, and significant efforts have been made to identify targets and develop therapies against these targets to re - activate the immune system to recognize and treat cancer.

[0005] Cytokine therapy is an effective strategy for stimulating the immune system to induce anti - tumor cytotoxicity. Specifically, Proleukin (aldesleukin), a recombinant form of interleukin - 2 (IL - 2), has been approved by the FDA for the treatment of metastatic renal cell carcinoma and melanoma. Unfortunately, cytokines administered to patients typically have a very short half - life, thus requiring frequent dosing. For example, the product label of aldesleukin sold under the trade name Proleukin indicates that the drug has an 85 - minute half - life in patients receiving a 5 - minute intravenous (IV) infusion. Additionally, the administration of high doses of cytokines can cause adverse health outcomes, such as vascular leak, through systemic immune activation. These findings illustrate the need to develop IL - 2 cytokine therapeutics that effectively target tumors without causing the side effects associated with systemic immune activation. Provided herein are targeted, masked IL - 2 cytokines, cleavage products of the targeted IL - 2 cytokines and compositions thereof, and methods for addressing this need. Summary of the Invention

[0006] The present invention particularly provides methods and compositions for treating cancer. The present invention is in part based on the surprising discovery that the targeted masked cytokines of the present invention specifically target cells of interest to effectively treat cancer without causing undesirable side effects. The targeted cytokines of the present invention specifically bind to target cells of interest, accumulate in tumors, and are activated by tumor proteases. In addition, treatment with the targeted masked cytokines of the present invention increases the percentage of CD8 T cells in the tumor microenvironment and significantly inhibits tumor growth with minimal in vivo toxicity.

[0007] In one aspect, the present invention particularly provides a targeted cytokine, which comprises a targeting moiety, a cytokine or a fragment thereof, a masking moiety, and an Fc domain, the Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide being linked to the cytokine or a fragment thereof through a first linker, and the second Fc polypeptide being linked to the masking moiety through a second linker.

[0008] In one aspect, the present invention particularly provides a targeted cytokine, which comprises a targeting moiety, a cytokine or a fragment thereof, a masking moiety, and an Fc domain, the Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide being linked to the IL-2 cytokine or a fragment thereof through a first linker, and the second Fc polypeptide being linked to the masking moiety through a second linker, wherein the masking moiety binds to the IL-2 cytokine or a fragment thereof, wherein the first linker or the second linker is a cleavable linker such that the masking moiety releases the IL-2 cytokine or a fragment thereof upon cleavage, wherein the cleavable linker comprises a sequence selected from Table 1, and wherein the targeting moiety is linked to the Fc domain through one or both of the first Fc polypeptide and the second Fc polypeptide.

[0009] In one aspect, the present invention particularly provides a targeted cytokine, which comprises a targeting moiety, a cytokine or a fragment thereof, a masking moiety, and an Fc domain, the Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide being linked to the IL-2 cytokine or a fragment thereof through a first linker, and the second Fc polypeptide being linked to the masking moiety through a second linker, wherein the masking moiety binds to the IL-2 cytokine or a fragment thereof, wherein the first linker or the second linker is a cleavable linker such that the masking moiety releases the IL-2 cytokine or a fragment thereof upon cleavage, wherein the cleavable linker comprises MPYDLYHP (SEQ ID NO:34) or VPLSLYSG (SEQ ID NO:42), and wherein the targeting moiety is linked to the Fc domain through one or both of the first Fc polypeptide and the second Fc polypeptide.

[0010] In some embodiments, the cleavable linker comprises PVSLRSGS (SEQ ID NO:196), or GMPKDLYHAS (SEQ ID NO:197), or RPLALWRS (SEQ ID NO:193), or TQKPLGLS (SEQ ID NO:194), or APAGLIVPYN (SEQ ID NO:195), or PANLVAPDP (SEQ ID NO:183), or IVGRPRHQGV (SEQ ID NO:199) or RSKYLATA (SEQ ID NO:198).

[0011] In some embodiments, the cleavable linker comprises MPYDLYHP (SEQ ID NO:34), RAAAVKSP or VPLSLYSG (SEQ ID NO:42).

[0012] In some embodiments, the masking moiety binds to a cytokine or a fragment thereof. In some embodiments, the masking moiety comprises an agent that binds to a cytokine or a fragment thereof. In some embodiments, the masking moiety comprises a peptide that binds to a cytokine or a fragment thereof. In some embodiments, the masking moiety comprises a polypeptide that binds to a cytokine or a fragment thereof. In some embodiments, the masking moiety comprises a protein that binds to a cytokine or a fragment thereof. In some embodiments, the masking moiety comprises a nucleic acid that binds to a cytokine or a fragment thereof.

[0013] In some embodiments, the masking moiety comprises a Fab. In some embodiments, the masking moiety comprises a single-chain Fv (scFv). In some embodiments, the masking moiety comprises a single-domain antibody (VHH). In some embodiments, the masking moiety comprises one or more CDRs. In some embodiments, the masking moiety comprises a variable heavy chain (VH). In some embodiments, the masking moiety comprises a variable light chain (VL). In some embodiments, the masking moiety comprises a Fab-like bispecific antibody (bsFab). In some embodiments, the masking moiety comprises a single-domain antibody-linked Fab (s-Fab). In some embodiments, the masking moiety comprises an antibody. In some embodiments, the masking moiety comprises a combination thereof. In some embodiments, the masking moiety comprises a Fab that binds to a cytokine. In some embodiments, the masking moiety comprises a single-chain Fv (scFv) that binds to a cytokine. In some embodiments, the masking moiety comprises a single-domain antibody (VHH) that binds to a cytokine. In some embodiments, the masking moiety comprises one or more CDRs that bind to a cytokine. In some embodiments, the masking moiety comprises a variable heavy chain (VH) that binds to a cytokine. In some embodiments, the masking moiety comprises a variable light chain (VL) that binds to a cytokine. In some embodiments, the masking moiety comprises a Fab-like bispecific antibody (bsFab) that binds to a cytokine. In some embodiments, the masking moiety comprises a single-domain antibody-linked Fab (s-Fab) that binds to a cytokine. In some embodiments, the masking moiety comprises an antibody or a fragment thereof that binds to a cytokine. In some embodiments, the masking moiety comprises an antibody or a binding fragment of an antibody against a cytokine.

[0014] In some embodiments, the masking moiety is a receptor of the cytokine. In some embodiments, the masking moiety is a fragment of the receptor of the cytokine. In some embodiments, the masking moiety is an extracellular domain (ECD) of the receptor of the cytokine.

[0015] In some embodiments, the masking portion is CD121. In some embodiments, the masking portion is IL-18Rα. In some embodiments, the masking portion is IL-18Rβ. In some embodiments, the masking portion is CD25. In some embodiments, the masking portion is CD132. In some embodiments, the masking portion is CD124. In some embodiments, the masking portion is CD213a13. In some embodiments, the masking portion is CD132. In some embodiments, the masking portion is CD127. In some embodiments, the masking portion is IL-9R. In some embodiments, the masking portion is CD213a1. In some embodiments, the masking portion is CD213a2. In some embodiments, the masking portion is CD1243. In some embodiments, the masking portion is CD132. In some embodiments, the masking portion is IL-15Ra. In some embodiments, the masking portion is CDw131. In some embodiments, the masking portion is CDw125. In some embodiments, the masking portion is CD131. In some embodiments, the masking portion is CD116. In some embodiments, the masking portion is CD126. In some embodiments, the masking portion is CD130. In some embodiments, the masking portion is IL-11Ra. In some embodiments, the masking portion is CD114. In some embodiments, the masking portion CD212. In some embodiments, the masking portion is LIFR. In some embodiments, the masking portion is OSMR. In some embodiments, the masking portion is IL-20Rα. In some embodiments, the masking portion is IL-20Rβ. In some embodiments, the masking portion is IL-14R. In some embodiments, the masking portion is CD4. In some embodiments, the masking portion is CDw127. In some embodiments, the masking portion is CD118. In some embodiments, the masking portion is CDw119. In some embodiments, the masking portion is CD40. In some embodiments, the masking portion is LTβR. In some embodiments, the masking portion is CD120a. In some embodiments, the masking portion is CD120b. In some embodiments, the masking portion is CDw137. In some embodiments, the masking portion is BCMA. In some embodiments, the masking portion is TACI. In some embodiments, the masking portion is CD27. In some embodiments, the masking portion is CD30. In some embodiments, the masking portion is CD95. In some embodiments, the masking portion is GITR. In some embodiments, the masking portion is LTbR. In some embodiments, the masking portion is HVEM. In some embodiments, the masking portion is OX40.In some embodiments, the masking moiety is TRAILR1-4. In some embodiments, the masking moiety is Apo3. In some embodiments, the masking moiety is RANK. In some embodiments, the masking moiety is OPG. In some embodiments, the masking moiety is TGF-βR1. In some embodiments, the masking moiety is TGF-βR2. In some embodiments, the masking moiety is TGF-βR3. In some embodiments, the masking moiety is CD115. In some embodiments, the masking moiety is CDw136.

[0016] In some embodiments, the masking moiety is CD122. In some embodiments, the CD122 is an engineered CD122 polypeptide or a fragment thereof, and the engineered CD122 polypeptide or a fragment thereof comprises one or more mutations relative to the wild-type CD122 amino acid sequence.

[0017] In some embodiments, the engineered CD122 comprises an F8C mutation. In some embodiments, the engineered CD122 comprises an A94C mutation. In some embodiments, the engineered CD122 comprises an L106C mutation. In some embodiments, the engineered CD122 comprises a C122S mutation. In some embodiments, the engineered CD122 comprises a C122V mutation. In some embodiments, the engineered CD122 comprises a C122A mutation. In some embodiments, the engineered CD122 comprises an N123C mutation. In some embodiments, the engineered CD122 comprises an N123Q mutation. In some embodiments, the engineered CD122 comprises a C168V mutation. In some embodiments, the engineered CD122 comprises a C168A mutation. In some embodiments, the engineered CD122 comprises a C168S mutation. In some embodiments, the engineered CD122 comprises an L169C mutation. In some embodiments, the engineered CD122 comprises a Q177C mutation. In some embodiments, the engineered CD122 comprises a V184C mutation. In some embodiments, the engineered CD122 comprises an S195C mutation. In some embodiments, the engineered CD122 comprises an R204C mutation.

[0018] In some embodiments, the engineered CD122 comprises C122S and C168S mutations. In some embodiments, the engineered CD122 comprises C122A and N123C mutations.

[0019] In some embodiments, the engineered CD122 includes C122V and C168V mutations. In some embodiments, the engineered CD122 includes C122A and C168V mutations. In some embodiments, the engineered CD122 includes C122V and C168A mutations. In some embodiments, the engineered CD122 includes C122V and N123C mutations. In some embodiments, the engineered CD122 includes C122A and C168A mutations. In some embodiments, the engineered CD122 includes V117C, N123Q and C168A mutations. In some embodiments, the engineered CD122 includes N123Q, C168A and L169C mutations. In some embodiments, the engineered CD122 includes L106C, C122A, C168A and S195C mutations. In some embodiments, the engineered CD122 includes L106C, C122A, C168A and V184C mutations. In some embodiments, the engineered CD122 includes C122A, C168A, V184C and S195C mutations. In some embodiments, the engineered CD122 includes C122A, C168A, Q177C and R204C mutations. In some embodiments, the engineered CD122 includes L106C, C122V, C168V and S195C mutations. In some embodiments, the engineered CD122 includes F8C, A94C, C122V and C168V mutations.

[0020] In some embodiments, the engineered CD122 polypeptide or fragment thereof includes mutations at amino acids 122 and 168 relative to SEQ ID NO:15, wherein the engineered CD122 has improved stability compared to wild-type CD122. In some embodiments, the engineered CD122 polypeptide or fragment thereof includes mutations that promote disulfide bond formation. In some embodiments, the engineered CD122 polypeptide or fragment thereof includes mutations that create salt bridges.

[0021] In some embodiments, the masking moiety includes the extracellular domain of human IL-12Rβ1 or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-12. In some embodiments, the masking moiety includes residues 24 to 237 of human IL-12Rβ1. In some embodiments, the masking moiety includes the sequence of SEQ ID NO:126. In some embodiments, the masking moiety includes residues 24 to 545 of human IL-12Rβ1. In some embodiments, the masking moiety includes the sequence of SEQ ID NO:127.

[0022] In some embodiments, the masking moiety comprises the extracellular domain of human IL-12Rβ2 or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-12. In some embodiments, the masking moiety comprises residues 24 to 212 of human IL-12Rβ2. In some embodiments, the masking moiety comprises the sequence of SEQ ID NO:128. In some embodiments, the masking moiety comprises residues 24 to 222 of human IL-12Rβ2. In some embodiments, the masking moiety comprises the sequence of SEQ ID NO:129. In some embodiments, the masking moiety comprises residues 24 to 227 of human IL-12Rβ2. In some embodiments, the masking moiety comprises the sequence of SEQ ID NO:133. In some embodiments, the masking moiety comprises residues 24 to 319 of human IL-12Rβ2. In some embodiments, the masking moiety comprises the sequence of SEQ ID NO:130.

[0023] In some embodiments, the masking moiety comprises at least one amino acid modification as compared to the sequence of SEQ ID NO:130. In some embodiments, the masking moiety comprises at least one amino acid modification as compared to the sequence of SEQ ID NO:130, optionally wherein the modification is a cysteine substitution mutation. In some embodiments, the masking moiety comprises SEQ ID NO:131. In some embodiments, the masking moiety comprises residues 24 to 622 of human IL-12Rβ2. In some embodiments, the masking moiety comprises the sequence of SEQ ID NO:132.

[0024] 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-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-18) or a variant thereof. In some embodiments, the cytokine is G-CSF or a variant thereof. In some embodiments, the cytokine is CXCL8 (IL-18) 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.

[0025] In some embodiments, the IL-2 is a modified IL-2 cytokine or a functional fragment thereof as compared to the sequence of mature IL-2 having SEQ ID NO:13. In some embodiments, the modified IL-2 cytokine or a functional fragment thereof comprises a modification R38A relative to the sequence of mature IL-2 having SEQ ID NO:13. In some embodiments, the modified IL-2 cytokine or a functional fragment thereof comprises a modification F42A relative to the sequence of mature IL-2 having SEQ ID NO:13. In some embodiments, the modified IL-2 cytokine or a functional fragment thereof comprises a modification Y45A relative to the sequence of mature IL-2 having SEQ ID NO:13. In some embodiments, the modified IL-2 cytokine or a functional fragment thereof comprises a modification E62A relative to the sequence of mature IL-2 having 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 mature IL-2 having 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 mature IL-2 having SEQ ID NO:13.

[0026] 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 mature IL-2 having SEQ ID NO:13. In some embodiments, the modified IL-2 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:14.

[0027] In some embodiments, the modified IL-2 cytokine or a functional fragment thereof comprises a modification that reduces or eliminates binding to CD25.

[0028] In some embodiments, the modified IL-2 cytokine or a functional fragment thereof comprises a modification that enhances binding to CD25.

[0029] In some embodiments, the modified IL-2 cytokine or a functional fragment thereof comprises a modification that reduces or eliminates binding to CD122.

[0030] In some embodiments, the modified IL-2 cytokine or a functional fragment thereof comprises a modification that enhances binding to CD122.

[0031] In some embodiments, the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO:93 or an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO:93. 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 position D22 as compared to the amino acid sequence of SEQ ID NO:93. 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 position E46 as compared to the amino acid sequence of SEQ ID NO:93. 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 position E53 as compared to the amino acid sequence of SEQ ID NO:93. 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 position N71 as compared to the amino acid sequence of SEQ ID NO:93. 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 position N79 as compared to the amino acid sequence of SEQ ID NO:93. 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 position N112 as compared to the amino acid sequence of SEQ ID NO:93.

[0032] 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 position D22, E46 or E53 as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71 and N79 as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71 and N112 as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N79 and N112 as compared to the amino acid sequence of SEQ ID NO:93. 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 position D22, E46, E53, N71, N79 or N112 as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQID NO:94.

[0033] 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. In some embodiments, the length of the IL-12p40-IL-12p35 linker ranges from 2 amino acids to 40 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker ranges from 4 amino acids to 30 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker ranges from 5 amino acids to 20 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker ranges from 7 amino acids to 15 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker ranges from 10 amino acids to 12 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 2 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 2 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 2 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 2 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 2 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 2 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 3 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 4 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 5 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 6 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 7 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 8 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 9 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 10 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 11 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 12 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 13 amino acids.In some embodiments, the length of the IL-12p40-IL-12p35 linker is 14 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 15 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 16 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 17 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 18 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 19 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 20 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 21 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 22 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 23 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 24 amino acids. In some embodiments, the length of the IL-12p40-IL-12p35 linker is 25 amino acids.

[0034] In some embodiments, the IL-12p40-IL-12p35 linker comprises amino acid residues A, P, G, and S. In some embodiments, the IL-12p40-IL-12p35 linker is rich in amino acid residues G and S. In some embodiments, the IL-12p40-IL-12p35 linker comprises SEQ ID NO:119 (GGGGSGGGGSGGGGS). In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO:111. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO:111. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having one amino acid modification as compared to the amino acid sequence of SEQ ID NO:111. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having two amino acid modifications as compared to the amino acid sequence of SEQ ID NO:111. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having three amino acid modifications as compared to the amino acid sequence of SEQ ID NO:111. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having four amino acid modifications as compared to the amino acid sequence of SEQ ID NO:111. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having five amino acid modifications as compared to the amino acid sequence of SEQ ID NO:111.

[0035] In some embodiments, as compared to the amino acid sequence of SEQ ID NO:111, the IL-12p40 polypeptide comprises at least one amino acid modification to the GAG binding domain (KSKREKKDRV).

[0036] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO:115. In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO:116.

[0037] In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having one or more cysteine substitution mutations as compared to the amino acid sequence of SEQ ID NO:111. In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO:117. In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO:118.

[0038] In some embodiments, the IL-12p35 polypeptide comprises SEQ ID NO:112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO:112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having at least two amino acid modifications as compared to the amino acid sequence of SEQ ID NO:112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having at least three amino acid modifications as compared to the amino acid sequence of SEQ ID NO:112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having at least four amino acid modifications as compared to the amino acid sequence of SEQ ID NO:112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having at least five amino acid modifications as compared to the amino acid sequence of SEQ ID NO:112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having at least six amino acid modifications as compared to the amino acid sequence of SEQ ID NO:112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having at least seven amino acid modifications as compared to the amino acid sequence of SEQ ID NO:112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having at least eight amino acid modifications as compared to the amino acid sequence of SEQ ID NO:112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having at least nine amino acid modifications as compared to the amino acid sequence of SEQ ID NO:112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having at least ten amino acid modifications as compared to the amino acid sequence of SEQ ID NO:112.

[0039] In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having one amino acid modification as compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having two amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having three amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having four amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having five amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having six amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having seven amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having eight amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having nine amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having ten amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 112.

[0040] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ IDNO:150)

[0041] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTERVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ IDNO:151)

[0042] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ IDNO:152)

[0043] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ IDNO:153)

[0044] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ ID NO:154)

[0045] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO:150. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO:151. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO:152. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO:153. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO:154.

[0046] In some embodiments, the first linker or the second linker is a cleavable linker such that the masking moiety releases the cytokine or its fragment upon cleavage.

[0047] In some embodiments, the cleavable linker comprises MPYDLYHP. In some embodiments, the cleavable linker comprises VPLSLYSG (SEQ ID NO:42). In some embodiments, the cleavable linker comprises MPYDLYHP (SEQ ID NO:34) and VPLSLYSG (SEQ ID NO:42). In some embodiments, the cleavable linker comprises DSGGFMLT (SEQ ID NO:35). In some embodiments, the cleavable linker comprises HEQLTV (SEQ ID NO:36). In some embodiments, the cleavable linker comprises RAAAVKSP (SEQ ID NO:37). In some embodiments, the cleavable linker comprises VPLSLY (SEQ ID NO:38). In some embodiments, the cleavable linker comprises DLLAVVAAS (SEQ ID NO:39). In some embodiments, the cleavable linker comprises ISSGLLSGRS (SEQ ID NO:40).

[0048] In some embodiments, the cleavable linker comprises 6 - 60 amino acid residues. In some embodiments, the cleavable linker comprises 8 - 50 amino acid residues. In some embodiments, the cleavable linker comprises 8 - 30 amino acid residues. In some embodiments, the cleavable linker comprises 8 - 25 amino acid residues. In some embodiments, the cleavable linker comprises 8 amino acid residues. In some embodiments, the cleavable linker comprises 9 amino acid residues. In some embodiments, the cleavable linker comprises 10 amino acid residues. In some embodiments, the cleavable linker comprises 11 amino acid residues. In some embodiments, the cleavable linker comprises 12 amino acid residues. In some embodiments, the cleavable linker comprises 13 amino acid residues. In some embodiments, the cleavable linker comprises 14 amino acid residues. In some embodiments, the cleavable linker comprises 15 amino acid residues. In some embodiments, the cleavable linker comprises 16 amino acid residues. In some embodiments, the cleavable linker comprises 17 amino acid residues. In some embodiments, the cleavable linker comprises 18 amino acid residues. In some embodiments, the cleavable linker comprises 19 amino acid residues. In some embodiments, the cleavable linker comprises 20 amino acid residues. In some embodiments, the cleavable linker comprises 21 amino acid residues. In some embodiments, the cleavable linker comprises 22 amino acid residues. In some embodiments, the cleavable linker comprises 23 amino acid residues. In some embodiments, the cleavable linker comprises 24 amino acid residues. In some embodiments, the cleavable linker comprises 25 amino acid residues.

[0049] In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide. In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide flanked by a spacer domain at the N-terminus. In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide flanked by a spacer domain at the C-terminus. In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide flanked by spacer domains on both sides. In some embodiments, the length of the spacer domain ranges from 2 amino acids to 20 amino acids. In some embodiments, the length of the spacer domain ranges from 3 amino acids to 10 amino acids. In some embodiments, the length of the spacer domain ranges from 3 amino acids to 6 amino acids. In some embodiments, the length of the spacer domain is 2 amino acids. In some embodiments, the length of the spacer domain is 3 amino acids. In some embodiments, the length of the spacer domain is 4 amino acids. In some embodiments, the length of the spacer domain is 5 amino acids. In some embodiments, the length of the spacer domain is 6 amino acids. In some embodiments, the length of the spacer domain is 7 amino acids. In some embodiments, the length of the spacer domain is 8 amino acids. In some embodiments, the length of the spacer domain is 9 amino acids. In some embodiments, the length of the spacer domain is 10 amino acids. In some embodiments, the length of the spacer domain is 11 amino acids. In some embodiments, the length of the spacer domain is 12 amino acids. In some embodiments, the length of the spacer domain is 13 amino acids. In some embodiments, the length of the spacer domain is 14 amino acids. In some embodiments, the length of the spacer domain is 15 amino acids.

[0050] In some embodiments, the targeting moiety comprises at least one antigen-binding domain. In some embodiments, the targeting moiety comprises a first antigen-binding domain and a second antigen-binding domain. In some embodiments, the targeting moiety comprises a first antigen-binding domain, a second antigen-binding domain, and a third antigen-binding domain. In some embodiments, the targeting moiety comprises a first antigen-binding domain, a second antigen-binding domain, a third antigen-binding domain, and a fourth antigen-binding domain. In some embodiments, the first antigen-binding domain and the second antigen-binding domain specifically bind to the same target. In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain specifically bind to the same target. In some embodiments, the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, and the fourth antigen-binding domain specifically bind to the same target. In some embodiments, the first antigen-binding domain and the second antigen-binding domain specifically bind to different targets. In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain each specifically bind to a different target. In some embodiments, the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, and the fourth antigen-binding domain each specifically bind to a different target.

[0051] In some embodiments, the first antigen-binding domain and the second antigen-binding domain comprise different amino acid sequences. In some embodiments, the first antigen-binding domain and the second antigen-binding domain comprise the same amino acid sequence. In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain comprise different amino acid sequences. In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain comprise the same amino acid sequence. In some embodiments, the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, and the fourth antigen-binding domain comprise different amino acid sequences. In some embodiments, the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, and the fourth antigen-binding domain comprise the same amino acid sequence.

[0052] In some embodiments, the first Fc polypeptide comprises a first IgG1 Fc domain or a fragment thereof. In some embodiments, the first Fc polypeptide comprises a first IgG2 Fc domain or a fragment thereof. In some embodiments, the first Fc polypeptide comprises a first IgG3 Fc domain or a fragment thereof. In some embodiments, the first Fc polypeptide comprises a first IgG4 Fc domain or a fragment thereof. In some embodiments, the second Fc polypeptide comprises a second IgG1 Fc domain or a fragment thereof. In some embodiments, the second Fc polypeptide comprises a second IgG2 Fc domain or a fragment thereof. In some embodiments, the second Fc polypeptide comprises a second IgG3 Fc domain or a fragment thereof. In some embodiments, the second Fc polypeptide comprises a second IgG4 Fc domain or a fragment thereof.

[0053] In some embodiments, the first Fc domain contains one or more modifications that promote non-covalent association of the first Fc polypeptide and the second Fc polypeptide. In some embodiments, the second Fc domain contains one or more modifications that promote non-covalent association of the first Fc polypeptide and the second Fc polypeptide. In some embodiments, each of the first Fc domain and / or the second Fc domain contains one or more modifications that promote non-covalent association of the first Fc polypeptide and the second Fc polypeptide. In some embodiments, each of the first Fc domain and the second Fc domain contains one or more modifications that promote non-covalent association of the first Fc polypeptide and the second Fc polypeptide.

[0054] In some embodiments, the first Fc domain comprises a Y349C mutation. In some embodiments, the first Fc domain comprises a T366S mutation. In some embodiments, the first Fc domain comprises an L368A mutation. In some embodiments, the first Fc domain comprises a Y407V mutation. In some embodiments, the first Fc domain comprises an N297A mutation. In some embodiments, the first Fc domain comprises an S354C mutation. In some embodiments, the first Fc domain comprises a T366W mutation. In some embodiments, the first Fc domain comprises an I253A mutation. In some embodiments, the second Fc domain comprises a Y349C mutation. In some embodiments, the second Fc domain comprises a T366S mutation. In some embodiments, the second Fc domain comprises an L368A mutation. In some embodiments, the second Fc domain comprises a Y407V mutation. In some embodiments, the second Fc domain comprises an N297A mutation. In some embodiments, the second Fc domain comprises an S354C mutation. In some embodiments, the second Fc domain comprises a T366W mutation. In some embodiments, the second Fc domain comprises an I253A mutation.

[0055] In some embodiments, the first Fc domain comprises Y349C and T366S mutations. In some embodiments, the first Fc domain comprises Y349C, T366S, and L368A mutations. In some embodiments, the first Fc domain comprises Y349C and L368A mutations. In some embodiments, the first Fc domain comprises Y349C, T366S, L368A, and Y407V mutations. In some embodiments, the first Fc domain comprises Y349C, T366S, and Y407V mutations. In some embodiments, the first Fc domain comprises T366S, L368A, and Y407V mutations. In some embodiments, the first Fc domain comprises Y349C, L368A, and Y407V mutations. In some embodiments, the first Fc domain comprises Y349C, T366S, L368A, Y407V, and N297A mutations. In some embodiments, the first Fc domain comprises S354C and T366W mutations. In some embodiments, the first Fc domain comprises S354C and N297A mutations. In some embodiments, the first Fc domain comprises T366W and N297A mutations. In some embodiments, the first Fc domain comprises S354C, T366W, and N297A mutations. In some embodiments, the second Fc domain comprises Y349C and T366S mutations. In some embodiments, the second Fc domain comprises Y349C, T366S, and L368A mutations. In some embodiments, the second Fc domain comprises Y349C and L368A mutations. In some embodiments, the second Fc domain comprises Y349C, T366S, L368A, and Y407V mutations. In some embodiments, the second Fc domain comprises Y349C, T366S, and Y407V mutations. In some embodiments, the second Fc domain comprises T366S, L368A, and Y407V mutations. In some embodiments, the second Fc domain comprises Y349C, L368A, and Y407V mutations. In some embodiments, the second Fc domain comprises Y349C, T366S, L368A, Y407V, and N297A mutations. In some embodiments, the second Fc domain comprises S354C and T366W mutations. In some embodiments, the second Fc domain comprises S354C and N297A mutations. In some embodiments, the second Fc domain comprises T366W and N297A mutations. In some embodiments, the second Fc domain comprises S354C, T366W, and N297A mutations.

[0056] In some embodiments, the first Fc domain comprises Y349C, T366S, L368A, Y407V, and N297A mutations, and the second Fc domain comprises S354C, T366W, and N297A mutations. In some embodiments, the second Fc domain comprises Y349C, T366S, L368A, Y407V, and N297A mutations, and the first Fc domain comprises S354C, T366W, and N297A mutations.

[0057] In some embodiments, the first Fc domain comprises Y349C, T366S, L368A, Y407V, and I253A mutations, and the second Fc domain comprises S354C, T366W, and I253A mutations. In some embodiments, the second Fc domain comprises Y349C, T366S, L368A, Y407V, and I253A mutations, and the first Fc domain comprises S354C, T366W, and I253A mutations.

[0058] In some embodiments, 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. In some embodiments, the second Fc domain comprises Y349C, T366S, L368A, Y407V, N297A, I253A mutations, and the first Fc domain comprises S354C, T366W, N297A, and I253A mutations.

[0059] In some embodiments, the first Fc polypeptide comprises a first CH3 domain, and the first CH3 domain comprises a modification that reduces or eliminates binding to Protein A. In some embodiments, the second Fc polypeptide comprises a second CH3 domain, and the second CH3 domain comprises a modification that reduces or eliminates binding to Protein A. In some embodiments, the first Fc polypeptide comprises a first CH3 domain that binds to Protein A. In some embodiments, the second Fc polypeptide comprises a second CH3 domain that binds to Protein A.

[0060] In some embodiments, the first Fc polypeptide comprises a first CH3 domain, and the first CH3 domain comprises a modification that reduces or eliminates binding to Protein A, and the second Fc polypeptide comprises a second CH3 domain that binds to Protein A. In some embodiments, the second Fc polypeptide comprises a second CH3 domain, and the second CH3 domain comprises a modification that reduces or eliminates binding to Protein A, and the first Fc polypeptide comprises a first CH3 domain that binds to Protein A.

[0061] In some embodiments, the CH3 domain that binds to Protein A is a human IgG1 sequence. In some embodiments, the CH3 domain that binds to Protein A is a human IgG2 sequence. In some embodiments, the CH3 domain that binds to Protein A is a human IgG4 sequence.

[0062] In some embodiments, the first CH3 domain is a human IgG1 sequence comprising a modification at position H435 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG1 sequence comprising a modification at position Y436 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG1 sequence comprising modifications at positions H435 and Y436 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG1 sequence comprising the H435R modification according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG1 sequence comprising the Y436F modification. In some embodiments, the first CH3 domain is a human IgG1 sequence comprising the modifications H435R and Y436F according to Kabat numbering.

[0063] In some embodiments, the first CH3 domain is a human IgG2 sequence comprising a modification at position H435 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising a modification at position Y436 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising modifications at positions H435 and Y436 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising the H435R modification according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising the Y436F modification. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising the modifications H435R and Y436F according to Kabat numbering.

[0064] In some embodiments, the first CH3 domain is a human IgG3 sequence comprising a modification at position H435 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising a modification at position Y436 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising modifications at positions H435 and Y436 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising the H435R modification according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising the Y436F modification. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising the modifications H435R and Y436F according to Kabat numbering.

[0065] In some embodiments, the second CH3 domain is a human IgG1 sequence comprising a modification at position H435 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG1 sequence comprising a modification at position Y436 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG1 sequence comprising modifications at positions H435 and Y436 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG1 sequence comprising the H435R modification according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG1 sequence comprising the Y436F modification. In some embodiments, the second CH3 domain is a human IgG1 sequence comprising the modifications H435R and Y436F according to Kabat numbering.

[0066] In some embodiments, the second CH3 domain is a human IgG2 sequence comprising a modification at position H435 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising a modification at position Y436 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising modifications at positions H435 and Y436 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising the H435R modification according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising the Y436F modification. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising the modifications H435R and Y436F according to Kabat numbering.

[0067] In some embodiments, the second CH3 domain is a human IgG3 sequence comprising a modification at position H435 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising a modification at position Y436 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising modifications at positions H435 and Y436 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising the H435R modification according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising the Y436F modification. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising the modifications H435R and Y436F according to Kabat numbering.

[0068] In some embodiments, the first Fc domain comprises SEQ ID NO:85. In some embodiments, the first Fc domain comprises SEQ ID NO:80. In some embodiments, the second Fc domain comprises SEQ ID NO:85. In some embodiments, the second Fc domain comprises SEQ ID NO:80. In some embodiments, the first Fc domain comprises SEQ ID NO:85 and the second Fc domain comprises SEQ ID NO:80. In some embodiments, the second Fc domain comprises SEQ ID NO:85 and the first Fc domain comprises SEQ ID NO:80.

[0069] In some embodiments, the first CH3 domain comprises a human IgG3 sequence. In some embodiments, the second CH3 domain comprises a human IgG3 sequence.

[0070] In some embodiments, the first Fc domain comprises SEQ ID NO:84. In some embodiments, the first Fc domain comprises SEQ ID NO:80. In some embodiments, the second Fc domain comprises SEQ ID NO:84. In some embodiments, the second Fc domain comprises SEQ ID NO:80. In some embodiments, the first Fc domain comprises SEQ ID NO:84 and the second Fc domain comprises SEQ ID NO:80. In some embodiments, the second Fc domain comprises SEQ ID NO:84 and the first Fc domain comprises SEQ ID NO:80.

[0071] In some embodiments, the first antigen-binding domain comprises a VH, and the second antigen-binding domain comprises a VL. In some embodiments, the first antigen-binding domain comprises three HCDRs, and the second antigen-binding domain comprises three LCDRs.

[0072] In some embodiments, the targeting moiety is linked to the Fc domain via one or both of the first Fc polypeptide and the second Fc polypeptide. In some embodiments, the targeting moiety is linked to the Fc domain via the first Fc polypeptide. In some embodiments, the targeting moiety is linked to the Fc domain via the second Fc polypeptide. In some embodiments, the targeting moiety is linked to the Fc domain via both the first Fc polypeptide and the second Fc polypeptide. In some embodiments, the targeting moiety is linked to the N-terminus of the Fc domain via one or both of the first Fc polypeptide and the second Fc polypeptide. In some embodiments, the targeting moiety is linked to the N-terminus of the Fc domain via the first Fc polypeptide. In some embodiments, the targeting moiety is linked to the N-terminus of the Fc domain via the second Fc polypeptide. In some embodiments, the targeting moiety is linked to the N-terminus of the Fc domain via both the first Fc polypeptide and the second Fc polypeptide. In some embodiments, the targeting moiety is linked to the C-terminus of the Fc domain via one or both of the first Fc polypeptide and the second Fc polypeptide. In some embodiments, the targeting moiety is linked to the C-terminus of the Fc domain via the first Fc polypeptide. In some embodiments, the targeting moiety is linked to the C-terminus of the Fc domain via the second Fc polypeptide. In some embodiments, the targeting moiety is linked to the C-terminus of the Fc domain via both the first Fc polypeptide and the second Fc polypeptide.

[0073] In some embodiments, the targeting moiety comprises a variable heavy chain (VH) and is linked to the Fc domain by one or both of the first Fc polypeptide and the second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain (VH) and is linked to the Fc domain by the first Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain (VH) and is linked to the Fc domain by the second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain (VH) and is linked to the Fc domain by both the first Fc polypeptide and the second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain (VH) and is linked to the N-terminus of the Fc domain by one or both of the first Fc polypeptide and the second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain (VH) and is linked to the N-terminus of the Fc domain by the first Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain (VH) and is linked to the N-terminus of the Fc domain by the second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain (VH) and is linked to the N-terminus of the Fc domain by both the first Fc polypeptide and the second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain (VH) and is linked to the C-terminus of the Fc domain by one or both of the first Fc polypeptide and the second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain (VH) and is linked to the C-terminus of the Fc domain by the first Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain (VH) and is linked to the C-terminus of the Fc domain by the second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain (VH) and is linked to the C-terminus of the Fc domain by both the first Fc polypeptide and the second Fc polypeptide.

[0074] In some embodiments, the targeting moiety comprises a Fab. In some embodiments, the targeting moiety comprises a Fab having HCDR1 of SEQ ID NO:4 (GYTFTNYY), HCDR2 of SEQ ID NO:5 (INPSNGGT), HCDR3 of SEQ ID NO:6 (ARRDYRFDMGFDY), LCDR1 of SEQ ID NO:9 (KGVSTSGYSY), LCDR2 of SEQ ID NO:10 (LAS), and LCDR3 of SEQ ID NO:11 (QHSRDLPLT).

[0075] In some embodiments, the targeting moiety comprises a variable heavy chain (VH) of SEQ ID NO:1 and a variable light chain (VL) of SEQ ID NO:7. In some embodiments, the targeting moiety comprises a heavy chain of SEQ ID NO:2 and a light chain of SEQ ID NO:8.

[0076] In some embodiments, the cytokine is linked to the first Fc polypeptide via an uncleavable linker. In some embodiments, the N-terminus of the cytokine is linked to the C-terminus of the first Fc polypeptide via an uncleavable linker. In some embodiments, the C-terminus of the cytokine is linked to the N-terminus of the first Fc polypeptide via an uncleavable linker. In some embodiments, the cytokine is linked to the first Fc polypeptide via a cleavable linker. In some embodiments, the N-terminus of the cytokine is linked to the C-terminus of the first Fc polypeptide via a cleavable linker. In some embodiments, the C-terminus of the cytokine is linked to the N-terminus of the first Fc polypeptide via a cleavable linker.

[0077] In some embodiments, the masking moiety is linked to the second Fc polypeptide via an uncleavable linker. In some embodiments, the N-terminus of the masking moiety is linked to the C-terminus of the second Fc polypeptide via an uncleavable linker. In some embodiments, the C-terminus of the masking moiety is linked to the N-terminus of the second Fc polypeptide via an uncleavable linker. In some embodiments, the masking moiety is linked to the second Fc polypeptide via a cleavable linker. In some embodiments, the N-terminal masking moiety is linked to the C-terminus of the second Fc polypeptide via a cleavable linker. In some embodiments, the C-terminal masking moiety is linked to the N-terminus of the second Fc polypeptide via a cleavable linker.

[0078] In some embodiments, the C-terminus of the first antigen-binding domain is linked to the N-terminus of the first Fc polypeptide. In some embodiments, the C-terminus of the first Fc polypeptide is linked to the N-terminus of the cytokine or a fragment thereof. In some embodiments, the C-terminus of the second antigen-binding domain is linked to the N-terminus of the second Fc polypeptide. In some embodiments, the C-terminus of the second Fc polypeptide is linked to the N-terminus of the masking moiety.

[0079] In some embodiments, the targeting moiety comprises an agent that specifically binds to a target. In some embodiments, the targeting moiety comprises a peptide that specifically binds to a target. In some embodiments, the targeting moiety comprises a polypeptide that specifically binds to a target. In some embodiments, the targeting moiety comprises a polypeptide that specifically binds to a target. In some embodiments, the targeting moiety comprises a protein that specifically binds to a target. In some embodiments, the targeting moiety comprises a nucleic acid that specifically binds to a target.

[0080] In some embodiments, the targeting moiety includes a Fab, a single-chain Fv (scFv), a single-domain antibody (VHH), one or more complementarity-determining regions (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. In some embodiments, the targeting moiety includes a Fab. In some embodiments, the targeting moiety includes a single-chain Fv (scFv). In some embodiments, the targeting moiety includes a single-domain antibody (VHH). In some embodiments, the targeting moiety includes one or more CDRs. In some embodiments, the targeting moiety includes a variable heavy chain (VH). In some embodiments, the targeting moiety includes a variable light chain (VL). In some embodiments, the targeting moiety includes a Fab-like bispecific antibody (bsFab). In some embodiments, the targeting moiety includes a single-domain antibody-linked Fab (s-Fab). In some embodiments, the targeting moiety includes an antibody or a fragment thereof.

[0081] In some embodiments, the targeting moiety comprises an antigen-binding moiety, wherein the antigen is expressed on immune cells. In some embodiments, the targeting moiety specifically binds to PD-1. In some embodiments, the targeting moiety specifically binds to PD-L1. In some embodiments, the targeting moiety specifically binds to PD-L2. In some embodiments, the targeting moiety specifically binds to CTLA-4. In some embodiments, the targeting moiety specifically binds to TIGIT. In some embodiments, the targeting moiety specifically binds to TIM-3. In some embodiments, the targeting moiety specifically binds to LAG-3. In some embodiments, the targeting moiety specifically binds to CD25. In some embodiments, the targeting moiety specifically binds to CD16a. In some embodiments, the targeting moiety specifically binds to CD16b. In some embodiments, the targeting moiety specifically binds to NKG2D. In some embodiments, the targeting moiety specifically binds to NKP44. In some embodiments, the targeting moiety specifically binds to NKP3. In some embodiments, the targeting moiety specifically binds to CD19. In some embodiments, the targeting moiety specifically binds to CD20. In some embodiments, the targeting moiety specifically binds to CD30. In some embodiments, the targeting moiety specifically binds to CD38. In some embodiments, the targeting moiety specifically binds to BMCA. In some embodiments, the targeting moiety specifically binds to HER2. In some embodiments, the targeting moiety specifically binds to HER3. In some embodiments, the targeting moiety specifically binds to DLL3. In some embodiments, the targeting moiety specifically binds to DLL4. In some embodiments, the targeting moiety specifically binds to EGFR. In some embodiments, the targeting moiety specifically binds to GPC3. In some embodiments, the targeting moiety specifically binds to c-MET. In some embodiments, the targeting moiety specifically binds to VEGF-R1. In some embodiments, the targeting moiety specifically binds to VEGF-R2. In some embodiments, the targeting moiety specifically binds to OX40. In some embodiments, the targeting moiety specifically binds to DR5. In some embodiments, the targeting moiety specifically binds to ICOS. In some embodiments, the targeting moiety specifically binds to GITR. In some embodiments, the targeting moiety specifically binds to CD73. In some embodiments, the targeting moiety specifically binds to CD39. In some embodiments, the targeting moiety specifically binds to CD25. In some embodiments, the targeting moiety specifically binds to CD16a. In some embodiments, the targeting moiety specifically binds to CD8. In some embodiments, the targeting moiety specifically binds to KLRC1.In some embodiments, the targeting moiety specifically binds to KLRD1. In some embodiments, the targeting moiety specifically binds to KLRB1. In some embodiments, the targeting moiety specifically binds to CD40. In some embodiments, the targeting moiety specifically binds to CD137. In some embodiments, the targeting moiety specifically binds to CD28.

[0082] In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing from about 1% to 99% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing from about 5% to 80% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing from about 8% to 60% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing from about 10% to 40% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing from about 10% to 20% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 3% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 5% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 8% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 10% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 12% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 15% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 20% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 25% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 30% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 35% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 40% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 45% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 50% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 60% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 70% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency of producing at least 80% active cytokine.

[0083] On the one hand, the present invention particularly provides a targeting cytokine, the targeting cytokine comprising a targeting moiety, a cytokine or a fragment thereof, a masking moiety, and an Fc domain, the Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide being linked to the cytokine or a fragment thereof through a first linker, the second Fc polypeptide being linked to the masking moiety through a second linker, wherein the masking moiety binds to the cytokine or a fragment thereof, wherein the first linker or the second linker is a cleavable linker such that the masking moiety releases the cytokine or a fragment thereof upon cleavage, wherein the cleavable linker comprises MPYDLYHP (SEQ ID NO:34) or VPLSLYSG (SEQ ID NO:42), and wherein the targeting moiety is linked to the Fc domain through one or both of the first Fc polypeptide and the second Fc polypeptide.

[0084] On the one hand, the present invention particularly provides a targeting cytokine, the targeting cytokine comprising a targeting moiety, a cytokine or a fragment thereof, a masking moiety, and an Fc domain, the Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide being linked to the cytokine or a fragment thereof through a first linker, the second Fc polypeptide being linked to the masking moiety through a second linker, wherein the masking moiety binds to the cytokine or a fragment thereof, wherein the first linker or the second linker is a tumor-specific cleavable linker such that the masking moiety releases the cytokine or a fragment thereof upon cleavage by a tumor-specific protease, wherein the tumor-specific cleavable linker comprises 8 - 50 amino acid residues, and wherein the targeting moiety is linked to the Fc domain through one or both of the first Fc polypeptide and the second Fc polypeptide.

[0085] On the one hand, the present invention particularly provides a targeting cytokine, the targeting cytokine comprising a targeting moiety, a cytokine or a fragment thereof, a masking moiety, and an Fc domain, the Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide being linked to the cytokine or a fragment thereof through a first linker, the second Fc polypeptide being linked to the masking moiety through a second linker, wherein the masking moiety binds to the cytokine or a fragment thereof, wherein the first linker or the second linker is a cleavable linker such that the masking moiety releases the cytokine or a fragment thereof upon cleavage to produce an active cytokine, wherein the cleavable linker has an in vitro cleavage efficiency of producing at least 10% active cytokine, and wherein the targeting moiety is linked to the Fc domain through one or both of the first Fc polypeptide and the second Fc polypeptide.

[0086] On the one hand, the present invention particularly provides a targeting cytokine, which comprises a targeting moiety; a cytokine or a fragment thereof; a masking moiety, which comprises CD122 or a fragment thereof, and the CD122 or the fragment thereof comprises 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, R204C; and an Fc domain, which comprises a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide is connected to the cytokine or the fragment thereof through a first linker, the second Fc polypeptide is connected to the masking moiety through a second linker, wherein the masking moiety binds to the cytokine or the fragment thereof, wherein the first linker or the second linker is a cleavable linker, such that the masking moiety releases the cytokine or the fragment thereof upon cleavage, and wherein the targeting moiety is connected to the Fc domain through one or two of the first Fc polypeptide and the second Fc polypeptide.

[0087] On the one hand, the present invention particularly provides a targeting cytokine, which comprises a targeting moiety, a masking moiety, a cytokine or a fragment thereof, and an Fc domain, the Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide comprises a CH3 domain, and the CH3 domain comprises a modification that reduces or eliminates binding to protein A, and the second Fc polypeptide comprises a CH3 domain that binds to protein A.

[0088] On the one hand, the present invention particularly provides a targeting cytokine, which comprises a targeting moiety, a cytokine or a fragment thereof, a masking moiety, and an Fc domain, the Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide is connected to the cytokine or the fragment thereof through a first linker, the second Fc polypeptide is connected to the masking moiety through a second linker, wherein the masking moiety binds to the cytokine or the fragment thereof, wherein the first linker or the second linker is a cleavable linker, such that the masking moiety releases the cytokine or the fragment thereof upon cleavage, wherein the cleavable linker comprises MPYDLYHP (SEQ ID NO:34) or VPLSLYSG (SEQ ID NO:42), and wherein the targeting moiety is connected to the Fc domain through one or two of the first Fc polypeptide and the second Fc polypeptide.

[0089] On the one hand, the present invention particularly provides a targeting cytokine, which comprises a targeting moiety that specifically binds to PD-1. The targeting moiety comprises HCDR1 of SEQ ID NO:4 (GYTFTNYY), HCDR2 of SEQ ID NO:5 (INPSNGGT), HCDR3 of SEQ ID NO:6 (ARRDYRFDMGFDY), LCDR1 of SEQ ID NO:9 (KGVSTSGYSY), LCDR2 of SEQ ID NO:10 (LAS), and LCDR3 of SEQ ID NO:11 (QHSRDLPLT); a cytokine or a fragment thereof; and an Fc domain.

[0090] On the one hand, the present invention particularly provides a targeting IL-2 cytokine, which comprises SEQ ID NO:87.

[0091] In some embodiments, the targeting IL-2 cytokine comprises SEQ ID NO:88.

[0092] In some embodiments, the targeting IL-2 cytokine comprises SEQ ID NO:89.

[0093] In some embodiments, the targeting IL-2 cytokine comprises SEQ ID NO:90.

[0094] In some embodiments, the targeting IL-2 cytokine comprises SEQ ID NO:91.

[0095] On the one hand, the present invention particularly provides a targeting IL-2 cytokine comprising SEQ ID NO:87 and SEQ ID NO:88, and a light chain comprising SEQ ID NO:8.

[0096] On the one hand, the present invention particularly provides a targeting IL-2 cytokine comprising SEQ ID NO:87 and SEQ ID NO:89, and a light chain comprising SEQ ID NO:8.

[0097] On the one hand, the present invention particularly provides a targeting IL-2 cytokine comprising SEQ ID NO:87 and SEQ ID NO:90, and a light chain comprising SEQ ID NO:8.

[0098] On the one hand, the present invention particularly provides a targeting IL-2 cytokine comprising SEQ ID NO:87 and SEQ ID NO:91, and a light chain comprising SEQ ID NO:8.

[0099] On the one hand, the present invention particularly provides a nucleic acid encoding the target cytokine of the present invention. On the one hand, the present invention particularly provides a vector comprising the nucleic acid encoding the target cytokine of the present invention. On the one hand, the present invention particularly provides a host cell comprising the nucleic acid encoding the target cytokine of the present invention.

[0100] On the one hand, the present invention particularly provides a method for producing a targeted cytokine, the method comprising culturing a host cell comprising the nucleic acid encoding the target cytokine of the present invention under conditions for producing the targeted cytokine.

[0101] On the one hand, the present invention particularly provides a composition comprising the targeted cytokine of the present invention.

[0102] On the one hand, the present invention particularly provides a pharmaceutical composition comprising the targeted cytokine of the present invention and a pharmaceutically acceptable carrier.

[0103] On the one hand, the present invention particularly provides a kit comprising the targeted cytokine of the present invention.

[0104] On the one hand, the present invention particularly provides a method for treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of the targeted cytokine of the present invention.

[0105] On the one hand, the present invention particularly provides a method for treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of a composition comprising the targeted cytokine of the present invention.

[0106] On the one hand, the present invention particularly provides a method for treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the targeted cytokine of the present invention.

[0107] On the one hand, the present invention particularly provides a method for treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of the targeted cytokine of the present invention.

[0108] On the one hand, the present invention particularly provides a method for treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of a composition comprising the targeted cytokine of the present invention.

[0109] On the one hand, the present invention particularly provides a method for treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the targeted cytokine of the present invention. Brief Description of the Drawings

[0110] The accompanying drawings are for illustrative purposes only and not for limitation.

[0111] Figure 1 Shows an exemplary schematic diagram of the Fc domain of the IL-2 cytokine targeting for the present invention. The first Fc polypeptide comprises CH2 and CH3 domains derived from IgG1. The second chain of the "chimeric Fc" comprises a CH2 domain derived from IgG1 and a CH3 domain derived from IgG3. The second chain of the "CH3 mutant" comprises CH2 and CH3 domains derived from IgG1 having a "RF mutation" (H435R and Y436F) in the CH3 domain.

[0112] Figure 2A Is a series of exemplary SDS-PAGE analyses of the purified masked IL-2 cytokine comprising a native Fc, CH3 mutant or chimeric Fc domain. Each construct was purified by protein A chromatography and size exclusion chromatography (SEC) at different ratios of the first Fc polypeptide and the second Fc polypeptide. Figure 2B Is a series of exemplary EC50 plots of the purified masked IL-2 cytokine comprising a native Fc, CH3 mutant or chimeric Fc domain. The EC50 values were determined in a cell-based reporter gene assay (HEK Blue IL-2 assay) that measures the calculated active cytokine %.

[0113] Figure 3 Shows an exemplary schematic diagram of the IL-2 cytokine targeting (TC) molecule of the present invention, and its purity determined by SEC, non-reducing capillary electrophoresis SDS (CE-SDS) and reducing CE-SDS. All of the TC molecules tested in this experiment comprise a bivalent Fab that binds to PD-1, the first Fc polypeptide having a "mortar mutation" (Y349C; T366S; L368A; and Y407V), and the second Fc polypeptide having a "pestle mutation" (S354C and T366W) fused to the IL-2 cytokine. TC1 is "unmasked" and has no masking portion that binds to IL-2. TC2, TC3 and TC4 comprise CD122 as a masking portion fused to the first Fc polypeptide. TC3 and TC4 have a cleavable linker between the first Fc polypeptide and the masking portion, the cleavable linker comprising the cleavage peptides MPYDLYHP (SEQ ID NO:34) and VPLSLYSG (SEQ ID NO:42), respectively.

[0114] Figure 4AShows exemplary schematic diagrams of different IL-2 cytokine-targeting of the present invention. The cytokine-targeting can be in a bivalent targeting form (Form A), which includes a bivalent Fab that binds to a specific antigen, or in a monovalent targeting form (Forms B1, B2, and C), which includes a monovalent Fab. Figure 4B is Figure 4A A series of exemplary EC50 graphs of the purified IL-2 cytokine-targeting shown in. The EC50 values were determined in a cell-based reporter gene assay (HEKBlue IL-2 assay), which measures the calculated percentage of active cytokine.

[0115] Figure 5A Is a series of exemplary binding graphs showing the binding of the PD-1 multi-functional construct to CD8+ T cells within Raji cells expressing PD-1 and CD3 / CD28-activated PBMCs. Figure 5B Is a series of exemplary graphs showing the surface residence time of the PD-1 multi-functional construct on CD8+ T cells within CD3 / CD28-activated PBMCs at 37 °C and 4 °C. Figure 5C Is an exemplary graph and table showing the proliferation of CD3- and CD28-sensitized human PBMCs in response to stimulation with up to 1000 nM of the indicated construct.

[0116] Figure 6A Is a series of exemplary graphs showing the changes in tumor weight and tumor volume over time after treatment with the IL-2 cytokine-targeting of the present invention. Figure 6B Is a series of exemplary graphs showing the changes in the amount of CD8+ T cells and CD8 proliferation in tumors, spleens, and peripheral blood after treatment with the IL-2 cytokine-targeting of the present invention. Figure 6C Is a series of exemplary graphs showing the fold change in CD8+ T cell expansion in the tumor microenvironment compared to the periphery after treatment with the IL-2 cytokine-targeting. Figure 6D Is a series of exemplary graphs showing the changes in the number of antigen-specific CD8+ T cells after treatment with the IL-2 cytokine-targeting. Figure 6E Is a series of exemplary graphs showing the potential increase in drug accumulation in tumors after treatment with the IL-2 cytokine-targeting. Figure 6F Is a series of exemplary graphs showing the half-life, maximum plasma concentration, AUC, volume of distribution, and clearance rate data of the IL-2 cytokine-targeting after treatment.

[0117] Figure 7A Is showing Figure 3Exemplary tables of binding affinity data and statistics for the targeted IL-2 cytokine shown. Pembrolizumab (anti-PD-1 antibody; "pembro") and anti-human PD-L1-hFc fusion were used as controls. Figure 7B Are a series of exemplary EC50 plots of purified targeted IL-2 cytokines as performed in a cell-based reporter assay (HEK Blue IL-2 assay), which determines the calculated % active cytokine. Each plot shows the binding curves in the absence and presence of various matrix metalloproteinases (MMPs). Recombinant human IL-2 (rhIL-2) and TC0 (unmasked Fc domain fused to IL-2) were used as positive controls. Figure 7C Is an exemplary graph and table showing the proliferation of CD3- and CD28-sensitized human PBMCs in response to stimulation with up to 3000 nM of the indicated constructs. Figure 7D Is an exemplary graph and table showing STAT5 phosphorylation of resting PBMCs using an uncleaved targeted cytokine. Figure 7E Is an exemplary graph and table showing STAT5 phosphorylation of resting PBMCs using a cleaved targeted cytokine.

[0118] Figure 8A Are a series of bar graphs showing the cleavage of the targeted IL-2 cytokine in in vivo tumors. Figure 8B Shows the cleavage of the targeted IL-2 cytokine in tumors compared to plasma, demonstrating in vivo tumor-specific cleavage.

[0119] Figure 9A Is an exemplary graph of tumor volume after treatment with the targeted IL-2 cytokine of the present invention. Pembrolizumab (anti-PD-1 antibody; "pembro") was used as a negative control. The table shows the percentage of tumor growth inhibition for each targeted cytokine. Figure 9B Are a series of exemplary graphs showing the percentage of CD8+ T cells in peripheral blood and spleen cells compared to tumor cells, which shows that the targeted cytokines of the present invention increase the percentage of CD8+ T cells in the tumor microenvironment. Figure 9C Shows the % of CD8+ T cells after treatment with the targeted IL-2 cytokine. Figure 9D Are a series of exemplary graphs showing the % change in body weight and pulmonary edema after treatment with the targeted cytokine, demonstrating the minimal toxicity caused by the targeted IL-2 cytokine of the present invention.

[0120] Figure 10A Is an exemplary table showing the engineered cleavable linker sequences incorporated into the targeted cytokines of the present invention and the yield and purity after purification by protein A chromatography and size exclusion chromatography (SEC). Figure 10BA series of exemplary tables showing the cleavage efficiency of different matrix metalloproteinases (MMPs) on targeted molecules with various engineered cleavable linker sequences. Figure 10C An exemplary graph showing the frequency of protease cleavage of targeted cytokines secreted from human tumor tissue conditioned medium. Figure 10D An exemplary graph showing the average percentage of cleaved targeted cytokines in human plasma from lung cancer patients (n = 8), as determined by Western blotting.

[0121] Figure 11A An exemplary graph and table showing the EC50 values determined in a cell-based reporter assay (HEK Blue IL-2 assay), which measures the calculated active cytokine % of targeted cytokines including the MPY sequence as a cleavable substrate. Figure 11B An exemplary graph and table showing the EC50 values determined in a cell-based reporter assay (HEK Blue IL-2 assay), which measures the calculated active cytokine % of targeted cytokines including the VPL sequence as a cleavable substrate. Figure 11C An exemplary graph and table showing the EC50 values of targeted cytokines including the MPY sequence as a cleavable substrate after complete cleavage with MMP protease. Figure 11D An exemplary graph and table showing the EC50 values of targeted cytokines including the VPL sequence as a cleavable substrate after complete cleavage with MMP protease. Figure 11E An exemplary graph and table showing the EC50 values of targeted cytokines including the MPY sequence as a cleavable substrate after minimal cleavage with MMP protease. Figure 11F A series of exemplary spider graphs showing the cleavage efficiency of different MMP proteases on various targeted cytokines.

[0122] Figure 12 An exemplary graph showing the binding of frequency anti-hIgG+ targeted PD-1 cytokines to CD8+ T cells within CD3-activated PBMCs.

[0123] Figure 13A A series of exemplary graphs showing the average tumor volume and average body weight change curves of unmasked PD-1 targeted TC29 against pembrolizumab. Figure 13B A series of exemplary graphs showing the average tumor volume and average body weight change curves of PD-1 targeted TC3 against pembrolizumab.

[0124] Figure 14A A series of exemplary graphs showing the average tumor volume and average body weight change curves of various targeted cytokines and pembrolizumab. Figure 14Bare a series of exemplary graphs showing the amount of peripheral spleen tumor-infiltrating CD8+ T cells. Figure 14C is an exemplary graph showing the amount of tumor antigen-specific CD8 T cells after administration of the targeted cytokines of the present invention. Figure 14D are a series of exemplary graphs showing the construct concentrations in plasma and tumor lysates.

[0125] Figure 15 is an exemplary graph showing the results of the RGA assay of various targeted masked cytokines before protease cleavage.

[0126] Figure 16A-16D is showing with MMP1( Figure 14A ), MMP7( Figure 16B ), MMP9( Figure 16C ) and MMP10( Figure 16D ) a series of exemplary graphs of the RGA assay results of various targeted masked cytokines cleaved.

[0127] Figure 17 is an exemplary graph demonstrating the average cleavage efficiency of each cleavage site based on kinetic analysis of various constructs.

[0128] Figure 18A-18F shows exemplary ELISA results demonstrating the binding affinity of various constructs for PD-1. Exemplary constructs tested include pembrolizumab scFv in VHVL form( Figure 18A ), pembrolizumab scFv in VLVH form( Figure 18B ), nivolumab (nivo) scFv( Figure 18C ), pembrolizumab Fab( Figure 18D ) or different masking moieties( Figure 18F ). Figure 18E shows the exemplary Kd values for each construct.

[0129] Figure 19A-19F shows exemplary results of PD1 / PD-L1 checkpoint blockade (induction fold) demonstrated by PD-1 RGA assay. Exemplary constructs tested include pembrolizumab scFv in VHVL form( Figure 19A ), pembrolizumab scFv in VLVH form( Figure 19B ), nivolumab (nivo) scFv( Figure 19C ), pembrolizumab Fab( Figure 19D ) or different masking moieties( Figure 19F ). Figure 19E shows the exemplary Kd values for each construct.

[0130] Figure 20A-20EShows exemplary results of the cell surface binding of various constructs to Raji cells expressing PD1. Exemplary constructs tested include pembrolizumab Fab( Figure 20A ), pembrolizumab scFv in VHVL form( Figure 20B ), pembrolizumab scFv in VLVH form( Figure 20C ), or nivolumab (nivo) scFv( Figure 20D ). Figure 20E Shows the exemplary EC50 for each construct.

[0131] Figure 21A-21D Shows exemplary results of peripheral T cell expansion( Figure 21A ), peripheral T cell activation( Figure 21B ), peripheral NK cell expansion( Figure 21C ), and peripheral NK cell activation( Figure 21D ) after administration of various targeted cytokine constructs.

[0132] Figure 22A-22B Shows the exemplary tumor growth curves( Figure 22A ) and overall survival rates( Figure 22B ) of subcutaneous MC38 tumors in human hPD1 transgenic mice after treatment with exemplary masking cytokines.

[0133] Figure 23A-23F Shows the exemplary tumor growth curves( Figure 23A-23C ), overall survival rates( Figure 23D-23F ), and body weight changes( Figure 23A , Figure 23D ) of subcutaneous MC38 tumors in human hPD1 transgenic mice after treatment with exemplary masking cytokines at a dose of 8 mg / kg( Figure 23B , Figure 23E ) or 2 mg / kg( Figure 23C , Figure 23E ) of each construct administered.

[0134] Figure 24A-24E Shows the exemplary tumor growth curves( Figure 24A ), overall survival rates( Figure 24B ), and body weight changes( Figure 24C ) of subcutaneous MC38 tumors in human hPD1 transgenic mice after treatment with exemplary masking cytokines. Figure 24D And Figure 24E Show a schematic diagram of the exemplary constructs tested.

[0135] Figure 25A Shows exemplary results of construct exposure in plasma over time after administration to NHP. Figure 25BShows the changes in lymphocytes after administration of an exemplary construct to NHP.

[0136] Definitions

[0137] To make the present invention more readily understandable, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification. Publications and other reference materials mentioned herein that describe the background of the invention and provide additional details regarding its practice are hereby incorporated by reference.

[0138] It should be understood that the present invention is not limited to specific compositions or biological systems which may of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0139] As used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "IL-2 polypeptide" optionally includes combinations of two or more such polypeptides, and the like.

[0140] As used herein, the term "about" refers to the common error range of the corresponding value that is readily known to those skilled in the art. Reference herein to a "about" value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0141] It should be understood that the aspects and embodiments of the invention described herein include "comprising aspects and embodiments", "consisting of aspects and embodiments", and "consisting essentially of aspects and embodiments".

[0142] As used herein, the term "and / or" refers to any one of the items associated with the term, any combination of the items, or all of the items. 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).

[0143] The term "antibody" includes polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions having multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab')2, and Fv). The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody".

[0144] The term "bispecific antibody" refers to a small antibody fragment having two antigen-binding sites, the antigen-binding sites comprising a heavy-chain variable (VH) domain linked to a light-chain variable (VL) domain in the same polypeptide chain (VH-VL).

[0145] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units together with an additional polypeptide called the J chain, and contain ten antigen-binding sites, while IgA antibodies comprise two to five basic four-chain units which can polymerize to combine with the J chain to form multivalent aggregates. In the case of IgG, the four-chain unit is typically about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while according to the H-chain isotype, the two H chains are linked to each other by one or more disulfide bonds. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) in each of the a and y chains, and four CH domains in the p and s isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain at its other end. The VL aligns with the VH, and the CL aligns 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 together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (Eds.), Appleton & Lange, Norwalk, CT, 1994, pages 71 and chapter 6.

[0146] L chains from any vertebrate species can be assigned to one of two distinct types called κ and λ based on the amino acid sequence of their constant domains. Immunoglobulins can be assigned to different classes or isotypes depending on the amino acid sequence of the constant domain (CH) of their heavy chains. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains named α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function. For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies can exist in multiple polymorphic variants called allotypes (reviewed in Jefferis and Lefranc 2009. Monoclonal Antibodies (mAbs) 1(4):1-7), all of which are applicable to the present invention. Common allotypic variants in the human population are variants designated by the letters a, f, n, z.

[0147] An "isolated" antibody is one that has been identified, separated, and / or recovered (e.g., naturally or recombinantly) from the components of its production environment. In some embodiments, an isolated polypeptide is free of all other components in its production environment. Contaminant components in its production environment, such as those produced by recombinant transfected cells, are materials that would typically interfere with the research, diagnostic, or therapeutic use of the antibody and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide is purified: (1) to greater than 95% by weight of the antibody, as determined, for example, by the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to an extent sufficient to obtain at least 15 N-terminal residues or an internal amino acid sequence by using a spinning cup sequencer, or (3) to homogeneity by SDS-PAGE using Coomassie blue or silver staining, under non-reducing or reducing conditions. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component in the natural environment of the antibody will not be present. However, isolated polypeptides are generally prepared by at least one purification step.

[0148] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., each antibody comprising the population is identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. In some embodiments, the monoclonal antibody has a C-terminal cleavage at the heavy chain and / or light chain. For example, at the C-terminus of the heavy chain and / or light chain, there is a cleavage of 1, 2, 3, 4, or 5 amino acid residues. In some embodiments, the C-terminal cleavage removes the C-terminal lysine from the heavy chain. In some embodiments, the monoclonal antibody has an N-terminal cleavage at the heavy chain and / or light chain. For example, at the N-terminus of the heavy chain and / or light chain, there is a cleavage of 1, 2, 3, 4, or 5 amino acid residues. In some embodiments, a truncated form of the monoclonal antibody can be prepared by recombinant techniques. In some embodiments, the monoclonal antibody has high specificity for a single antigenic site. In some embodiments, the monoclonal antibody has high specificity for multiple antigenic sites (such as bispecific or multispecific antibodies). The modifier "monoclonal" indicates that the properties of the antibody are obtained from a substantially homologous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to the present invention can be prepared by a variety of techniques, including, for example, the hybridoma method, recombinant DNA methods, phage display techniques, and techniques for producing human or human-like antibodies in animals having part or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0149] The terms "full-length antibody", "intact antibody", or "complete antibody" may be used interchangeably to refer to an antibody in a substantially intact form relative to an antibody fragment. Specifically, a complete antibody comprises an antibody having a heavy chain and a light chain that includes 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, an intact antibody may have one or more effector functions.

[0150] "Antibody fragment" includes a portion of a full antibody, such as the antigen-binding region and / or variable region of a full antibody, and / or the constant region of a full antibody. Examples of antibody fragments include the Fc region of an antibody, a portion of the Fc region, or a portion of an antibody that includes the Fc region. Examples of antigen-binding antibody fragments include domain antibodies (dAbs), Fab, Fab', F(ab')2, and Fv fragments; bispecific 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 from antibody fragments, single-chain Fv (scFv), single-domain antibodies (VHH), one or more CDRs, variable heavy chain (VH), variable light chain (VL), Fab-like bispecific antibodies (bsFab), single-domain antibody-linked Fab (s-Fab), and combinations thereof. Single heavy-chain antibodies or single light-chain antibodies can be engineered, or in the case of heavy chains, can be isolated from camels, sharks, libraries, or mice that have been engineered to produce single-chain molecules.

[0151] Papain digestion of an antibody produces two identical antigen-binding fragments (referred to as "Fab" fragments) and a residual "Fc" fragment, a name that reflects the ability to crystallize readily. The Fab fragment consists of the entire L chain together with the variable domain of the H chain (VH) and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still able to cross-link antigens. The Fab' fragment differs from the Fab fragment in having several additional residues at the carboxyl terminus of the CHI domain that include one or more cysteines from the antibody hinge region. Fab'-SH is the name used herein for Fab' in which the cysteine residue of the constant domain carries a free thiol group. The F(ab')2 antibody fragment is initially produced as a pair of Fab' fragments with a hinge cysteine between them. Other chemical conjugations of antibody fragments are also known. The Fc fragment includes the carboxyl-terminal portions of two H chains held together by disulfide bonds. The effector functions of an antibody are determined by sequences and glycans in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain types of cells.

[0152] "Percent amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. The alignment for the purposes of determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. For example, the percent amino acid sequence identity of a given amino acid sequence A with, and / or against, a given amino acid sequence B (which can alternatively be stated as a given amino acid sequence A that includes a certain percent amino acid sequence identity with, and / or against, a given amino acid sequence B) is calculated as follows:

[0153] 100 times the fraction X / Y

[0154] where X is the number of amino acid residues scored as identical matches in the alignment of A and B in the program, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A with B will not be equal to the percent amino acid sequence identity of B with A.

[0155] "Antibody effector function" refers to biological activities attributable to the antibody Fc region (either the 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.

[0156] As used herein, "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., a cytokine) and its binding partner (e.g., a cytokine receptor). In some embodiments, the affinity of a binding protein (e.g., a cytokine) can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including the methods described herein.

[0157] An "isolated" nucleic acid molecule encoding a cytokine polypeptide described herein is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule that is ordinarily associated with it in the environment in which it is produced. In some embodiments, the isolated nucleic acid is free of all components associated with the production environment. 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, the isolated nucleic acid molecules are distinct from the nucleic acids encoding the polypeptides and cytokine polypeptides that are naturally present in cells herein.

[0158] The term "pharmaceutical formulation" refers to a preparation in a form that permits the bioactivity of an active ingredient to be effective and that contains no additional components that are toxic to the subject to which the formulation is to be administered.

[0159] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers are usually aqueous pH buffered solutions. Examples of physiologically acceptable carriers include buffers such as phosphoric, citric, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN TM , polyethylene glycol (PEG), and PLURONICS TM .

[0160] As used herein, the term "treatment" refers to a clinical intervention that is designed to alter the natural course of the individual or cell being treated during a clinical pathological process. Desirable therapeutic effects include reducing the rate of disease progression, ameliorating or alleviating the disease state, and relieving or improving the prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a disorder (e.g., a neoplastic disease) are alleviated or eliminated. For example, an individual is successfully "treated" if the treatment can improve the quality of life of an individual suffering from a disease, reduce the dosage of other medications required to treat the disease, reduce the frequency of disease recurrence, alleviate the severity of the disease, retard the development or progression of the disease, and / or extend the survival of the individual.

[0161] As used herein, "in combination with" or "in conjunction with" refers to the administration of another mode of treatment in addition to one mode of treatment. Thus, "in combination with" or "in conjunction with" refers to the administration of another mode of treatment before, during, or after the administration of one mode of treatment to an individual.

[0162] As used herein, the term "prevention" encompasses providing prophylaxis against the occurrence or recurrence of a disease in an individual. The individual may be predisposed to, susceptible to, or at risk of developing a disease, but has not been diagnosed with the disease. In some embodiments, the targeted cytokines described herein are used to delay the progression of the disease.

[0163] As used herein, an individual "at risk of developing a disease" may or may not have a detectable disease or disease symptoms prior to the treatment methods described herein, and may or may not exhibit detectable disease or disease symptoms. "At risk" means that the individual has one or more risk factors, which are measurable parameters known in the art to be associated with the development of a disease. An individual having one or more of these risk factors has a higher likelihood of developing the disease compared to an individual not having one or more of these risk factors.

[0164] "Effective amount" means an amount that is effective to achieve the desired or indicated effect, including a therapeutic or prophylactic result, at least at the required dosage and for the required period of time.

[0165] An effective amount may 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 particular disease state. In the present context, the therapeutically effective amount may vary depending on factors such as the disease state, the age, sex and weight of the patient, and the ability of the antibody to elicit the desired response in the individual. The therapeutically effective amount may also be an amount where the therapeutically beneficial effect exceeds any toxic or detrimental effect of the targeted cytokine. A "prophylactically effective amount" means an amount that is effective to achieve the desired prophylactic result at the required dosage and for the required period of time. Generally but not necessarily, since the prophylactic dose is administered to a subject prior to or at an earlier stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.

[0166] "Chronic" administration refers to the administration of a drug in a continuous mode as opposed to an acute mode, in order to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is non - continuous without interruption, but is cyclic in nature.

[0167] As used herein, "individual" or "subject" is a mammal. For therapeutic purposes, "mammal" includes humans, domestic and farm animals, and zoo, sports or pet animals such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual or subject is a human.

[0168] Amino acid: As used herein, the term "amino acid" in its broadest sense refers 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 one of the twenty standard l-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or obtained from natural sources. 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, which may alter the circulatory half-life of the peptide without adversely affecting its activity. Amino acids can participate in disulfide bonds. Amino acids can include one or more post-translational modifications, such as being associated with one or more chemical entities (e.g., methyl, acetate group, acetyl, phosphate group, formyl moiety, isoprenoid group, sulfate group, polyethylene glycol moiety, lipid moiety, carbohydrate moiety, biotin moiety, etc.). The terms "amino acid" and "amino acid residue" are used interchangeably and can refer to free amino acids and / or amino acid residues 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.

[0169] 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., rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include but are not limited to mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the animal can be a transgenic animal, a genetically engineered animal, and / or a clone.

[0170] Bioactivity: As used herein, the phrase "bioactivity" refers to the property of any agent that is active in a biological system, particularly in a living organism. For example, an agent that has a biological effect on the organism when administered to the organism is considered to be bioactive.

[0171] Delivery: As used herein, the term "delivery" encompasses both local delivery and systemic delivery. For example, delivery of mRNA encompasses cases where the mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as "local distribution" or "local delivery"), and cases where the mRNA is delivered to a 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").

[0172] Dosing interval: As used herein, in the context of a method for treating a disease, the dosing interval is the frequency at which a therapeutic composition (e.g., an mRNA composition) is administered to a subject in need (a mammal) at an effective dose of mRNA 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 over the dosing interval period. In the present disclosure, the terms "dosing frequency" and "dosing interval" may be used interchangeably.

[0173] Expression: As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into a polypeptide, the assembly of multiple polypeptides into a complete protein (e.g., an enzyme), and / or post-translational modification of the polypeptide or fully assembled protein (e.g., an enzyme). In the present application, the terms "expression" and "production" and grammatical equivalents are used interchangeably.

[0174] Effective dose: As used herein, an effective dose is the dose of mRNA in a pharmaceutical composition that, when administered to a subject in need, effectively produces an expected result in the subject, e.g., alleviates symptoms associated with a disease, according to the methods of the present invention.

[0175] Improve, increase, or decrease: As used herein, the terms "improve", "increase", or "decrease" or grammatical equivalents refer to a value relative to a baseline measurement, such as a measurement in the same individual before initiation of treatment as described herein, or a measurement in a control subject (or control subjects) in the absence of treatment as described herein. A "control subject" is a subject having the same form of disease as the treated subject and being of approximately the same age as the treated subject.

[0176] 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 within a multicellular organism.

[0177] In vivo: As used herein, the term "in vivo" refers to events occurring within a multicellular organism such as a human and non-human animals. In the context of a cell-based system, the term can be used to refer to events occurring within a living cell (as opposed to, for example, an in vitro system).

[0178] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition can be administered, for example for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. Humans include pre- and post-natal forms.

[0179] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to a substance that, within the scope of sound medical judgment, is suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0180] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. described in detail pharmaceutically acceptable salts in Journal of Pharmaceutical Sciences (1977) 66:1-19. The pharmaceutically acceptable salts of the compounds of the present invention include those salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed by the reaction 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 salts formed by using other methods used in the art (such as ion exchange). Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, sulfonates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactates, lactobionates, laurates, dodecyl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, sodium p-toluenesulfonates, undecanoates, valerates, etc. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N+(C1-4 alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. In appropriate cases, 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 the quaternization of amines, which is carried out using a suitable electrophile (e.g., an alkyl halide) to form a quaternized alkylated ammonium salt.

[0181] 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 pre-birth and post-birth forms. In many embodiments, the subject is a human. A subject can be a patient, where a patient is a person presenting to a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient". A subject may have or be susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0182] Substantially: As used herein, the term "substantially" refers to a qualitative condition of exhibiting the total range or near 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, achieve completion and / or continue to completion or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the inherent lack of potential completeness of many biological and chemical phenomena.

[0183] 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-related pathology, symptoms, or characteristics.

[0184] 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 having or susceptible to the disease, disorder, and / or condition. One of ordinary skill in the art will understand that a therapeutically effective amount is typically administered via a dosing regimen that includes at least one unit dose.

[0185] Treat: As used herein, the terms "treat", "treatment", or "treating" refer to any method used to partially or completely alleviate, ameliorate, mitigate, inhibit, prevent, delay the onset, reduce the severity and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment can be administered to a subject who does not exhibit signs of the disease and / or who exhibits only early signs of the disease in order to reduce the risk of developing disease-related pathology.

[0186] Aspects of the invention are described in detail in the following sections. The use of sections is not intended to limit the invention. Each section can apply to any aspect of the invention. In this application, unless otherwise indicated, the use of "or" means "and / or". Detailed Description

[0187] Targeted cytokine

[0188] The present invention particularly provides a targeted cytokine, which comprises a targeting moiety, a cytokine or its variant, a masking moiety, and an Fc domain. The targeted cytokine of the present invention becomes active at the disease site and can specifically target the cells of interest to effectively treat cancer without causing undesired side effects.

[0189] In some embodiments, the cytokine is linked to the Fc domain via a cleavable or non-cleavable linker. In some embodiments, the masking moiety is linked to the Fc domain via a cleavable or non-cleavable linker. In some embodiments, the targeting moiety is linked to the Fc domain with or without a cleavable or non-cleavable linker. Figure 3 and Figure 4A Exemplary targeted cytokines of the present invention are shown in. In some embodiments, the masking moiety is linked to the Fc polypeptide via a cleavable linker. In some embodiments, the masking moiety is linked to the Fc polypeptide via a non-cleavable linker. In some embodiments, the cytokine or its variant is linked to the Fc polypeptide via a cleavable linker. In some embodiments, the cytokine or its variant is linked to the Fc polypeptide via a non-cleavable linker.

[0190] The targeted IL-2 cytokine according to the present disclosure can combine the IL-2 cytokine or its functional fragment as described anywhere herein; the masking moiety as described anywhere herein; the first Fc domain and the second Fc domain as described anywhere herein; the cleavable and non-cleavable linkers as described anywhere herein; and the targeting moiety as described anywhere herein.

[0191] Targeted cytokine form

[0192] In some embodiments, the targeted cytokine is in a bivalent targeting form, which comprises 1) a first chain, which comprises a variable heavy region with "mortar mutations" (Y349C; T366S; L368A; and Y407V) and an IgG1 or IgG4 heavy constant region; 2) a second chain, which comprises a variable heavy region with "pestle mutations" (S354C and T366W) fused to a cytokine or its variant, 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, which comprises a variable light region and an immunoglobulin κ or λ constant region.

[0193] In some embodiments, the targeted cytokine is in a bivalent targeting form, the bivalent targeting form comprising: 1) a first chain comprising a variable heavy region having a "mortar mutation" (Y349C; T366S; L368A; and Y407V) and an IgG1 or IgG4 heavy constant region; 2) a second chain comprising a variable heavy region having a "pestle mutation" (S354C and T366W) and an "RF mutation" (H435R and Y436F) and a heavy constant region from IgG1 or IgG4 fused to the cytokine or a variant thereof; and 3) a third chain comprising a variable light region and an immunoglobulin kappa or lambda constant region.

[0194] In some embodiments, the targeted cytokine is in a bivalent targeting form, the bivalent targeting form comprising: 1) a first chain comprising a variable heavy region having a "mortar mutation" (Y349C; T366S; L368A; and Y407V) and an IgG1 or IgG4 heavy constant region fused to the cytokine or a variant thereof; 2) a second chain comprising a variable heavy region having a "pestle mutation" (S354C and T366W) and a heavy constant region fused to a masking moiety, 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.

[0195] In some embodiments, the targeted cytokine is in a bivalent targeting form, the bivalent targeting form comprising: 1) a first chain comprising a variable heavy region having a "mortar mutation" (Y349C; T366S; L368A; and Y407V) and an IgG1 or IgG4 heavy constant region fused to the cytokine or a variant thereof; 2) a second chain comprising a variable heavy region having a "pestle mutation" (S354C and T366W) and an "RF mutation" (H435R and Y436F) and a heavy constant region from IgG1 or IgG4 fused to a masking moiety; and 3) a third chain comprising a variable light region and an immunoglobulin kappa or lambda constant region.

[0196] In some embodiments, the targeted cytokine is in a bivalent targeting form, the bivalent targeting form comprising: 1) a first chain comprising a variable heavy region having a "mortar mutation" (Y349C; T366S; L368A; and Y407V) and an IgG1 or IgG4 heavy constant region fused to a masking moiety; 2) a second chain comprising a variable heavy region having a "pestle mutation" (S354C and T366W) and a heavy constant region fused to the 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.

[0197] In some embodiments, the targeted cytokine is a bivalent targeting form, and the bivalent targeting form includes: 1) a first chain including a variable heavy region with a "mortar mutation" (Y349C; T366S; L368A; and Y407V) fused to a masking portion and an IgG1 or IgG4 heavy constant region; 2) a second chain including a variable heavy region with a "pestle mutation" (S354C and T366W) and an "RF mutation" (H435R and Y436F) and a heavy constant region from IgG1 or IgG4 fused to a cytokine or its variant; and 3) a third chain including a variable light region and an immunoglobulin kappa or lambda constant region.

[0198] In some embodiments, the targeted cytokine is a monovalent targeting form, and the monovalent targeting form includes: 1) a first chain including a Fab fused to a first Fc polypeptide chain from IgG1 or IgG4 with a "mortar mutation" (Y349C; T366S; L368A; and Y407V); and 2) a second chain including a second Fc polypeptide chain from IgG1 or IgG4 with a "pestle mutation" (S354C and T366W) and an "RF mutation" (H435R and Y436F).

[0199] In some embodiments, the targeted cytokine is a monovalent targeting form, and the monovalent targeting form includes: 1) a first chain including a Fab fused to a first Fc polypeptide chain from IgG1 or IgG4 with a "mortar mutation" (Y349C; T366S; L368A; and Y407V); and 2) a second chain including a second Fc polypeptide chain with a "pestle mutation" (S354C and T366W), wherein the CH1 and CH2 domains are from IgG1 or IgG4 and the CH3 domain is from IgG3.

[0200] In some embodiments, the targeted cytokine is a monovalent targeting form, and the monovalent targeting form includes: 1) a first chain including a first Fc polypeptide chain from IgG1 or IgG4 with a "mortar mutation" (Y349C; T366S; L368A; and Y407V); and 2) a second chain including a Fab fused to a second Fc polypeptide chain from IgG1 or IgG4 with a "pestle 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 portion is fused to the first Fc polypeptide chain. In some embodiments, the masking portion is fused to the second Fc polypeptide chain.

[0201] In some embodiments, the targeted cytokine is a monovalent targeting form, the monovalent targeting form comprising 1) a first chain comprising a first Fc polypeptide chain from IgG1 or IgG4 having a "mortar mutation" (Y349C; T366S; L368A; and Y407V); and 2) a second chain comprising a Fab fused to a second Fc polypeptide chain having a "pestle 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.

[0202] In some embodiments, the targeted cytokine is a monovalent targeting form, the monovalent targeting form comprising 1) a first chain comprising a variable light region and an IgGκ or λ constant region fused to a heavy constant region of IgG1 or IgG4 having a "mortar mutation" (Y349C; T366S; L368A; and Y407V); and 2) a second chain comprising a variable heavy region fused to a heavy constant region of IgG1 or IgG4 having a "pestle mutation" (S354C and T366W) and a "RF mutation" (H435R and Y436F), wherein the C-terminus of the CH3 domain is fused to the cytokine or a variant thereof. 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.

[0203] In some embodiments, the targeted cytokine is a monovalent targeting form, the monovalent targeting form comprising 1) a first chain comprising a variable light region and an IgGκ or λ constant region fused to a heavy constant region of IgG1 or IgG4 having a "mortar mutation" (Y349C; T366S; L368A; and Y407V); and 2) a second chain comprising a variable heavy region fused to a heavy constant region having a "pestle mutation" (S354C and T366W) fused to the cytokine or a variant thereof, wherein the CH1 and CH2 domains are from IgG1 or IgG4 and the CH3 domain is from IgG3.

[0204] In some embodiments, the targeted cytokine is a monovalent targeting form, the monovalent targeting form comprising 1) a first chain comprising a variable heavy region fused to an IgG1 or IgG4 heavy constant region having a "mortar mutation" (Y349C; T366S; L368A; and Y407V); and 2) a second chain comprising a variable light region and an IgGκ or λ constant region fused to an IgG1 or IgG4 heavy constant region having a "pestle mutation" (S354C and T366W) and an "RF mutation" (H435R and Y436F), wherein the C-terminus of the CH3 domain is fused to the cytokine or a variant thereof.

[0205] In some embodiments, the targeted cytokine is a monovalent targeting form, the monovalent targeting form comprising 1) a first chain comprising a variable heavy region fused to an IgG1 or IgG4 heavy constant region having a "mortar mutation" (Y349C; T366S; L638A; and Y407V); and 2) a second chain comprising a variable light region and an IgGκ or λ constant region fused to a heavy constant region having a "pestle mutation" (S354C and T366W) fused to the cytokine or a variant thereof, wherein the CH1 and CH2 domains are from IgG1 or IgG4, and the CH3 domain is from IgG3.

[0206] Targeting moiety

[0207] Provided herein is a targeted cytokine, the targeted cytokine comprising a targeting moiety. In some embodiments, the targeting moiety comprises an antigen-binding moiety that binds to an antigen expressed on the surface of a target cell.

[0208] In some embodiments, the targeting moiety comprises an antigen-binding moiety, wherein the antigen is expressed on an immune cell. In some embodiments, the targeting moiety comprises an antigen-binding moiety, wherein the antigen is selected from PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, OX40, DR5, ICOS, GITR, CD73, CD39, CD25, CD16a, CD8, KLRC1, KLRD1, KLRB1, CD40, CD137, CD28, and CD16b.

[0209] 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, OX40, DR5, ICOS, GITR, NKG2D, KLRC1, KLRD1, KLRB1, NKP44, NKP30, BCMA, human epidermal growth factor receptor 2 (HER2), MICA, DLK1, human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epidermal growth factor receptor (EGFR), glypican-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), nectin-4, Liv-1, glycoprotein NMB (GPNMB), prostate-specific membrane antigen (PSMA), Trop-2, carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), six-transmembrane epithelial antigen of the prostate 1 (STEAP1), NAPI2B, folate receptor alpha (FR-α), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), ectonucleotide pyrophosphatase / phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG), CD19, CD8, CD20, CD22, CD28, CD33, CD39, CD40, CD56, CD66e, CD70, CD73, CD74, CD79b, CD98, CD123, CD137, CD138, CD352, CD47, signal regulatory protein alpha (SIRPα), Claudin 18.2, Claudin 6, 5T4, fibroblast activation protein alpha (FAPα), fibronectin, melanoma-associated chondroitin sulfate proteoglycan (MCSP), epithelial cell adhesion molecule (EPCAM), or a combination thereof.

[0210] In some embodiments, the targeting moiety binds to a tumor-associated antigen. In some embodiments, the targeting moiety is an antibody or antigen-binding fragment that binds to a tumor-associated antigen. In some embodiments, the targeting moiety is a bispecific antibody or antigen-binding fragment that binds to a tumor-associated antigen. In some embodiments, the targeting moiety is an anti-alpha-fetoprotein (AFP) antibody or a fragment thereof. In some embodiments, the targeting moiety is an anti-B2M antibody or a fragment thereof. In some embodiments, the targeting moiety is an anti-beta-human chorionic gonadotropin (β-hCG) antibody or a fragment thereof. In some embodiments, the targeting moiety is an anti-CD117 antibody or a fragment thereof. In some embodiments, the targeting moiety is an anti-CD19 antibody or a fragment thereof. In some embodiments, the targeting moiety is an anti-CD20 antibody or a fragment thereof. In some embodiments, the targeting moiety is an anti-CD22 antibody or a fragment thereof. In some embodiments, the targeting moiety is an anti-CD25 antibody or a fragment thereof. In some embodiments, the targeting moiety is an anti-CD30 antibody or a fragment thereof. In some embodiments, the targeting moiety is an anti-CD33 antibody or a fragment thereof. In some embodiments, the targeting moiety is an anti-CD151 antibody or a fragment thereof. In some embodiments, the targeting moiety is an anti-MUC-1 antibody or a fragment thereof.

[0211] In some embodiments, the targeting moiety binds specifically to PD-1. In some embodiments, the targeting moiety is an anti-PD1 Fab. In some embodiments, the targeting moiety is an anti-PD1 scFv.

[0212] In some embodiments, the targeting moiety is derived from an anti-PD1 antibody. In some embodiments, the targeting moiety is derived from pembrolizumab or nivolumab.

[0213] In some embodiments, the targeting moiety binds to PD-L1.

[0214] In some embodiments, the targeting moiety comprises an agent, peptide, or polypeptide that binds specifically to a target.

[0215] In some embodiments, the targeting moiety includes 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. In some embodiments, the targeting moiety includes a Fab. In some embodiments, the targeting moiety includes a single-chain Fv (scFv). In some embodiments, the targeting moiety includes a single-domain antibody (VHH). In some embodiments, the targeting moiety includes one or more CDRs. In some embodiments, the targeting moiety includes a variable heavy chain (VH). In some embodiments, the targeting moiety includes a variable light chain (VL). In some embodiments, the targeting moiety includes a Fab-like bispecific antibody (bsFab). In some embodiments, the targeting moiety includes a single-domain antibody-linked Fab (s-Fab). In some embodiments, the targeting moiety includes an antibody or a fragment thereof.

[0216] In some embodiments, the targeting moiety includes

[0217] The heavy chain variable region of QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO:1).

[0218] In some embodiments, the targeting moiety includes an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:1. In some embodiments, the targeting moiety includes an amino acid sequence having at least about 85% sequence identity with SEQ ID NO:1. In some embodiments, the targeting moiety includes an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:1. In some embodiments, the targeting moiety includes an amino acid sequence having at least about 92% sequence identity with SEQ ID NO:1. In some embodiments, the targeting moiety includes an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:1. In some embodiments, the targeting moiety includes an amino acid sequence having at least about 96% sequence identity with SEQ ID NO:1. In some embodiments, the targeting moiety includes an amino acid sequence having at least about 97% sequence identity with SEQ ID NO:1. In some embodiments, the targeting moiety includes an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:1. In some embodiments, the targeting moiety includes an amino acid sequence having at least about 99% sequence identity with SEQ ID NO:1.

[0219] In some embodiments, the targeting moiety comprises

[0220] the heavy chain of QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO:2).

[0221] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity with SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity with SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity with SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO:2.

[0222] In some embodiments, the targeting moiety comprises

[0223] The heavy chain of QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:3).

[0224] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity with SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity with SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity with SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO:3.

[0225] In some embodiments, the targeting moiety comprises the heavy chain CDR1 sequence of GYTFTNYY (SEQ ID NO:4). In some embodiments, the targeting moiety comprises the heavy chain CDR2 sequence of INPSNGGT (SEQ ID NO:5). In some embodiments, the targeting moiety comprises the heavy chain CDR3 sequence of ARRDYRFDMGFDY (SEQ ID NO:6). In some embodiments, the targeting moiety comprises HCDR1 of SEQ ID NO:4, HCDR2 of SEQ ID NO:5, and HCDR3 of SEQ ID NO:6.

[0226] In some embodiments, the targeting moiety comprises

[0227] the light chain variable region of EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKTSENLYFQ (SEQ ID NO:7).

[0228] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity with SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity with SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity with SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO:7.

[0229] In some embodiments, the targeting moiety comprises the light chain of EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:8).

[0230] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity with SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity with SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity with SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO:8.

[0231] In some embodiments, the targeting moiety comprises the light chain CDR1 sequence of KGVSTSGYSY (SEQ ID NO:9). In some embodiments, the targeting moiety comprises the light chain CDR2 sequence of LAS (SEQ ID NO:10). In some embodiments, the targeting moiety comprises the light chain CDR3 sequence of QHSRDLPLT (SEQ ID NO:11). In some embodiments, the targeting moiety comprises LCDR1 of SEQ ID NO:9, LCDR2 of SEQ ID NO:10, and LCDR3 of SEQ ID NO:11.

[0232] In some embodiments, the targeting moiety comprises HCDR1 of SEQ ID NO:4, HCDR2 of SEQ ID NO:5, HCDR3 of SEQ ID NO:6, LCDR1 of SEQ ID NO:9, LCDR2 of SEQ ID NO:10, and LCDR3 of SEQ ID NO:11.

[0233] In some embodiments, the targeting moiety comprises the heavy chain variable region or the light chain variable region of nivolumab.

[0234] In some embodiments, the targeting moiety comprises

[0235] the heavy chain variable region of QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS (SEQ ID NO:159).

[0236] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO:159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity with SEQ ID NO:159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity with SEQ ID NO:159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity with SEQ ID NO:159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO:159.

[0237] In some embodiments, the targeting moiety comprises the heavy chain of QVQLVESGGG VVQPGRSLRLDCKASGITFS NSGMHWVRQAPGKGLEWVAV IWYDGSKRYYADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATNDDYWGQGTLVTVSSASTKGPS VFPLAPCSRS TSESTAALGCLVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQ VYTLPPSQEEMTKNQVSLTC LVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYTQKSLSLSLGK (SEQ ID NO:157).

[0238] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO:157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity with SEQ ID NO:157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity with SEQ ID NO:157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity with SEQ ID NO:157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO:157.

[0239] In some embodiments, the targeting moiety comprises the heavy chain CDR1 sequence of GITFSNSG (SEQ ID NO:161). In some embodiments, the targeting moiety comprises the heavy chain CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO:162). In some embodiments, the targeting moiety comprises the heavy chain CDR3 sequence of ATNDDY (SEQ ID NO:163). In some embodiments, the targeting moiety comprises the HCDR1 of GITFSNSG (SEQ ID NO:161), the HCDR2 of VIWYDGSKRYYADSVKG (SEQ ID NO:162), and the HCDR3 of ATNDDY (SEQ ID NO:163).

[0240] In some embodiments, the targeting moiety comprises the heavy chain variable region and the CH1 domain comprising SEQ ID NO:264.

[0241] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC(SEQID NO:264).

[0242] In some embodiments, the targeting moiety comprises

[0243] the light chain variable region of EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK (SEQ ID NO:160).

[0244] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity with SEQ ID NO:160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity with SEQ ID NO:160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity with SEQ ID NO:160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity with SEQ ID NO:160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO:160.

[0245] In some embodiments, the targeting moiety comprises the light chain of EIVLTQSPAT LSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQGTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGE (SEQ ID NO:158).

[0246] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:158. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:158. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:158. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO:158. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:158. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO:158. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:158. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:158. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:158.

[0247] In some embodiments, the targeting moiety comprises the light chain CDR1 sequence of QSVSSY (SEQ ID NO:164). In some embodiments, the targeting moiety comprises the light chain CDR2 sequence of DAS (SEQ ID NO:165). In some embodiments, the targeting moiety comprises the light chain CDR3 sequence of QQSSNWPRT (SEQ ID NO:166). In some embodiments, the targeting moiety comprises LCDR1 of SEQ ID NO:164, LCDR2 of SEQ ID NO:165, and LCDR3 of SEQ ID NO:166.

[0248] In some embodiments, the targeting moiety comprises HCDR1 of SEQ ID NO:161, HCDR2 of SEQ ID NO:162, HCDR3 of SEQ ID NO:163, LCDR1 of SEQ ID NO:164, LCDR2 of SEQ ID NO:165, and LCDR3 of SEQ ID NO:166.

[0249] In some embodiments, the targeting moiety comprises a heavy chain variable region and a CH1 domain. In some embodiments, the heavy chain variable region and the CH1 domain comprise the amino acid sequence (SEQ ID NO:264) QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO:264).

[0250] In some embodiments, the targeting domain is fused to an Fc polypeptide. In some embodiments, the C-terminus of the targeting domain is fused to the N-terminus of the Fc polypeptide. In some embodiments, the heavy chain of the Fab is fused to the Fc polypeptide. In some embodiments, the C-terminus of the heavy chain of the Fab is fused to the N-terminus of the Fc polypeptide. In some embodiments, the Fc polypeptide comprises a cleavage site.

[0251] In some embodiments, the targeting moiety comprises a heavy chain variable region, a light chain variable region, and a CH1 domain. In some embodiments, the CH1 domain is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the CH1 domain is derived from IgG1. In some embodiments, the CH1 domain is derived from IgG4.

[0252] In some embodiments, the targeting moiety comprises a CH1 domain that comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical to SEQ ID NO:156. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO:156).

[0253] Cytokine

[0254] The immune system is good at communication and is designed to respond quickly, specifically, and globally to protect the organism from foreign invaders and diseases. The cytokine superfamily of proteins is part of the signaling network between cells and is essential in the generation and regulation of the immune system. These interacting biological signals have significant capabilities, such as affecting growth and development, hematopoiesis, lymphocyte recruitment, T cell subset differentiation, and inflammation.

[0255] Cytokines can be part of a larger immune program (e.g., T cell subset differentiation). Mature CD4 and CD8 T cells leave the thymus with a naïve phenotype and produce various cytokines. In the periphery, these T cells encounter antigen-presenting cells (APCs) that display major histocompatibility complex (MHC) class I molecules (presenting peptides generated in the cytosol to CD8 T cells) or MHC class II molecules (presenting peptides degraded in intracellular vesicles to CD4 T cells). Upon activation, the characteristic cytokine and chemokine secretion profiles allow the classification of CD4 T helper (Th) cells into two major subsets in mice and humans. 3-7 Th1 cells mainly secrete IL-2, interferon-γ (IFN-γ), and tumor necrosis factor-β (TNF-β), while Th2 cells mainly secrete IL-4, IL-5, IL-6, IL-10, and IL-13. Th1 cells support cell-mediated immunity and thus promote inflammation, cytotoxicity, and delayed-type hypersensitivity (DTH). Th2 cells support humoral immunity and serve to downregulate the inflammatory actions of Th1 cells. This paradigm is a good example of an integrated biological network and is very useful in simplifying the understanding of typical and pathogenic immune responses. For example, the inability to "self-communicate" may lead to the loss of tolerance to self-antigens and prompt a destructive immune response against self-tissues and autoimmune diseases. Autoimmunity (the main focus of this book) is the underlying mechanism for a group of conditions such as type 1 diabetes, multiple sclerosis, and rheumatoid arthritis. Autoimmune diseases can be caused in part by a dysregulation of Th cell subset differentiation mediated by cytokines and chemokines. In addition to the context of presenting antigens and co-stimulatory signals, the main factors affecting Th subset development are the 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.

[0256] The targeting cytokine of the present invention may include any cytokine known in the art or variants thereof. See, for example, Cameron MJ, Kelvin DJ. Cytokines, Chemokines and Their Receptors. In: Madame Curie Bioscience Database. Austin (TX): Landes Bioscience; 2000 - 2013, the content of which is incorporated herein by reference in its entirety. For example, the cytokine incorporated into the targeting cytokine may be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-20, TNF-α, TNF-β, CXCL8 (IL-18), 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 fragments thereof.

[0257] 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-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-18) or a variant thereof. In some embodiments, the cytokine is G-CSF or a variant thereof. In some embodiments, the cytokine is CXCL8 (IL-18) 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.

[0258] Interleukin 2 (IL-2)

[0259] Provided herein is an IL-2 cytokine or a functional fragment thereof, which is used to target a cytokine or a cleavage product thereof. Cytokines play 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 applicable to the present invention can be any IL-2 or a functional fragment thereof. In some embodiments, IL-2 is a naturally occurring IL-2, an IL-2 including one or more substitutions (e.g., an IL-2 mutant protein or an IL-2 variant), or a truncated IL-2. In some embodiments, IL-2 is a polypeptide that retains at least one property of the biological activity of IL-2.

[0260] In some embodiments, IL-2 is a naturally occurring IL-2. In some embodiments, IL-2 includes a C125A substitution of mature IL-2 (SEQ ID NO:13).

[0261] In some embodiments, the amino acid substitution reduces the affinity of the IL-2 polypeptide or its functional fragment for CD25 (IL-2Rα).

[0262] In some embodiments, the IL-2 polypeptide or its functional fragment includes 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 substitution increases 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 includes 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 (CD122) as compared to the amino acid sequence of wild-type IL-2. In some embodiments, the IL-2 polypeptide or its functional fragment includes an amino acid sequence having one or more amino acid substitutions that reduce the affinity of the IL-2 peptide or its functional fragment for IL-2Ra (CD25) and one or more amino acid substitutions that enhance the affinity of the IL-2 polypeptide or its functional fragment for IL-2R (CD122) as compared to the amino acid sequence of wild-type IL-2.

[0263] In some embodiments, IL-2 binds to IL-2Ra with an affinity similar to or higher than wild-type IL-2. In some embodiments, IL-2 preferentially binds to CD25 (e.g., α-biased). In some embodiments, IL-2 has a reduced affinity for CD122 and / or CD132. In some embodiments, IL-2 includes N88D and C125A relative to SEQ ID NO:13. In some embodiments, IL-2 includes SEQ ID NO:167.

[0264] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:167)

[0265] In eukaryotic cells, native IL-2 is synthesized as a 153 amino acid precursor polypeptide having SEQ ID NO:12.

[0266] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:12)

[0267] It is then processed to mature IL-2 by removal of amino acid residues 1-20. This results in the mature form of IL-2 consisting of 133 amino acids (amino acid residues 21-153) having SEQ ID NO:13.

[0268] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:13)

[0269] "Functional fragments" of the IL-2 cytokine include a portion of the full-length cytokine protein that retains or has 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 demonstrated, for example, by the ability of the cytokine to bind to the cognate receptor of the cytokine or its components (e.g., one or more chains of a heterotrimeric receptor complex).

[0270] In some embodiments, the IL-2 cytokine or a functional fragment thereof is any naturally occurring interleukin-2 (IL-2) protein or a modified variant thereof that is capable of binding to the interleukin-2 receptor, particularly the IL-2Rα chain. In the case of IL-2 cytokine binding, the target protein can be IL-2R (including the IL-2Rα, IL-2Rβ and IL-2Rγ chains), the IL-2Rα chain, the IL-2Rβ chain or the IL-2Rα / β dimer complex. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises the amino acid sequence of amino acid residues 21-153 of SEQ ID NO:13. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises mature IL-2, the amino acid sequence of SEQ ID NO:12.

[0271] In some embodiments, the IL-2 cytokine or a functional fragment thereof 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 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:13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 5 amino acid substitutions compared to the amino acid sequence of SEQ ID NO:13.

[0272] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 91% sequence identity with SEQ ID NO:13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 92% sequence identity with SEQ ID NO:13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 93% sequence identity with SEQ ID NO:13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 94% sequence identity with SEQ ID NO:13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 96% sequence identity with SEQ ID NO:13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO:13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO:13.

[0273] 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 a functional fragment thereof for IL-2Rα (CD25) as compared to the amino acid sequence of wild-type IL-2 of SEQ ID NO:13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO:13 such that one or more of the amino acid residues at positions 38, 42, 45, and 62 is alanine (A). In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO:13 such that the amino acid residues at positions 38, 42, 45, and 62 are alanine (A).

[0274] In some embodiments, compared to the amino acid sequence of SEQ ID NO:13, the IL-2 cytokine or a functional fragment thereof comprises the amino acid sequence substitution C125A.

[0275] 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) and amino acid residue 125 is alanine (A). In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid residues R38, F42, Y45, and E62 substituted with alanine in the amino acid sequence of SEQ ID NO:13. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid residues R38, F42, Y45, and E62 substituted with alanine (A) and amino acid residue C125 substituted with alanine (A) in the amino acid sequence of SEQ ID NO:13.

[0276] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:14.

[0277] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:14)

[0278] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 80% identity to SEQ ID NO:14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 85% identity to SEQ ID NO:14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 90% identity to SEQ ID NO:14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 92% identity to SEQ ID NO:14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 95% identity to SEQ ID NO:14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 97% identity to SEQ ID NO:14. In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises an amino acid sequence having at least about 99% identity to SEQ ID NO:14.

[0279] In some embodiments, for the purpose of removing O-glycosylation sites, compared to the amino acid sequence of mature IL-2 of SEQ ID NO:12, the IL-2 cytokine or a functional fragment thereof removes one or more amino acid residues, such as residues 1-3. In some embodiments, for the purpose of removing O-glycosylation sites, compared to the amino acid sequence of mature IL-2 of SEQ ID NO:12, the IL-2 cytokine or a functional fragment thereof has one or more substituted amino acid residues. In some embodiments, for the purpose of removing O-glycosylation sites, compared to the amino acid sequence of mature IL-2 of SEQ ID NO:12, the IL-2 cytokine or a functional fragment thereof has one or more amino acid residues inserted in a region such as residues 1-3. In some embodiments, the IL-2 cytokine or a functional fragment thereof does not have an O-glycosylation site within residues 1-3.

[0280] Interleukin 15 (IL-15)

[0281] The present invention provides an IL-15 cytokine or a functional fragment thereof, which is used to target a cytokine or a cleavage product thereof. Cytokines play a role in cell signaling, especially 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.

[0282] In eukaryotic cells, IL-15 is synthesized as a precursor polypeptide of 162 amino acids (SEQ ID NO:92), and then it is processed into mature IL-15 by removing amino acid residues 1-48. This results in the mature form of IL-15 consisting of 114 amino acids (amino acid residues 49-162), which is secreted in a mature active form (see SEQ ID NO:93).

[0283] IL-15 precursor polypeptide (SEQ ID NO:92):

[0284] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0285] IL-15 mature polypeptide (SEQ ID NO:93):

[0286] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0287] 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 native 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., the 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., the mature form of IL-15 consisting of amino acid residues 49-162). Thus, the term encompasses proteins encoded by the amino acid sequence of SEQ ID NO:93 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 truncated, deleted, forms in which IL-15 is linked to another molecule, and variants caused by at least one amino acid change in the amino acid sequence (e.g., by substitution, addition, or deletion). In some aspects, compared to a naturally occurring IL-15 polypeptide (such as an IL-15 polypeptide encoded by the amino acid sequence of SEQ ID NO:92 or 93), the variant or homolog 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-consecutive amino acid portion). Thus, the term "IL-15" or "IL-15 polypeptide" includes an IL-15 protein comprising the amino acid sequence of SEQ ID NO:92 or 93, including variants thereof, such as variants produced by one or more amino acid substitutions in the amino acid sequence of SEQ ID NO:92 or 93).

[0288] A "functional fragment" of the IL-15 cytokine includes a portion of the full-length cytokine protein that retains or has 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 demonstrated, for example, by the ability of the cytokine to bind to the cytokine's cognate receptor or a component thereof (e.g., one or more chains of a heterotrimeric receptor complex).

[0289] In some embodiments, the IL-15 cytokine or a functional fragment thereof is any native 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.

[0290] In some embodiments, the IL-15 cytokine or a fragment thereof includes SEQ ID NO:93 or a functional fragment thereof.

[0291] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:93.

[0292] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO:93. 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 a functional fragment thereof comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acid substitutions as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 5 amino acid substitutions as compared to the amino acid sequence of SEQ ID NO:93. 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:93.

[0293] 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:93. 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:93. 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:93. 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:93. 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:93. 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:93. 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:93. 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:93. 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:93. 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:93.

[0294] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO:93.

[0295] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22, E46, and E53 as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22, E46, E53, N71, N79, and N112 as compared to the amino acid sequence of SEQ ID NO:93.

[0296] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position D22 as compared to the amino acid sequence of SEQ ID NO:93.

[0297] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position E46 as compared to the amino acid sequence of SEQ ID NO:93.

[0298] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position E53 as compared to the amino acid sequence of SEQ ID NO:93.

[0299] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N71 as compared to the amino acid sequence of SEQ ID NO:93.

[0300] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N79 as compared to the amino acid sequence of SEQ ID NO:93.

[0301] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N112 as compared to the amino acid sequence of SEQ ID NO:93.

[0302] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions E46 and E53 as compared to the amino acid sequence of SEQ ID NO:93.

[0303] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71 and N79 as compared to the amino acid sequence of SEQ ID NO:93.

[0304] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71 and N112 as compared to the amino acid sequence of SEQ ID NO:93.

[0305] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N79 and N112 as compared to the amino acid sequence of SEQ ID NO:93.

[0306] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71, N79 and N112 as compared to the amino acid sequence of SEQ ID NO:93.

[0307] In some embodiments, the amino acid substitution at position D22 is D22A.

[0308] In some embodiments, the amino acid substitution at position E46 is E46A.

[0309] In some embodiments, the amino acid substitution at position E46 is E46R.

[0310] In some embodiments, the amino acid substitution at position E46 is E46S.

[0311] In some embodiments, the amino acid substitution at position E53 is E53A.

[0312] In some embodiments, the amino acid substitution at position E53 is E53R.

[0313] In some embodiments, the amino acid substitution at position E53 is E53S.

[0314] In some embodiments, the amino acid substitution at position N71 is N71Q.

[0315] In some embodiments, the amino acid substitution at position N79 is N79Q.

[0316] In some embodiments, the amino acid substitution at position N112 is N112Q.

[0317] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution D22A as compared to the amino acid sequence of SEQ ID NO:93.

[0318] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:94.

[0319] NWVNVISDLKKIEDLIQSMHIAATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:94)

[0320] 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:94.

[0321] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution E46A as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:95.

[0322] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLALQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:95)

[0323] 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:95.

[0324] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46A and E53A as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:96. 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:96.

[0325] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLALQVISLASGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:96)

[0326] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46R and E53R as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:97. 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:6.

[0327] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLRLQVISLRSGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:97)

[0328] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46S and E53S as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:98. 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:98.

[0329] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLSLQVISLSSGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:98)

[0330] 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:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:99. 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:99.

[0331] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLASGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:99)

[0332] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequences of SEQ ID NO:100 and SEQ ID NO:101. 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:100 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:101.

[0333] NWVNVISDLKKIEDLIQS(SEQ ID NO:100)

[0334] KVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:101)

[0335] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution N71Q as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:102. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:102.

[0336] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAQNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:102)

[0337] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution N79Q as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:103. 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:103.

[0338] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:103)

[0339] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having an amino acid substitution N112Q as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:104. 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:104.

[0340] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS(SEQ ID NO:104)

[0341] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions N71Q and N79Q as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:105. 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:105.

[0342] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAQNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:105)

[0343] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions N71Q and N112Q as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:106. 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:106. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAQNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS(SEQ ID NO:106)

[0344] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions N79Q and N112Q as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:107. 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:107. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS(SEQ ID NO:107)

[0345] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises an amino acid sequence having amino acid substitutions N71Q, N79Q, and N112Q as compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:108. 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:108. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAQNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS(SEQ ID NO:108)

[0346] In some embodiments, additional mutations can 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.

[0347] In some embodiments, additional mutations can be included in any of the above sequences at position S73. In some embodiments, the mutation is S73A, S73W, S73V, or S73M.

[0348] In some embodiments, additional mutations can be included in any of the above sequences at one or more amino acid positions 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 can be included in any of the above sequences.

[0349] In some embodiments, additional mutations can be included in any of the above sequences at one or more amino acid positions 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 can be included in any of the above sequences.

[0350] In some embodiments, for the purpose of removing O-glycosylation sites, compared to the amino acid sequence of mature IL-15 of SEQ ID NO:93, the IL-15 cytokine or its functional fragment removes one or more amino acid residues, such as residues 1-3s. In some embodiments, for the purpose of removing O-glycosylation sites, compared to the amino acid sequence of mature IL-15 of SEQ ID NO:93, the IL-15 cytokine or its functional fragment has one or more substituted amino acid residues. In some embodiments, for the purpose of removing O-glycosylation sites, compared to the amino acid sequence of mature IL-15 of SEQ ID NO:93, the IL-15 cytokine or its functional fragment has one or more amino acid residues inserted in a region such as residues 1-3. In some embodiments, the IL-15 cytokine or its functional fragment does not have an O-glycosylation site within residues 1-3.

[0351] Interleukin 12 (IL-12)

[0352] Provided herein is an IL-12 cytokine or its functional fragment, which is used to target a cytokine or its cleavage product. 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.

[0353] Endogenous IL-12 exists as two different molecules, IL-12p40 and IL-12p35, which dimerize in cells during biosynthesis.

[0354] The full sequences of IL-12p40 and IL-12p35 are in bold (the prepeptides that are cleaved during biosynthesis are shown):

[0355] IL-12p40 subunit:

[0356] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS(SEQ ID NO:109)

[0357] IL-12 p35 subunit:

[0358] MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ ID NO:110)

[0359] The mature form is as follows:

[0360] IL-12 p40 subunit:

[0361] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS(SEQ ID NO:111)

[0362] IL-12 p35 subunit:

[0363] RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ ID NO:112)

[0364] They are expressed as two chains that covalently dimerize via a disulfide bond between the two subunits during biosynthesis: Cysteine C199 of the p40 subunit associates with Cysteine C96 of the p35 subunit.

[0365] A "functional fragment" of the IL-12 cytokine includes a portion of the full-length cytokine protein that retains or has 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 demonstrated, for example, by the ability of the cytokine to bind to the cytokine's cognate receptor or its components.

[0366] In some embodiments, the IL-12 cytokine or its functional fragment is any naturally occurring interleukin-2 (IL-12) protein or a modified variant thereof that is capable of binding to the interleukin-12 receptor.

[0367] 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.

[0368] The IL-12p40 polypeptide or a functional fragment thereof can be linked to a first half-life extending domain such that the first polypeptide chain comprises:

[0369] N'HL1-L1-MM C'

[0370] And the second polypeptide chain comprises:

[0371] N'HL2-L2-[IL-12p40-linker-IL-12p35]C'

[0372] Where '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.

[0373] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO:111. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO:111. 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 a functional fragment thereof comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acid substitutions as compared to the amino acid sequence of SEQ ID NO:111. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 5 amino acid substitutions as compared to the amino acid sequence of SEQ ID NO:111.

[0374] The IL-12p40 polypeptide comprises 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 a high local concentration of cytokines. Second, GAGs can regulate the biological activity of growth factors and cytokines through multiple mechanisms, which include dimerization and prevention of proteolytic degradation.

[0375] The GAG-binding domain in the mature form of the IL-12p40 subunit is shown in bold as follows: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO:113)

[0376] Modifications to the GAG-binding domain KSKREKKDRV (SEQ ID NO:114) have been shown herein to increase the PK characteristics of constructs of IL-12 cytokines comprising the IL-12 cytokine having a mutated GAG-binding domain without any decrease in cytokine activity. Accordingly, 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.

[0377] 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:115.

[0378] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTERVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS(SEQ ID NO:115)

[0379] In some embodiments, the GAG-binding domain comprises the amino acid sequence KDNTEGRV. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO:116.

[0380] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS(SEQ ID NO:116)

[0381] In some embodiments, the GAG-binding domain consists of the amino acid sequence KDNTERV. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO:115. In some embodiments, the GAG-binding domain consists of the amino acid sequence KDNTEGRV. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO:116.

[0382] In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having one or more cysteine substitutions as compared to the amino acid sequence of SEQ ID NO:111. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having an amino acid substitution at position C252 as compared to the amino acid sequence of SEQ ID NO:111. 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:117. 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:117. In some embodiments, the IL-12p40 polypeptide consists of the amino acid sequence of SEQ ID NO:117.

[0383] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEW ASVPCS(SEQ ID NO:117)

[0384] 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 in the GAG binding domain as compared to the amino acid sequence of SEQ ID NO:111. In some embodiments, as compared to the amino acid sequence of SEQ ID NO:111, the IL-12p40 polypeptide comprises an amino acid substitution at position C252S, and the GAG binding domain comprises the amino acid sequence KDNTERV. In some embodiments, as compared to the amino acid sequence of SEQ ID NO:111, the IL-12p40 polypeptide comprises an amino acid substitution at position C252S, and the GAG binding domain comprises the amino acid sequence KDNTEGRV. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO:118. 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:118. In some embodiments, the IL-12p40 polypeptide consists of the amino acid sequence of SEQ ID NO:118.

[0385] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS(SEQ ID NO:118)

[0386] In some embodiments, the IL-12p35 polypeptide comprises SEQ ID NO:112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO:112. 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 a functional fragment thereof comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid substitutions as compared to the amino acid sequence of SEQ ID NO:112. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 5 amino acid substitutions as compared to the amino acid sequence of SEQ ID NO:112.

[0387] In some embodiments, the length of the IL-12p40-IL-12p35 linker ranges from 5 amino acids to 20 amino acids.

[0388] In some embodiments, the IL-12p40-IL-12p35 linker is rich in amino acid residues G and S.

[0389] In some embodiments, the IL-12p40-IL-12p35 linker contains only amino acid residue types selected from the group consisting of G and S.

[0390] In some embodiments, the IL-12p40-IL-12p35 linker contains [(G)nS], where n = 4 or 5.

[0391] In some embodiments, the IL-12p40-IL-12p35 linker contains a (GGGGS) repeat sequence.

[0392] In some embodiments, the IL-12p40-IL-12p35 linker comprises SEQ ID NO:119. (GGGGSGGGGSGGGGS)

[0393] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO:120. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having at least one amino acid modification as compared to the amino acid sequences of SEQ ID NOs:111 and 112. 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 a functional fragment thereof comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acid substitutions as compared to the amino acid sequences of SEQ ID NOs:111 and 112. In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises an amino acid sequence having at least 5 amino acid substitutions as compared to the amino acid sequences of SEQ ID NOs:111 and 112.

[0394] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMA(SEQ ID NO:120)

[0395] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO:120. 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:120.

[0396] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 121. 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: 121.

[0397] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTERVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ IDNO:121)

[0398] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 122. 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: 122.

[0399] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ ID NO:122)

[0400] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 123. 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: 123.

[0401] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ IDNO:123)

[0402] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 124. 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: 124.

[0403] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALC(SEQ ID NO:124)

[0404] Masking moiety

[0405] The present disclosure provides a masking moiety for targeting a cytokine. It should be understood that the masking moiety is cleaved from the targeted cytokine to form its cleavage product. The masking moiety binds to 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.

[0406] In some embodiments, the masking moiety comprises an agent, peptide, or polypeptide that binds to the cytokine.

[0407] In some embodiments, the masking moiety comprises a Fab, a single-chain Fv (scFv), a single-domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibody (bsFab), a single-domain antibody-linked Fab (s-Fab), an antibody, or a combination thereof. In some embodiments, the masking moiety comprises a Fab that binds to a cytokine. In some embodiments, the masking moiety comprises a single-chain Fv (scFv) that binds to a cytokine. In some embodiments, the masking moiety comprises a single-domain antibody (VHH) that binds to a cytokine. In some embodiments, the masking moiety comprises one or more CDRs that bind to a cytokine. In some embodiments, the masking moiety comprises a variable heavy chain (VH) that binds to a cytokine. In some embodiments, the masking moiety comprises a variable light chain (VL) that binds to a cytokine. In some embodiments, the masking moiety comprises a Fab-like bispecific antibody (bsFab) that binds to a cytokine. In some embodiments, the masking moiety comprises a single-domain antibody-linked Fab (s-Fab) that binds to a cytokine. In some embodiments, the masking moiety comprises an antibody or a fragment thereof that binds to a cytokine. In some embodiments, the masking moiety comprises an antibody or a binding fragment of an antibody against a cytokine.

[0408] 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.

[0409] 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 against 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.

[0410] 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 against 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.

[0411] 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 against IL-18. In some embodiments, the cytokine is IL-18; and wherein the masking moiety is IL-18Rα, IL-18Rβ, or a fragment thereof.

[0412] In some embodiments, the cytokine is IL-4; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-4. In some embodiments, the cytokine is IL-4; and wherein the masking moiety is CD124, CD213a13, CD132, or a fragment thereof.

[0413] In some embodiments, the cytokine is IL-7; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-7. In some embodiments, the cytokine is IL-7; and wherein the masking moiety is CD127, CD132, or a fragment thereof.

[0414] In some embodiments, the cytokine is IL-9; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-9. In some embodiments, the cytokine is IL-9, and wherein the masking moiety is IL-9R, CD132, or a fragment thereof.

[0415] In some embodiments, the cytokine is IL-13; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-13. In some embodiments, the cytokine is IL-13; and wherein the masking moiety is CD213a1, CD213a2, or a fragment thereof.

[0416] In some embodiments, the cytokine is IL-15; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-15. In some embodiments, the cytokine is IL-15; and wherein the masking moiety is IL-15Ra, CD122, CD132, or a fragment thereof.

[0417] In some embodiments, the cytokine is IL-3; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-3. In some embodiments, the cytokine is IL-3; and wherein the masking moiety is CD123, CDw131, or a fragment thereof.

[0418] In some embodiments, the cytokine is IL-5; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-5. In some embodiments, the cytokine is IL-5; and wherein the masking moiety is CDw125, CD131, or a fragment thereof.

[0419] In some embodiments, the cytokine is GM-CSF; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against GM-CSF. In some embodiments, the cytokine is GM-CSF; and wherein the masking moiety is CD116, CDw131, or a fragment thereof.

[0420] In some embodiments, the cytokine is IL-6; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-6. In some embodiments, the cytokine is IL-6; and wherein the masking moiety is CD126, CD130, or a fragment thereof.

[0421] In some embodiments, the cytokine is IL-11; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-11. In some embodiments, the cytokine is IL-11; and wherein the masking moiety is IL-11Ra, CD130, or a fragment thereof.

[0422] In some embodiments, the cytokine is G-CSF; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against G-CSF. In some embodiments, the cytokine is G-CSF; and wherein the masking moiety is CD114 or a fragment thereof.

[0423] In some embodiments, the cytokine is IL-12; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-12. In some embodiments, the cytokine is IL-12; and wherein the masking moiety is CD212 or a fragment thereof.

[0424] In some embodiments, the cytokine is LIF; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against LIF. In some embodiments, the cytokine is LIF; and wherein the masking moiety is LIFR, CD130, or a fragment thereof.

[0425] In some embodiments, the cytokine is OSM; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against OSM. In some embodiments, the cytokine is OSM; and wherein the masking moiety is OSMR, CD130, or a fragment thereof.

[0426] In some embodiments, the cytokine is IL-10; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-10. In some embodiments, the cytokine is IL-10; and wherein the masking moiety is CDw210 or a fragment thereof.

[0427] In some embodiments, the cytokine is IL-20; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-20. In some embodiments, the cytokine is IL-20; and wherein the masking moiety is IL-20Rα, IL-20Rβ, or a fragment thereof.

[0428] In some embodiments, the cytokine is IL-14; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-14. In some embodiments, the cytokine is IL-14; and wherein the masking moiety is IL-14R or a fragment thereof.

[0429] In some embodiments, the cytokine is IL-16; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-16. In some embodiments, the cytokine is IL-16; and wherein the masking moiety is CD4 or a fragment thereof.

[0430] In some embodiments, the cytokine is IL-17; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-17. In some embodiments, the cytokine is IL-17; and wherein the masking moiety is CDw217 or a fragment thereof.

[0431] In some embodiments, the cytokine is IFN-α; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IFN-α. In some embodiments, the cytokine is IFN-α; and wherein the masking moiety is CD118 or a fragment thereof.

[0432] In some embodiments, the cytokine is IFN-β; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IFN-β. In some embodiments, the cytokine is IFN-β; and wherein the masking moiety is CD118 or a fragment thereof.

[0433] In some embodiments, the cytokine is IFN-γ; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IFN-γ. In some embodiments, the cytokine is IFN-γ; and wherein the masking moiety is CDw119 or a fragment thereof.

[0434] In some embodiments, the cytokine is CD154; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against CD154. In some embodiments, the cytokine is CD154; and wherein the masking moiety is CD40 or a fragment thereof.

[0435] In some embodiments, the cytokine is LT-β; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against LT-β. In some embodiments, the cytokine is LT-β; and wherein the masking moiety is LT-βR or a fragment thereof.

[0436] In some embodiments, the cytokine is TNF-α; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TNF-α. In some embodiments, the cytokine is TNF-α; and wherein the masking moiety is CD120a, CD120b, or a fragment thereof.

[0437] In some embodiments, the cytokine is TNF-β; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TNF-β. In some embodiments, the cytokine is TNF-β; and wherein the masking moiety is CD120a, CD120b, or a fragment thereof.

[0438] In some embodiments, the cytokine is 4-1BBL; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against 4-1BBL. In some embodiments, the cytokine is 4-1BBL; and wherein the masking moiety is CDw137, 4-1BB, or a fragment thereof.

[0439] In some embodiments, the cytokine is APRIL; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against APRIL. In some embodiments, the cytokine is APRIL; and wherein the masking moiety is BCMA, TACI, or a fragment thereof.

[0440] In some embodiments, the cytokine is CD70; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against CD70. In some embodiments, the cytokine is CD70; and wherein the masking moiety is CD27 or a fragment thereof.

[0441] In some embodiments, the cytokine is CD153; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against CD153. In some embodiments, the cytokine is CD153; and wherein the masking moiety is CD30 or a fragment thereof.

[0442] In some embodiments, the cytokine is CD178; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against CD178. In some embodiments, the cytokine is CD178; and wherein the masking moiety is CD95 or a fragment thereof.

[0443] In some embodiments, the cytokine is GITRL; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against GITRL. In some embodiments, the cytokine is GITRL; and wherein the masking moiety is GITR or a fragment thereof.

[0444] In some embodiments, the cytokine is LIGHT; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against LIGHT. In some embodiments, the cytokine is LIGHT; and wherein the masking moiety is LTβR, HVEM, or a fragment thereof.

[0445] In some embodiments, the cytokine is OX40; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against OX40. In some embodiments, the cytokine is OX40; and wherein the masking moiety is OX40 or a fragment thereof.

[0446] In some embodiments, the cytokine is TALL-1; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TALL-1. In some embodiments, the cytokine is TALL-1; and wherein the masking moiety is BCMA, TACI, or a fragment thereof.

[0447] In some embodiments, the cytokine is TRAIL; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TRAIL. In some embodiments, the cytokine is TRAIL; and wherein the masking moiety is TRAILR1-4 or a fragment thereof.

[0448] In some embodiments, the cytokine is TWEAK; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TWEAK. In some embodiments, the cytokine is TWEAK; and wherein the masking moiety is Apo3 or a fragment thereof.

[0449] In some embodiments, the cytokine is TRANCE; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TRANCE. In some embodiments, the cytokine is TRANCE; and wherein the masking moiety is RANK, OPG, or a fragment thereof.

[0450] In some embodiments, the cytokine is TGF-β1; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TGF-β1. In some embodiments, the cytokine is TGF-β1; and wherein the masking moiety is TGF-βR1 or a fragment thereof.

[0451] In some embodiments, the cytokine is TGF-β2; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TGF-β2. In some embodiments, the cytokine is TGF-β2; and wherein the masking moiety is TGF-βR2 or a fragment thereof.

[0452] In some embodiments, the cytokine is TGF-β3; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against TGF-β3. In some embodiments, the cytokine is TGF-β3; and wherein the masking moiety is TGF-βR3 or a fragment thereof.

[0453] In some embodiments, the cytokine is Epo; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against Epo. In some embodiments, the cytokine is Epo; and wherein the masking moiety is EpoR or a fragment thereof.

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

[0455] In some embodiments, the cytokine is Flt-3L; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against Flt-3L. In some embodiments, the cytokine is Flt-3L; and wherein the masking moiety is Flt-3 or a fragment thereof.

[0456] In some embodiments, the cytokine is SCF; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against SCF. In some embodiments, the cytokine is SCF; and wherein the masking moiety is CD117 or a fragment thereof.

[0457] In some embodiments, the cytokine is M-CSF; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against M-CSF. In some embodiments, the cytokine is M-CSF; and wherein the masking moiety is CD115 or a fragment thereof.

[0458] In some embodiments, the cytokine is MSP; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against MSP. In some embodiments, the cytokine is MSP; and wherein the masking moiety is CDw136 or a fragment thereof.

[0459] In some embodiments, the cytokine is an IL-15 agonist polypeptide; and wherein the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-15. In some embodiments, the cytokine moiety comprises an IL-15 agonist polypeptide, wherein the chimeric molecule further comprises the sushi domain of IL-15 receptor a (IL-15Ra sushi domain).

[0460] 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 analogue 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, single-chain Fv (scFv), or single-domain antibody against IL-21.

[0461] 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 analogue 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, single-chain Fv (scFv), or single-domain antibody against IL-21.

[0462] CD122 (masking moiety of IL-2 and IL-15)

[0463] A masking moiety masks a cytokine or a functional fragment thereof in a target cytokine, thereby reducing or preventing the binding of the cytokine or its functional fragment to its cognate receptor. In some embodiments, the masking moiety reduces or prevents the binding of the IL-2 cytokine or its functional fragment to IL-2Rα (CD25). In some embodiments, the masking moiety as provided herein refers to a moiety capable of binding to the IL-2 cytokine or its functional fragment or otherwise exhibiting an affinity for the IL-2 cytokine or its functional fragment (such as an anti-IL-2 antibody or an IL-2 cognate receptor protein). In some embodiments, the masking moiety reduces or prevents the binding of the IL-15 cytokine or its functional fragment to IL-15Rα. In some embodiments, the masking moiety as provided herein refers to a moiety capable of binding to the IL-15 cytokine or its functional fragment or otherwise exhibiting an affinity for the IL-15 cytokine or its functional fragment (such as an anti-IL-15 antibody or an IL-15 cognate receptor protein). Methods for determining the degree of binding of a protein (e.g., a cytokine) to a cognate protein (e.g., a cytokine receptor) are well known in the art.

[0464] In some embodiments, the masking moiety comprises an IL-2 cytokine receptor or a subunit or functional fragment thereof.

[0465] In some embodiments, the masking moiety comprises CD122 (also known as IL-2Rβ) or a fragment, portion or variant thereof that retains or otherwise displays an affinity for IL-2 and IL-15.

[0466] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:15.

[0467] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:15)

[0468] In some embodiments, the masking portion comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:15. In some embodiments, the masking portion comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:15, wherein the amino acid sequence has one to four amino acid substitutions. In some embodiments, the masking portion comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:15, wherein the amino acid sequence has one or two amino acid substitutions.

[0469] In some embodiments, compared to CD122 of SEQ ID NO:15, CD122 or a fragment, portion or variant thereof has a mutation at amino acid position C122.

[0470] In some embodiments, compared to CD122 of SEQ ID NO:15, CD122 or a fragment, portion or variant thereof has a mutation C122S at amino acid position 122.

[0471] In some embodiments, the masking portion comprises an amino acid sequence of SEQ ID NO:15 having a C122 mutation.

[0472] In some embodiments, the masking portion comprises an amino acid sequence of SEQ ID NO:15 having a C122S mutation.

[0473] In some embodiments, compared to CD122 of SEQ ID NO:15, CD122 or a fragment, portion or variant thereof has a mutation at amino acid position C168.

[0474] In some embodiments, compared to CD122 of SEQ ID NO:15, CD122 or a fragment, portion or variant thereof has a mutation C168S at amino acid position 168.

[0475] In some embodiments, the masking portion comprises an amino acid sequence of SEQ ID NO:15 having a C168 mutation.

[0476] In some embodiments, the masking portion comprises an amino acid sequence of SEQ ID NO:15 having a C168S mutation.

[0477] In some embodiments, compared to CD122 of SEQ ID NO:15, CD122 or a fragment, portion or variant thereof has mutations at amino acid positions C122 and C168.

[0478] In some embodiments, compared to CD122 of SEQ ID NO:15, CD122 or a fragment, portion or variant thereof has mutations C122S and C168S.

[0479] In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO:16.

[0480] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRSNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWISLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:16)

[0481] In some embodiments, the masking portion comprises an amino acid sequence having at least about 80% identity to SEQ ID NO:16. In some embodiments, the masking portion comprises an amino acid sequence having at least about 85% identity to SEQ ID NO:16. In some embodiments, the masking portion comprises an amino acid sequence having at least about 90% identity to SEQ ID NO:16. In some embodiments, the masking portion comprises an amino acid sequence having at least about 91% identity to SEQ ID NO:16. In some embodiments, the masking portion comprises an amino acid sequence having at least about 92% identity to SEQ ID NO:16. In some embodiments, the masking portion comprises an amino acid sequence having at least about 93% identity to SEQ ID NO:16. In some embodiments, the masking portion comprises an amino acid sequence having at least about 94% identity to SEQ ID NO:16. In some embodiments, the masking portion comprises an amino acid sequence having at least about 95% identity to SEQ ID NO:16. In some embodiments, the masking portion comprises an amino acid sequence having at least about 96% identity to SEQ ID NO:16. In some embodiments, the masking portion comprises an amino acid sequence having at least about 97% identity to SEQ ID NO:16. In some embodiments, the masking portion comprises an amino acid sequence having at least about 98% identity to SEQ ID NO:16. In some embodiments, the masking portion comprises an amino acid sequence having at least about 99% identity to SEQ ID NO:16.

[0482] In some embodiments, the masking moiety has a mutation at amino acid position F8 compared to CD122 of SEQ ID NO:15. In some embodiments, the masking moiety has the mutation F8C compared to CD122 of SEQ ID NO:15.

[0483] In some embodiments, the masking moiety has a mutation at amino acid position A94 compared to CD122 of SEQ ID NO:15. In some embodiments, the masking moiety has the mutation A94C compared to CD122 of SEQ ID NO:15.

[0484] In some embodiments, the masking moiety has a mutation at amino acid position L106 compared to CD122 of SEQ ID NO:15. In some embodiments, the masking moiety has the mutation L106C compared to CD122 of SEQ ID NO:15.

[0485] In some embodiments, the masking moiety has a mutation at amino acid position V117 compared to CD122 of SEQ ID NO:15. In some embodiments, the masking moiety has the mutation V117C compared to CD122 of SEQ ID NO:15.

[0486] In some embodiments, the masking moiety has a mutation at amino acid position C122 compared to CD122 of SEQ ID NO:15. In some embodiments, the masking moiety has the mutation C122S compared to CD122 of SEQ ID NO:15. In some embodiments, the masking moiety has the mutation C122V compared to CD122 of SEQ ID NO:15. In some embodiments, the masking moiety has the mutation C122A compared to CD122 of SEQ ID NO:15.

[0487] In some embodiments, the masking moiety has a mutation at amino acid position N123 compared to CD122 of SEQ ID NO:15. In some embodiments, the masking moiety has the mutation N123C compared to CD122 of SEQ ID NO:15. In some embodiments, the masking moiety has the mutation N123Q compared to CD122 of SEQ ID NO:15.

[0488] In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has a mutation at amino acid position C168. In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has the mutation C168S. In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has the mutation C168V. In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has the mutation C168A.

[0489] In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has a mutation at amino acid position L169. In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has the mutation L169C.

[0490] In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has a mutation at amino acid position Q177. In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has the mutation Q177C.

[0491] In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has a mutation at amino acid position V184. In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has the mutation V184C.

[0492] In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has a mutation at amino acid position S195. In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has the mutation S195C.

[0493] In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has a mutation at amino acid position R204. In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has the mutation R204C.

[0494] In some embodiments, compared to the CD122 of SEQ ID NO:15, the masking portion has the mutation C122V / C168V. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO:17.

[0495] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:17)

[0496] In some embodiments, compared to CD122 of SEQ ID NO:15, the masking moiety has the mutations C122A / C168V. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:18.

[0497] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:18)

[0498] In some embodiments, compared to CD122 of SEQ ID NO:15, the masking moiety has the mutation C168V. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:19.

[0499] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:19)

[0500] In some embodiments, the masking moiety has the mutations C122V / C168A compared to CD122 of SEQ ID NO:15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:20.

[0501] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:20)

[0502] In some embodiments, the masking moiety has the mutations C122A / N123C compared to CD122 of SEQ ID NO:15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:21.

[0503] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRACISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:21)

[0504] In some embodiments, the masking portion comprises an amino acid sequence having at least about 80% identity to SEQ ID NO:21. In some embodiments, the masking portion comprises an amino acid sequence having at least about 85% identity to SEQ ID NO:21. In some embodiments, the masking portion comprises an amino acid sequence having at least about 90% identity to SEQ ID NO:21. In some embodiments, the masking portion comprises an amino acid sequence having at least about 91% identity to SEQ ID NO:21. In some embodiments, the masking portion comprises an amino acid sequence having at least about 92% identity to SEQ ID NO:21. In some embodiments, the masking portion comprises an amino acid sequence having at least about 93% identity to SEQ ID NO:21. In some embodiments, the masking portion comprises an amino acid sequence having at least about 94% identity to SEQ ID NO:21. In some embodiments, the masking portion comprises an amino acid sequence having at least about 95% identity to SEQ ID NO:21. In some embodiments, the masking portion comprises an amino acid sequence having at least about 96% identity to SEQ ID NO:21. In some embodiments, the masking portion comprises an amino acid sequence having at least about 97% identity to SEQ ID NO:21. In some embodiments, the masking portion comprises an amino acid sequence having at least about 98% identity to SEQ ID NO:21. In some embodiments, the masking portion comprises an amino acid sequence having at least about 99% identity to SEQ ID NO:21.

[0505] In some embodiments, compared to CD122 of SEQ ID NO:15, the masking portion has the mutations C122V / N123C. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO:22.

[0506] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRVCISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:22)

[0507] In some embodiments, the masking moiety has the mutations C122A / C168A compared to CD122 of SEQ ID NO:15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:23.

[0508] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:23)

[0509] In some embodiments, the masking moiety has the mutations V117C / N123Q / C168A compared to CD122 of SEQ ID NO:15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:24.

[0510] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHCETHRCQISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:24)

[0511] In some embodiments, the masking moiety has the mutations N123Q / C168A / L169C compared to CD122 of SEQ ID NO:15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:25.

[0512] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCQISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIACETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:25)

[0513] In some embodiments, compared to CD122 of SEQ ID NO:15, the masking moiety has the mutations L106C / C122A / C168A / S195C. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:26.

[0514] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWCPWSQPLAFRTKPAALGKD(SEQ IDNO:26)

[0515] In some embodiments, compared to CD122 of SEQ ID NO:15, the masking moiety has the mutations L106C / C122A / C168A / V184C. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:27.

[0516] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRCKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:27)

[0517] In some embodiments, compared to CD122 of SEQ ID NO:15, the masking portion has the mutations C122A / C168A / V184C / S195C. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO:28.

[0518] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRCKPLQGEFTTWCPWSQPLAFRTKPAALGKD(SEQ IDNO:28)

[0519] In some embodiments, compared to CD122 of SEQ ID NO:15, the masking portion has the mutations C122A / C168A / Q177C / R204C. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO:29.

[0520] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTCYEFQVRVKPLQGEFTTWSPWSQPLAFCTKPAALGKD(SEQ IDNO:29)

[0521] In some embodiments, compared to CD122 of SEQ ID NO:15, the masking portion has the mutations L106C / C122V / C168V / S195C. In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO:30.

[0522] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMAPISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWCPWSQPLAFRTKPAALGKD(SEQ IDNO:30)

[0523] In some embodiments, compared to CD122 of SEQ ID NO:15, the masking moiety has the mutations F8C / A94C / C122V / C168V. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:31.

[0524] AVNGTSQCTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMCIQDFKPFENLRLMAPISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD(SEQ IDNO:31)

[0525] IL-12 receptor

[0526] The masking moiety masks the IL-12 cytokine or a functional fragment thereof in the target cytokine, thereby reducing or preventing the binding of the IL-cytokine or a functional fragment thereof to its cognate receptor.

[0527] 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 interleukin-12 (IL-12) with low affinity. This protein forms a disulfide-bonded oligomer, which is essential 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 results in the formation of high-affinity IL-12 binding sites.

[0528] In some embodiments, the masking moiety comprises the extracellular domain of the IL-12 cytokine receptor or a subunit or functional fragment thereof.

[0529] Interleukin-12 receptor subunit beta-1 (also known as CD212) has the following sequence:

[0530]

[0531]

[0532] Interleukin-12 receptor subunit beta-2 has the following sequence:

[0533]

[0534] Bold indicates the propeptide, underlined italics indicate the extracellular domain, italics indicate the transmembrane domain and underlined bold indicates the cytoplasmic domain.

[0535] 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.

[0536] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of human IL-12Rβ1 (SEQ ID NO:125) 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.

[0537] In some embodiments, the masking moiety comprises residues 24 to 237 of human IL-12Rβ1, i.e., a sequence having SEQ ID NO:126 or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-12.

[0538] CRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRQLGSQGSSWSKWSSPVCVPPENP(SEQ IDNO:126)

[0539] In some embodiments, the masking moiety comprises an IL-12Rβ1 having SEQ ID NO:126. 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:126. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:126. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:126. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO:126. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO:126. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO:126. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:126. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO:126. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:126. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:126. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:126.

[0540] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:126, 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 SEQ ID NO:126, the amino acid sequence having one or two amino acid substitutions.

[0541] In some embodiments, the masking moiety comprises residues 24 to 545 of human IL-12Rβ1, i.e., a sequence having SEQ ID NO:127 or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-12.

[0542] CRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRQLGSQGSSWSKWSSPVCVPPENPPQPQVRFSVEQLGQDGRRRLTLKEQPTQLELPEGCQGLAPGTEVTYRLQLHMLSCPCKAKATRTLHLGKMPYLSGAAYNVAVISSNQFGPGLNQTWHIPADTHTEPVALNISVGTNGTTMYWPARAQSMTYCIEWQPVGQDGGLATCSLTAPQDPDPAGMATYSWSRESGAMGQEKCYYITIFASAHPEKLTLWSTVLSTYHFGGNASAAGTPHHVSVKNHSLDSVSVDWAPSLLSTCPGVLKEYVVRCRDEDSKQVSEHPVQPTETQVTLSGLRAGVAYTVQVRADTAWLRGVWSQPQRFSIEVQVSD(SEQ ID NO:127)

[0543] In some embodiments, the masking moiety comprises an IL-12Rβ1 having SEQ ID NO:127. 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:127. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:127. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:127. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO:127. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO:127. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO:127. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:127. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO:127. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:127. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:127. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:127.

[0544] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:127, 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 SEQ ID NO:127, the amino acid sequence having one or two amino acid substitutions.

[0545] In some embodiments, the masking moiety comprises the extracellular domain of human IL-12Rβ2 or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-12. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of human IL-12Rβ2 (SEQ ID NO:155), 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.

[0546] MAHTFRGCSLAFMFIITWLLIKAKIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTGMLDVWYMKRHIDYSRQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGMDNILVTWQPPRKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSSILGNSKHKAPLSGPHINAITEEKGSILISWNSIPVQEQMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNSHPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKANWMAFVAPSICIAIIMVGIFSTHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML(SEQ ID NO:155)

[0547] In some embodiments, the masking portion comprises residues 24 to 212 of human IL-12Rβ2, i.e., having the sequence of SEQ ID NO:128.

[0548] KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSL(SEQ ID NO:128)

[0549] In some embodiments, the masking moiety comprises an IL-12Rβ1 having SEQ ID NO:128. 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:128. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:128. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:128. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO:128. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO:128. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO:128. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:128. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO:128. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:128. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:128. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:128.

[0550] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:128, which amino acid sequence has one to four amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:128, which amino acid sequence has one or two amino acid substitutions.

[0551] In some embodiments, the masking moiety comprises residues 24 to 222 of human IL-12Rβ2, i.e., having the sequence of SEQ ID NO:129.

[0552] KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIV (SEQ ID NO:129)

[0553] In some embodiments, the masking moiety comprises an IL-12Rβ1 having SEQ ID NO:129. 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:129. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:129. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:129. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO:129. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO:129. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO:129. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:129. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO:129. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:129. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:129. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:129.

[0554] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:129, wherein the amino acid sequence has one to four amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:129, wherein the amino acid sequence has one or two amino acid substitutions.

[0555] In some embodiments, the masking moiety comprises residues 24 to 319 of human IL-12Rβ2, i.e., having the sequence of SEQ ID NO:130.

[0556] KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEE(SEQ ID NO:130)

[0557] In some embodiments, the masking moiety comprises IL-12Rβ1 having SEQ ID NO:130. 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:130. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:130. 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:130. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO:130. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO:130. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:130. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO:130. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:130. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:130. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:130.

[0558] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 130, said 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 SEQ ID NO: 130, said amino acid sequence having one or two amino acid substitutions.

[0559] In some embodiments, the masking moiety comprises residues 24 to 319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 130, which has one or more cysteine substitutions. In some embodiments, the masking moiety comprises residues 24 to 319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 130, 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: 131.

[0560] KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRSTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEE (SEQ ID NO: 131)

[0561] 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: 131. In some embodiments, the masking moiety consists of the amino acid sequence of SEQ ID NO: 131.

[0562] In some embodiments, the masking moiety comprises residues 24 to 622 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 132.

[0563] KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTGMLDVWYMKRHIDYSRQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGMDNILVTWQPPRKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSSILGNSKHKAPLSGPHINAITEEKGSILISWNSIPVQEQMGCLLHYRIYWKER(SEQ ID NO:132)

[0564] In some embodiments, the masking moiety comprises an IL-12Rβ1 having SEQ ID NO:132. 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:132. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:132. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:132. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO:132. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO:132. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO:132. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:132. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO:132. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:132. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:132. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:132.

[0565] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:132, said 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 SEQ ID NO:132, said amino acid sequence having one or two amino acid substitutions.

[0566] In some embodiments, the masking moiety comprises residues 24 to 227 of human IL-12Rβ2, i.e., having the sequence of SEQ ID NO:133.

[0567] KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPP(SEQ ID NO:133)

[0568] In some embodiments, the masking moiety comprises an IL-12Rβ1 having SEQ ID NO:133. 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 SEQ ID NO:133. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:133. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:133. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO:133. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO:133. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO:133. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:133. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO:133. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:133. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:133. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:133.

[0569] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 133, which amino acid sequence has one to four amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 133, which amino acid sequence has one or two amino acid substitutions.

[0570] Antibody-based masking moiety

[0571] The masking moiety that masks a cytokine or a functional fragment thereof in a target cytokine can be an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment binds to the cytokine. The cytokine and the anti-cytokine antibody or binding fragment complex in the target cytokine thus reduces or prevents the binding of the cytokine or its functional fragment to its cognate receptor or ligand (e.g., masks the activity of the cytokine).

[0572] The cytokine-specific antibody can be a whole antibody or a binding fragment thereof. In some embodiments, the binding fragment derived from an antibody includes but is not limited to Fab, Fab', F(ab')2, scFv, and single-chain antibodies. In some embodiments, the antibody-based masking moiety is an scFv. In some embodiments, the antibody-based masking moiety is a Fab fragment.

[0573] Single-chain variable fragment (scFv)

[0574] In some embodiments, the masking moiety comprises an anti-IL-2 scFv having a variable heavy chain (VH) of SEQ ID NO: 172. In some embodiments, the masking moiety comprises an anti-IL-2 scFv having a variable light chain (VL) of SEQ ID NO: 173. In some embodiments, the masking moiety comprises an anti-IL-2 scFv having a VH of SEQ ID NO: 172 and a VL of SEQ ID NO: 173.

[0575] In some embodiments, the masking moiety comprises an anti-IL-2 scFv having an hCDR1 of SEQ ID NO:174, an hCDR2 of SEQ ID NO:175, and an hCDR3 of SEQ ID NO:176. In some embodiments, the masking moiety comprises an anti-IL-2 scFv having an lCDR1 of SEQ ID NO:177, an lCDR2 of SEQ ID NO:178, and an lCDR3 of SEQ ID NO:179. In some embodiments, the masking moiety comprises an anti-IL-2 scFv having an hCDR1 of SEQ ID NO:174, an hCDR2 of SEQ ID NO:175, an hCDR3 of SEQ ID NO:176, an lCDR1 of SEQ ID NO:177, an lCDR2 of SEQ ID NO:178, and an lCDR3 of SEQ ID NO:179.

[0576] Exemplary anti-IL-2 scFv sequences

[0577]

[0578] In some embodiments, the masking moiety comprises a VL linked to a VH via a non-cleavable linker. In some embodiments, the masking moiety comprises a VH linked to a VL via a non-cleavable linker. In some embodiments, the non-cleavable linker is GGGGG SGGGGS GGGGGS GGGGGS (SEQ ID NO:182).

[0579] In some embodiments, the scFv masking moiety is linked to the Fc domain via a cleavable linker. In some embodiments, the scFv masking moiety is linked to the Fc domain via a cleavable linker described herein (e.g., a linker comprising a cleavable peptide described in Table 1).

[0580] In some embodiments, the masking moiety comprises a scFv having an amino acid sequence with about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the amino acid sequences of SEQ ID NO: 180. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 180. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 180. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 180. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 180. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 180. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 180. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 180. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 180. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 180. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 180.

[0581] VHH antibody

[0582] A VHH antibody (or nanobody) is an antigen-binding fragment of an antibody consisting only of a heavy chain. In some embodiments, the masking moiety is an antibody consisting only of a heavy chain (VHH). In some embodiments, the masking moiety is an antibody consisting only of a heavy chain (VHH) that binds to a cytokine. VHHs can be derived from organisms that produce VHH antibodies, such as camels and sharks, for example. In some cases, the VHH can be a recombinant VHH. The VHH technology is based on fully functional antibodies from camels that lack light chains. These heavy chain antibodies contain a single variable domain (V H H) and two constant domains (CH2 and CH3). A VHH comprises a single-chain polypeptide having three CDRs and four framework regions (FR1-4). As used herein, a "framework region" or "FR" refers to the regions within the variable domain that lie between the CDRs. As used herein, a "complementary determining region" or "CDR" refers to the variable region of a VHH that contains an amino acid sequence capable of specifically binding to an antigen target (e.g., a cytokine).

[0583] In some embodiments, the VHH comprises three CDRs and four framework regions, named FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, the VHH may be truncated at the N-terminus or C-terminus such that it only includes a portion of FR1 and / or FR4 or lacks one or both of those framework regions, provided that the VHH substantially maintains cytokine binding and specificity.

[0584] VHH is not limited to a particular biological source or a particular method of preparation. For example, VHH can generally be obtained by: (1) isolating the V H H domain of a naturally occurring heavy chain antibody; (2) expressing the nucleotide sequence encoding the naturally occurring V H H domain; (3) "humanizing" the naturally occurring V H H domain or by expressing the nucleic acid encoding such a humanized V H H domain; (4) "camelizing" the naturally occurring VH domain from any animal species (such as from a mammalian species, such as from a human), or by expressing the nucleic acid encoding such a camelized VH domain; (5) "camelizing" a "domain antibody" or "Dab" as described in the art, or by expressing the nucleic acid encoding such a camelized VH domain; (6) using synthetic or semi-synthetic techniques for preparing proteins, polypeptides, or other amino acid sequences known in the art; (7) preparing the nucleic acid encoding VHH using techniques known in the art for nucleic acid synthesis and subsequently expressing the thus obtained nucleic acid; and / or (8) any combination of one or more of the foregoing.

[0585] In some embodiments, the IL-2 masking moiety is an anti-IL-2 VHH antibody. The IL-2 masking moiety comprises a VHH corresponding to the V H H domain of a naturally occurring heavy chain antibody against IL-2. Such anti-IL-2 V H H sequences can generally be produced or obtained by appropriately immunizing a camelid species with an IL-2 molecule. In some embodiments, the V H H sequence against the IL-2 molecule involves appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies.

[0586] In some embodiments, the masking moiety comprises an anti-IL-2 VHH having the amino acid sequence of SEQ ID NO:168. In some embodiments, the masking moiety comprises an anti-IL-2 VHH having hCDR1 of SEQ ID NO:169, hCDR2 of SEQ ID NO:170, and hCDR3 of SEQ ID NO:171.

[0587] Exemplary anti-IL-2 scFv sequences

[0588]

[0589] In some embodiments, the masking moiety comprises a VHH having an amino acid sequence with about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the amino acid sequences of SEQ ID NO: 168. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 168. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 168. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 168. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 168. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 168. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 168. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 168. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 168. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 168. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 168.

[0590] In some embodiments, the VHH masking moiety is linked to the Fc domain via a cleavable linker. In some embodiments, the VHH masking moiety is linked to the Fc domain via a cleavable linker described herein (e.g., a linker comprising a cleavable peptide described in Table 1).

[0591] Linker

[0592] Linkers for targeting cytokines or their cleavage products are provided herein. A linker as provided herein refers to a peptide having more than two amino acids for linking two functional components of the targeting cytokines described herein together.

[0593] The targeting cytokine comprises a first linker and a second linker, wherein at least the first linker or the second linker comprises a proteolytically cleavable peptide.

[0594] In some embodiments, the first Fc polypeptide is linked to the cytokine or a variant thereof via a first linker. In some embodiments, the second Fc polypeptide is linked to the masking moiety via a second linker. In some embodiments, the first Fc polypeptide is linked to the cytokine or a variant thereof via a cleavable linker. In some embodiments, the second Fc polypeptide is linked to the masking moiety via a cleavable linker. In some embodiments, the first Fc polypeptide is linked to the cytokine or a variant thereof via a cleavable linker and the second Fc polypeptide is linked to the masking moiety via a non-cleavable linker. In some embodiments, the first Fc polypeptide is linked to the cytokine or a variant thereof via a non-cleavable linker and the second Fc polypeptide is linked to the masking moiety via a cleavable linker. In some embodiments, the first Fc polypeptide is linked to the cytokine or a variant thereof via a cleavable linker and the second Fc polypeptide is linked to the masking moiety via a cleavable linker. In some embodiments, the first Fc polypeptide is linked to the cytokine or a variant thereof via a non-cleavable linker and the second Fc polypeptide is linked to the masking moiety via a non-cleavable linker.

[0595] Non-cleavable linker

[0596] In some embodiments, the length of the non-cleavable linker ranges from 2 amino acids to 25 amino acids. In some embodiments, the length of the non-cleavable linker ranges from 3 amino acids to 21 amino acids. In some embodiments, the length of the non-cleavable linker ranges from 3 amino acids to 18 amino acids. In some embodiments, the length of the non-cleavable linker ranges from 5 amino acids to 18 amino acids. In some embodiments, the length of the non-cleavable linker ranges from 3 amino acids to 8 amino acids. In some embodiments, the length of the non-cleavable linker ranges from 4 amino acids to 6 amino acids.

[0597] In some embodiments, the length of the non-cleavable linker is 15 amino acids. In some embodiments, the length of the non-cleavable linker is 16 amino acids. In some embodiments, the length of the non-cleavable linker is 17 amino acids. In some embodiments, the length of the non-cleavable linker is 18 amino acids. In some embodiments, the length of the non-cleavable linker is 19 amino acids. In some embodiments, the length of the non-cleavable linker is 20 amino acids.

[0598] In some embodiments, the non-cleavable linker is rich in amino acid residues G, S, and P. In some embodiments, the non-cleavable linker contains only amino acid residue types selected from the group consisting of G, S, and P. In some embodiments, the non-cleavable linker contains a 'GS' repeat sequence. In some embodiments, the non-cleavable linker contains an N'-terminal 'P' residue.

[0599] In some embodiments, the non-cleavable linker comprises the amino acid sequence as shown in SEQ ID NO:32 (PGSGS).

[0600] In some embodiments, the non-cleavable linker comprises the amino acid sequence as shown in SEQ ID NO:33 (GGSSPPGGGSSGGGSGP).

[0601] In some embodiments, the non-cleavable linker comprises the amino acid sequence GGS.

[0602] In some embodiments, the non-cleavable linker contains [(G) n S], where n = 4 or 5.

[0603] In some embodiments, the non-cleavable linker comprises the amino acid sequence as shown in SEQ ID NO:134 (GGGG S).

[0604] In some embodiments, the non-cleavable linker comprises the amino acid sequence as shown in SEQ ID NO:135 (GGGG SGGGG S).

[0605] In some embodiments, the non-cleavable linker comprises the amino acid sequence as shown in SEQ ID NO:136 (GGSGGGSGGGG S).

[0606] In some embodiments, the non-cleavable linker comprises the amino acid sequence as shown in SEQ ID NO:137 (GGSGGSGGSGGSGGSSGP).

[0607] In some embodiments, the non-cleavable linker comprises the amino acid sequence as shown in SEQ ID NO:138 (PGGSGP).

[0608] In some embodiments, the non-cleavable linker comprises the amino acid sequence as shown in SEQ ID NO:56 (GGSPG).

[0609] In some embodiments, the linker non-cleavable linker comprises the amino acid sequence as shown in SEQ ID NO:181 (GGSSGSGGSGGGSGSGGG).

[0610] In some embodiments, the second linker comprises a proteolytically cleavable peptide such that the second linker is a proteolytically cleavable linker, and the first linker does not comprise a proteolytically cleavable peptide such that the first linker is a non-proteolytically cleavable linker, and the length of the non-cleavable linker is between 3 and 18 amino acids. In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO:134 (GGGGS). In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO:135 (GGGGSGGGGS). In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO:136 (GGSGGGSGGGGGS). In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO:137 (GGSGGSGGSGGSGGSSGP). In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO:138 (PGGSGP). In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO:139 (GGSPG).

[0611] In some embodiments, it is desirable for the first polypeptide chain and the second polypeptide chain to have the same or similar lengths to facilitate the association of the first half-life extending domain with the second half-life extending domain and to mask the cytokine or its functional fragment in the assembled construct. Thus, in cases where the masking moiety is a shorter amino acid sequence than the cytokine or its functional fragment, the length difference can be fully or partially compensated for by using a longer linker L1.

[0612] Cleavable linker

[0613] In some embodiments, the length of the cleavable linker is between 2 and 25 amino acids. In some embodiments, the length of the cleavable linker is between 3 and 21 amino acids. In some embodiments, the length of the cleavable linker is between 3 and 18 amino acids. In some embodiments, the length of the cleavable linker is between 5 and 18 amino acids. In some embodiments, the length of the cleavable linker is between 3 and 8 amino acids. In some embodiments, the length of the cleavable linker is between 4 and 6 amino acids.

[0614] In some embodiments, the length of the cleavable linker is 15 amino acids. In some embodiments, the length of the cleavable linker is 16 amino acids. In some embodiments, the length of the cleavable linker is 17 amino acids. In some embodiments, the length of the cleavable linker is 18 amino acids. In some embodiments, the length of the cleavable linker is 19 amino acids. In some embodiments, the length of the cleavable linker is 20 amino acids.

[0615] In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide (CP) flanked on both sides by spacer domains (SD), as shown in the following formula:

[0616] SD-CP-SD

[0617] Cleavable peptide

[0618] The cleavable linker comprises a cleavable peptide.

[0619] The cleavable peptide is a polypeptide that contains a protease cleavage site such that the cleavable peptide is proteolytically cleavable. A protease is an enzyme that cleaves the peptide bond between two specific amino acid residues of a target substrate protein. As used herein, a "cleavage site" refers to a recognizable site for cleaving a portion of a cleavable peptide found in any linker including the cleavable peptides described herein. Thus, a cleavage site can be found in the sequence of a cleavable peptide as described herein. In some embodiments, the cleavage site is an amino acid sequence recognized and cleaved by a cleavage agent.

[0620] In some embodiments, the protease cleavage site is a tumor-associated protease cleavage site. As provided herein, a "tumor-associated protease cleavage site" is an amino acid sequence recognized by a protease whose expression is specific or upregulated for tumor cells or their tumor cell environment.

[0621] The tumor cell environment is complex and can include a variety of different proteases. Thus, the precise site at which a given cleavable peptide will be cleaved in the 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 the specific tumor cell environment. In addition, even after cleavage, further modification of the initial cleavage product, such as by removal of one or two terminal amino acids, can occur through further action of proteases in the tumor cell environment. Thus, upon administration of a single construct of a targeted cytokine as described herein, a distribution of cleavage products can be expected to form in the tumor cell environment of a patient.

[0622] It should be understood that a cleavage site, as referred to herein, is a site between two specific amino acid residues within a cleavable peptide, and the amino acid residues are targets of proteases known to be associated with the tumor cell environment. In this sense, there is more than one cleavage site in the cleavable peptides described herein, where different proteases cleave the cleavable peptide at different cleavage sites. It is also possible that more than one protease can act on the same cleavage site within the cleavable peptide. Discussions of protease cleavage sites can be found in the art.

[0623] Accordingly, the cleavable peptides disclosed herein can be cleaved by one or more proteases. In some embodiments, the protease cleavage site is a tumor-associated protease cleavage site. A tumor-associated protease cleavage site can be recognized by a tumor-associated protease. As a non-limiting example, tumor-associated proteases are matrix metalloproteinases (MMPs) 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 some embodiments, the tumor-associated protease is MMP7. In yet another embodiment, the tumor-associated protease is MMP9. In yet another embodiment, the tumor-associated protease is MMP10. Other disease-associated proteases and tissue-selective proteases include but are not limited to cathepsins (cathepsin B (catB), 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 (granzyme A, B, M, H, and K), fibroblast activation protein (FAP), plasmin, urokinase plasminogen activator (uPA), tryptase, caspases, thrombin, podoplanin, chymotrypsin, collagenase, nepenthesin A, and proteinase 1-2. In some embodiments, the disease-associated protease and / or tissue-selective protease is a cathepsin (cathepsin B (catB)).

[0624] In some embodiments, the cleavable peptide is a substrate of a protease that is co-localized to a region or tissue expressing a cytokine receptor.

[0625] In some embodiments, the cleavable peptide is a pentamer (i.e., a peptide of 5 amino acids in length), a hexamer (i.e., a peptide of 6 amino acids in length), a heptamer (i.e., a peptide of 7 amino acids in length), an octamer (i.e., a peptide of 8 amino acids in length), a nonamer (i.e., a peptide of 9 amino acids in length), a decamer (i.e., a peptide of 10 amino acids in length), an undecamer (i.e., a peptide of 11 amino acids in length), a dodecamer (i.e., a peptide of 12 amino acids in length), a tridecamer (i.e., a peptide of 13 amino acids in length), a tetradecamer (i.e., a peptide of 14 amino acids in length), a pentadecamer (i.e., a peptide of 15 amino acids in length), a hexadecamer (i.e., a peptide of 16 amino acids in length), a heptadecamer (i.e., a peptide of 17 amino acids in length), or an octadecamer (i.e., a peptide of 18 amino acids in length).

[0626] In some embodiments, the cleavable peptide has a length of 5 to 18 amino acids. In some emb...

Claims

1. A targeting cytokine, comprising: a) A targeting moiety; b) A cytokine or a fragment thereof; c) A masking moiety; and d) An Fc domain, the Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide being linked to the cytokine or a fragment thereof by a first linker, and the second Fc polypeptide being linked to the masking moiety by a second linker, wherein the masking moiety binds to the cytokine or a fragment thereof, wherein the first linker or the second linker is a cleavable linker such that the masking moiety releases the cytokine or a fragment thereof upon cleavage, wherein the cleavable linker comprises (i) MPYDLYHP (SEQ ID NO:34) or VPLSLYSG (SEQ ID NO:42), (ii) an amino acid sequence selected from Table 1, or (iii) between 8 - 50 amino acid residues and wherein the targeting moiety is linked to the Fc domain via one or both of the first Fc polypeptide and the second Fc polypeptide.

2. A targeting cytokine, comprising: a) A targeting moiety; b) A cytokine or a fragment thereof; c) A masking moiety; and d) An Fc domain, the Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide being linked to the cytokine or a fragment thereof by a first linker, and the second Fc polypeptide being linked to the masking moiety by a second linker; wherein the masking moiety binds to the cytokine or a fragment thereof, wherein the first linker or the second linker is a cleavable linker such that the masking moiety releases the cytokine or a fragment thereof upon cleavage to produce an active cytokine, wherein the cleavable linker has an in vitro cleavage efficiency of producing at least 10% active cytokine, and wherein the targeting moiety is linked to the Fc domain via one or both of the first Fc polypeptide and the second Fc polypeptide.

3. A targeting cytokine, comprising: a) A targeting moiety; b) A cytokine or a fragment thereof; c) A masking moiety, the masking moiety comprising CD122 or a fragment thereof, the CD122 or a fragment thereof comprising 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, R204C; and d) An Fc domain, the Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide being linked to the cytokine or a fragment thereof by a first linker, and the second Fc polypeptide being linked to the masking moiety by a second linker, wherein the masking moiety binds to the cytokine or a fragment thereof, wherein the first linker or the second linker is a cleavable linker such that the masking moiety releases the cytokine or a fragment thereof upon cleavage, and wherein the targeting moiety is linked to the Fc domain via one or both of the first Fc polypeptide and the second Fc polypeptide.

4. A targeted cytokine, comprising: a) a targeting moiety, b) a masking moiety, c) a cytokine or a fragment thereof, and d) an Fc domain, the Fc domain comprising i. a first Fc polypeptide, the first Fc polypeptide comprising a CH3 domain, the CH3 domain comprising a modification that reduces or eliminates binding to protein A, and ii. a second Fc polypeptide, the second Fc polypeptide comprising a CH3 domain that binds to protein A; and optionally, a) wherein the first Fc polypeptide is linked to the cytokine or a fragment thereof via a first linker, and the second Fc polypeptide is linked to the masking moiety via a second linker; or b) wherein the first Fc polypeptide is linked to the masking moiety via a first linker, and the second Fc polypeptide is linked to the cytokine or a fragment thereof via a second linker.

5. The targeted cytokine according to claim 4, wherein the targeting moiety is linked to the Fc domain via one or both of the first Fc polypeptide and the second Fc polypeptide.

6. The targeted cytokine according to any one of claims 1 to 5, wherein the targeting moiety specifically binds to the following: 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 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), nectin-4, Liv-1, glycoprotein NMB (GPNMB), prostate-specific membrane antigen (PSMA), Trop-2, carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), prostate six-transmembrane epithelial antigen 1 (STEAP1), folate receptor alpha (FR-α), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), ectonucleotide pyrophosphatase / phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG), CD19, CD20, CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD98, CD123, CD138, CD352, CD47, signal regulatory protein alpha (SIRPα), Claudin 18.2, Claudin 6, 5T4, fibroblast activation protein alpha (FAPα), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epithelial cell adhesion molecule (EPCAM) or a combination thereof.

7. A targeted cytokine, comprising: a) a targeting moiety that specifically binds to PD-1; b) a cytokine or a fragment thereof; c) a masking moiety; and d) an Fc domain, the Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide being linked to the cytokine or a fragment thereof through a first linker, and the second Fc polypeptide being linked to the masking moiety through a second linker, wherein the masking moiety binds to the cytokine or a fragment thereof, wherein the first linker or the second linker is a cleavable linker such that the masking moiety releases the cytokine or a fragment thereof upon cleavage, wherein the cleavable linker comprises MPYDLYHP (SEQ ID NO:34) or VPLSLYSG (SEQ ID NO:42), and wherein the targeting moiety is linked to the Fc domain through one or both of the first Fc polypeptide and the second Fc polypeptide.

8. A targeted cytokine, comprising: a) A targeting moiety that specifically binds to PD-1; b) A cytokine or a fragment thereof; c) A masking moiety; and d) An Fc domain, the Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide being linked to the cytokine or a fragment thereof via a first linker, and the second Fc polypeptide being linked to the masking moiety via a second linker, wherein the masking moiety binds to the cytokine or a fragment thereof, wherein the first linker or the second linker is a cleavable linker such that the masking moiety releases the cytokine or a fragment thereof upon cleavage, wherein the cleavable linker comprises an amino acid sequence selected from Table 1, and wherein the targeting moiety is linked to the Fc domain via one or both of the first Fc polypeptide and the second Fc polypeptide.

9. A targeted cytokine, comprising: a) A targeting moiety that specifically binds to PD-1, the targeting moiety comprising HCDR1 of SEQ ID NO:4 (GYTFTNYY), HCDR2 of SEQ ID NO:5 (INPSNGGT), HCDR3 SEQ ID NO:6 (ARRDYRFDMGFDY), LCDR1 of SEQ ID NO:9 (KGVSTSGYSY), LCDR2 of SEQ ID NO:10 (LAS), and LCDR3 of SEQ ID NO:11 (QHSRDLPLT); b) A cytokine or a fragment thereof, and c) An Fc domain.

10. A targeted cytokine, comprising: a) A targeting moiety that specifically binds to PD-1, the targeting moiety comprising HCDR1 GITFSNSG (SEQ ID NO:161), HCDR2 of VIWYDGSKRYYADSVKG (SEQ ID NO:162), HCDR3 ATNDDY (SEQ ID NO:163), LCDR1 QSVSSY (SEQ ID NO:164), LDR2 DAS (SEQ ID NO:165), and LCDR3 QQSSNWPRT (SEQ ID NO:166), b) A cytokine or a fragment thereof, and c) An Fc domain.

11. The targeted cytokine according to any one of claims 9 or 10, wherein the Fc domain comprises i. A first Fc polypeptide, the first Fc polypeptide comprising a CH3 domain, the CH3 domain comprising a modification that reduces or eliminates binding to protein A, and ii. A second Fc polypeptide, the second Fc polypeptide comprising a CH3 domain that binds to protein A.

12. A targeted cytokine, comprising: a) A targeting moiety; b) An IL-2 cytokine or a fragment thereof; c) A VHH masking moiety; and d) The Fc domain, wherein the Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide is linked to the cytokine or a fragment thereof via a first linker, and the second Fc polypeptide is linked to the masking moiety via a second linker, wherein the masking moiety binds to the cytokine or a fragment thereof, wherein the first linker or the second linker is a cleavable linker such that the masking moiety releases the cytokine or a fragment thereof upon cleavage, wherein the VHH masking moiety comprises hCDR1 of SEQ ID NO:169, hCDR2 of SEQ ID NO:170, and hCDR3 of SEQ ID NO:171, and wherein the targeting moiety is linked to the Fc domain via one or both of the first Fc polypeptide and the second Fc polypeptide.

13. The targeted cytokine according to any one of claims 9 to 12, wherein the first Fc polypeptide is linked to the cytokine or a fragment thereof via a first linker, and the second Fc polypeptide is linked to the masking moiety via a second linker, optionally wherein the targeting moiety is linked to the Fc domain via one or both of the first Fc polypeptide and the second Fc polypeptide.

14. The targeted cytokine according to any one of claims 9 to 13, wherein the masking moiety binds to the cytokine or a fragment thereof and / or wherein the first linker or the second linker is a cleavable linker such that the masking moiety releases the cytokine or a fragment thereof upon cleavage.

15. The targeted cytokine according to any one of claims 9 to 14, wherein the cleavable linker comprises MPYDLYHP (SEQ ID NO:34), VPLSLYSG (SEQ ID NO:42), PVSLRSGS (SEQ ID NO:196), GMPKDLYHAS (SEQ ID NO.197), RPLALWRS (SEQ ID NO:193), TQKPLGLS (SEQ ID NO:194), APAGLIVPYN (SEQ ID NO:195), PANLVAPDP (SEQ ID NO:183), IVGRPRHQGV (SEQ ID NO:199), RSKYLATA (SEQ ID NO:198), or RAAAVKSP (SEQ ID NO:37).

16. The targeted cytokine according to any one of the preceding claims, wherein the cleavable linker comprises a proteolytically cleavable peptide flanked by spacer domains on both sides, optionally wherein the length of the spacer domain ranges from 3 amino acids to 10 amino acids or from 3 amino acids to 6 amino acids.

17. The targeted cytokine according to any one of the preceding claims, wherein the first linker is the cleavable linker or the second linker is the cleavable linker.

18. The targeted cytokine according to any one of the preceding claims, wherein (i) the C-terminus of the targeting moiety is linked to the N-terminus of the first Fc polypeptide, (ii) the C-terminus of the first Fc polypeptide is linked to the N-terminus of the cytokine or a fragment thereof, (iii) the C-terminus of the targeting moiety is linked to the N-terminus of the second Fc polypeptide, and / or (iv) the C-terminus of the second Fc polypeptide is linked to the N-terminus of the masking moiety.

19. The targeted cytokine according to any one of the preceding claims, wherein the targeting moiety and / or the masking moiety comprises one or more antigen-binding domains, a peptide, polypeptide, protein, ligand or agent that specifically binds to the cytokine or a fragment thereof, or 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), antibodies and combinations thereof.

20. The targeted cytokine according to any one of the preceding claims, wherein the targeted cytokine comprises a first antigen-binding domain and a second antigen-binding domain, optionally wherein (i) the first antigen-binding domain and the second antigen-binding domain specifically bind to the same target; (ii) the first antigen-binding domain and the second antigen-binding domain comprise the same amino acid sequence; (iii) the first antigen-binding domain and the second antigen-binding domain specifically bind to different targets; or (iv) the first antigen-binding domain and the second antigen-binding domain comprise different amino acid sequences.

21. The targeted cytokine according to claim 20, wherein the C-terminus of the first antigen-binding domain is linked to the N-terminus of the first Fc polypeptide; wherein the C-terminus of the first Fc polypeptide is linked to the N-terminus of the cytokine or a fragment thereof; wherein the C-terminus of the second antigen-binding domain is linked to the N-terminus of the second Fc polypeptide; and wherein the C-terminus of the second Fc polypeptide is linked to the N-terminus of the masking moiety.

22. The targeted cytokine according to any one of the preceding claims, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises a first IgG1, IgG2 or IgG4 Fc domain or a fragment thereof.

23. The targeted cytokine according to any one of the preceding claims, wherein each of the first Fc domain and / or the second Fc domain contains one or more modifications that promote non-covalent association of the first Fc polypeptide and the second Fc polypeptide, optionally 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; c) 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 d) 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; or e) wherein the first Fc polypeptide comprises a first CH3 domain, the first CH3 domain comprises a modification that reduces or eliminates binding to protein A, and the second Fc polypeptide comprises a second CH3 domain that binds to protein A.

24. The targeted cytokine according to claim 23, wherein the CH3 domain that binds to protein A is a human IgG1, IgG2, or IgG4 sequence, optionally wherein the second CH3 domain is a human IgG1, IgG2, or IgG4 sequence comprising a modification at position H435 and / or Y436 according to Kabat numbering.

25. The targeted cytokine according to any one of the preceding claims, wherein (i) the first Fc domain or the second Fc domain comprises SEQ ID NO:85 or SEQ ID NO:80, or (ii) the first Fc domain or the second Fc domain comprises SEQ ID NO:84 or SEQ ID NO:

80.

26. The targeted cytokine according to any one of the preceding claims, wherein the cytokine or a fragment thereof 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-20, TNF-α, TNF-β, CXCL8 (IL-18), 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.

27. The targeted cytokine according to any one of claims 1 to 2, 4 to 11, or 13 to 26, wherein the masking moiety is a receptor of the cytokine or a fragment thereof, optionally, 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.

28. The masking moiety according to claim 3 or 27 is engineered CD122, and the engineered CD122 comprises a mutation selected from the following: a) C122S and C168S, b) C122V and C168V, c) C122A and C168V, d) C168V, e) C122V and C168A, f) C122A and N123C, g) C122V and N123C, h) C122A and C168A, i) V117C, N123Q and C168A, j) N123Q, C168A and L169C, k) L106C, C122A, C168A and S195C, l) L106C, C122A, C168A and V184C, m) C122A, C168A, V184C and S195C, n) C122A, C168A, Q177C and R204C, o) L106C, C122V, C168V and S195C, or p) F8C, A94C, C122V and C168V.

29. The targeted cytokine according to any one of the preceding claims, wherein the cytokine is a modified IL-2 cytokine or a functional fragment thereof as compared to the sequence of mature IL-2 having SEQ ID NO:

13.

30. The targeted cytokine according to claim 29, wherein the modified IL-2 cytokine or a functional fragment thereof comprises a modification that (i) reduces or eliminates binding to CD25, (ii) enhances binding to CD25, (iii) reduces or eliminates binding to CD122, or (iv) enhances binding to CD122. Optionally, wherein the modified IL-2 cytokine or a functional fragment thereof comprises (i) modifications R38A, F42A, Y45A, and E62A relative to the sequence of mature IL-2 having SEQ ID NO: 13, (ii) modification C125A relative to the sequence of mature IL-2 having SEQ ID NO: 13, (iii) R38A, F42A, Y45A, E62A, and C125A relative to the sequence of mature IL-2 having SEQ ID NO: 13, or (iv) the amino acid sequence of SEQ ID NO:

14.

31. The targeted cytokine according to any one of claims 1-11 or 13-28, wherein the cytokine comprises an IL-15 polypeptide, the IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 93 or an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO: 93, optionally wherein the modification comprises (i) one or more amino acid substitutions at positions D22, E46, or E53 as compared to the amino acid sequence of SEQ ID NO: 93, (ii) one or more amino acid substitutions at positions D22, E46, E53, N71, N79, or N112 as compared to the amino acid sequence of SEQ ID NO: 93, (iii) amino acid substitutions at positions N71 and N79 as compared to the amino acid sequence of SEQ ID NO: 93, (iv) amino acid substitutions at positions N71 and N112 as compared to the amino acid sequence of SEQ ID NO: 93, (v) amino acid substitutions at positions N79 and N112 as compared to the amino acid sequence of SEQ ID NO:

93.

32. The targeted cytokine according to any one of claims 1-11 or 13-28, wherein the cytokine comprises an IL-12 polypeptide or a functional fragment thereof, the IL-12 polypeptide or a functional fragment thereof comprising an IL-12p40 polypeptide or a functional fragment thereof covalently linked to an IL-12p35 polypeptide or a functional fragment thereof.

33. The targeted cytokine according to claim 32, wherein the length of the IL-12p40-IL-12p35 linker is between 5 and 20 amino acids, optionally wherein the IL-12p40-IL-12p35 linker (i) is rich in amino acid residues G and S, or (ii) comprises SEQ ID NO: 119 (GGGGSGGGGSGGGGS).

34. The targeted cytokine according to any one of claims 32 to 33, wherein the IL-12p40 polypeptide comprises SEQ ID NO: 111 or an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO: 111, optionally wherein as compared to the amino acid sequence of SEQ ID NO: 111, the IL-12p40 polypeptide comprises at least one amino acid modification to the GAG binding domain of SEQ ID NO: 114 (KSKREKKDRV).

35. The targeted cytokine according to any one of claims 32 - 34, wherein the IL-12p40 polypeptide comprises SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO:

118.

36. The targeted cytokine according to any one of claims 32 to 35, wherein the IL-12p35 polypeptide comprises SEQ ID NO: 112 or an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO:

11.

37. The targeted cytokine according to any one of claims 32 to 36, wherein the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153 or SEQ ID NO:

154.

38. The targeted cytokine according to any one of claims 32 to 37, wherein 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, optionally, the masking moiety comprises (i) a sequence having SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132 or SEQ ID NO: 133, or (ii) the extracellular domain of human IL-12Rβ2 or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-12.

39. A nucleic acid encoding the targeted cytokine according to any one of the preceding claims.

40. A vector comprising the nucleic acid according to claim 39.

41. A host cell comprising the nucleic acid according to claim 39 or the vector according to claim 40.

42. A method for producing a targeted cytokine, the method comprising culturing the host cell according to claim 41 under conditions for producing the targeted cytokine.

43. A composition comprising the targeted cytokine according to any one of claims 1 to 38.

44. A pharmaceutical composition comprising a targeting cytokine according to any one of claims 1 to 38 and a pharmaceutically acceptable carrier.

45. A kit comprising a targeting cytokine according to any one of claims 1 to 38, or the composition according to claim 43 or the pharmaceutical composition according to claim 44.

46. Use of a targeting cytokine according to any one of claims 1 to 38 in the manufacture of a medicament for a) use in medicine; b) the treatment of neoplastic diseases; c) the treatment or prevention of inflammatory diseases; or d) the treatment or prevention of autoimmune diseases.

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