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 comprises an antigen-binding moiety that specifically binds to an antigen expressed on the surface of a target cell. The masking moiety masks the cytokine or a functional fragment thereof, thereby reducing or preventing the binding of the cytokine or a functional fragment thereof to its cognate receptor, but upon proteolytic cleavage of the cleavable linker at the target site, the cytokine or a functional fragment thereof becomes activated, thereby enabling or even enabling the cytokine or a functional fragment thereof to bind to its cognate receptor.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 314,928, filed February 28, 2022, and U.S. Provisional Patent Application No. 63 / 314,926, filed February 28, 2022, the contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Cancer is the second leading cause of death in the United States, accounting for more deaths than the next five leading causes (chronic respiratory disease, stroke, accidents, Alzheimer's disease, and diabetes). While great advances have been made, particularly in targeted therapies, much research remains in this field. Immunotherapy and its branch, immuno-oncology, are yielding viable and exciting therapeutic options for treating malignancies. In particular, it is now recognized that one of the hallmarks of cancer is immune evasion, and that significant efforts have been made to identify targets and develop therapies directed at these targets to reactivate the immune system to recognize and treat cancer.

[0003] Cytokine therapy is an effective strategy for stimulating the immune system to induce antitumor cytotoxicity. In particular, 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 generally have a very short half-life and therefore require frequent administration. For example, the product label for aldesleukin, marketed under the brand name Proleukin, states that the drug has been shown to have a half-life of 85 minutes in patients receiving a 5-minute intravenous (IV) administration. Furthermore, administration of high-dose cytokines can cause health hazards, such as vascular leakage, due to systemic immune activation. These findings demonstrate the need for the development of IL-2 cytokine therapeutic agents that effectively target tumors without 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, as well as methods of use, to address this need. Summary of the Invention

[0004] The present invention provides, inter alia, methods and compositions for use in the treatment of cancer. The present invention is based, in part, on the surprising discovery that the targeted masked cytokines of the present invention specifically target target cells for effective cancer treatment without causing undesirable side effects. The targeted cytokines of the present invention specifically bind to the intended target cells, accumulate in tumors, and are activated by tumor proteases. Furthermore, 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 in vivo with minimal toxicity.

[0005] In one aspect, the present invention provides a targeted cytokine comprising, inter alia, a targeting moiety, a cytokine or fragment thereof, a masking moiety, and an Fc domain comprising a first Fc polypeptide linked to the cytokine or fragment thereof via a first linker and a second Fc polypeptide linked to the masking moiety via a second linker.

[0006] In one aspect, the invention provides a targeted cytokine comprising, inter alia, a targeting moiety, a cytokine or fragment thereof, a masking moiety, and an Fc domain comprising a first Fc polypeptide linked to an IL-2 cytokine or fragment thereof via a first linker and a second Fc polypeptide linked to the masking moiety via a second linker, wherein the masking moiety binds to the IL-2 cytokine or fragment thereof, the first or the second linker is a cleavable linker such that the masking moiety releases the IL-2 cytokine or fragment thereof upon cleavage, the cleavable linker comprising a sequence selected from Table 1, and the targeting moiety is linked to the Fc domain via one or both of the first and second Fc polypeptides.

[0007] In one aspect, the present invention provides a targeted cytokine comprising, inter alia, a targeting moiety, a cytokine or fragment thereof, a masking moiety, and an Fc domain comprising a first Fc polypeptide linked to an IL-2 cytokine or fragment thereof via a first linker and a second Fc polypeptide linked to the masking moiety via a second linker, wherein the masking moiety binds to the IL-2 cytokine or fragment thereof, the first or the second linker is a cleavable linker such that the masking moiety releases the IL-2 cytokine or fragment thereof upon cleavage, the cleavable linker comprising MPYDLYHP (SEQ ID NO: 34) or VPLSLYSG (SEQ ID NO: 42), and the targeting moiety is linked to the Fc domain via one or both of the first and second Fc polypeptides.

[0008] 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).

[0009] In some embodiments, the cleavable linker comprises MPYDLYHP (SEQ ID NO: 34), RAAAVKSP, or VPLSLYSG (SEQ ID NO: 42).

[0010] 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.

[0011] 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 binding 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 binding Fab (s-Fab) that binds to a cytokine. In some embodiments, the masking moiety comprises an antibody or fragment thereof that binds to a cytokine. In some embodiments, the masking moiety comprises an antibody against a cytokine, or a binding fragment of that antibody.

[0012] In some embodiments, the masking moiety is a cytokine receptor. In some embodiments, the masking moiety is a fragment of a cytokine receptor. In some embodiments, the masking moiety comprises the extracellular domain (ECD) of a cytokine receptor.

[0013] In some embodiments, the masking moiety is CD121. In some embodiments, the masking moiety is IL-18Rα. In some embodiments, the masking moiety is IL-18Rβ. In some embodiments, the masking moiety is CD25. In some embodiments, the masking moiety is CD132. In some embodiments, the masking moiety is CD124. In some embodiments, the masking moiety is CD213a13. In some embodiments, the masking moiety is CD132. In some embodiments, the masking moiety is CD127. In some embodiments, the masking moiety is IL-9R. In some embodiments, the masking moiety is CD213a1. In some embodiments, the masking moiety is CD213a2. In some embodiments, the masking moiety is CD1243. In some embodiments, the masking moiety is CD132. In some embodiments, the masking moiety is IL-15Ra. In some embodiments, the masking moiety is CDw131. In some embodiments, the masking moiety is CDw125. In some embodiments, the masking moiety is CD131. In some embodiments, the masking moiety is CD116. In some embodiments, the masking moiety is CD126. In some embodiments, the masking moiety is CD130. In some embodiments, the masking moiety is IL-11Ra. In some embodiments, the masking moiety is CD114. In some embodiments, the masking moiety is CD212. In some embodiments, the masking moiety is LIFR. In some embodiments, the masking moiety is OSMR. In some embodiments, the masking moiety is IL-20Rα. In some embodiments, the masking moiety is IL-20Rβ. In some embodiments, the masking moiety is IL-14R. In some embodiments, the masking moiety is CD4. In some embodiments, the masking moiety is CDw127. In some embodiments, the masking moiety is CD118. In some embodiments, the masking moiety is CDw119. In some embodiments, the masking moiety is CD40. In some embodiments, the masking moiety is LTβR.In some embodiments, the masking moiety is CD120a. In some embodiments, the masking moiety is CD120b. In some embodiments, the masking moiety is CDw137. In some embodiments, the masking moiety is BCMA. In some embodiments, the masking moiety is TACI. In some embodiments, the masking moiety is CD27. In some embodiments, the masking moiety is CD30. In some embodiments, the masking moiety is CD95. In some embodiments, the masking moiety is GITR. In some embodiments, the masking moiety is LTbR. In some embodiments, the masking moiety is HVEM. In some embodiments, the masking moiety 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 CDl 15. In some embodiments, the masking moiety is CDwl36.

[0014] In some embodiments, the masking moiety is CD 122. In some embodiments, the CD122 is an engineered CD122 polypeptide or fragment thereof that contains one or more mutations relative to the wild-type CD122 amino acid sequence.

[0015] 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 a 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 a S195C mutation. In some embodiments, the engineered CD122 comprises a R204C mutation.

[0016] In some embodiments, the engineered CD122 comprises a C122S mutation and a C168S mutation. In some embodiments, the engineered CD122 comprises a C122A mutation and a N123C mutation.

[0017] In some embodiments, the engineered CD122 comprises a C122V ​​mutation and a C168V mutation. In some embodiments, the engineered CD122 comprises a C122A mutation and a C168V mutation. In some embodiments, the engineered CD122 comprises a C122V ​​mutation and a C168A mutation. In some embodiments, the engineered CD122 comprises a C122V ​​mutation and an N123C mutation. In some embodiments, the engineered CD122 comprises a C122A mutation and a C168A mutation. In some embodiments, the engineered CD122 comprises a V117C mutation, an N123Q mutation, and a C168A mutation. In some embodiments, the engineered CD122 comprises an N123Q mutation, a C168A mutation, and an L169C mutation. In some embodiments, the engineered CD122 comprises an L106C mutation, a C122A mutation, a C168A mutation, and an S195C mutation. In some embodiments, the engineered CD122 comprises an L106C mutation, a C122A mutation, a C168A mutation, and a V184C mutation. In some embodiments, the engineered CD122 comprises a C122A mutation, a C168A mutation, a V184C mutation, and an S195C mutation. In some embodiments, the engineered CD122 comprises a C122A mutation, a C168A mutation, a Q177C mutation, and an R204C mutation. In some embodiments, the engineered CD122 comprises an L106C mutation, a C122V ​​mutation, a C168V mutation, and an S195C mutation. In some embodiments, the engineered CD122 comprises an F8C mutation, an A94C mutation, a C122V ​​mutation, and a C168V mutation.

[0018] In some embodiments, the engineered CD122 polypeptide or fragment thereof comprises mutations at amino acids 122 and 168 relative to SEQ ID NO: 15, and the engineered CD122 has improved stability compared to wild-type CD122. In some embodiments, the engineered CD122 polypeptide or fragment thereof comprises mutations to promote disulfide bond formation. In some embodiments, the engineered CD122 polypeptide or fragment thereof comprises mutations to create salt bridges.

[0019] 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. In some embodiments, the masking moiety comprises residues 24-237 of human IL-12Rβ1. In some embodiments, the masking moiety comprises the sequence having SEQ ID NO: 126. In some embodiments, the masking moiety comprises residues 24-545 of human IL-12Rβ1. In some embodiments, the masking moiety comprises the sequence having SEQ ID NO: 127.

[0020] 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 24-212 of human IL-12Rβ2. In some embodiments, the masking moiety comprises the sequence having SEQ ID NO: 128. In some embodiments, the masking moiety comprises residues 24-222 of human IL-12Rβ2. In some embodiments, the masking moiety comprises the sequence having SEQ ID NO: 129. In some embodiments, the masking moiety comprises residues 24-227 of human IL-12Rβ2. In some embodiments, the masking moiety comprises the sequence having SEQ ID NO: 133. In some embodiments, the masking moiety comprises residues 24-319 of human IL-12Rβ2. In some embodiments, the masking moiety comprises the sequence having SEQ ID NO: 130.

[0021] In some embodiments, the masking portion comprises at least one amino acid modification compared to the sequence of SEQ ID NO: 130. In some embodiments, the masking portion comprises at least one amino acid modification compared to the sequence of SEQ ID NO: 130, optionally, the modification is a cysteine ​​substitution mutation. In some embodiments, the masking portion comprises SEQ ID NO: 131. In some embodiments, the masking portion comprises residues 24-622 of human IL-12Rβ2. In some embodiments, the masking portion comprises the sequence having SEQ ID NO: 132.

[0022] 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 is 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.

[0023] In some embodiments, the IL-2 is an IL-2 cytokine or functional fragment thereof that is modified relative to the sequence of mature IL-2 having SEQ ID NO: 13. In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises the modification R38A relative to the sequence of mature IL-2 having SEQ ID NO: 13. In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises the modification F42A relative to the sequence of mature IL-2 having SEQ ID NO: 13. In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises the modification Y45A relative to the sequence of mature IL-2 having SEQ ID NO: 13. In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises the modification E62A relative to the sequence of mature IL-2 having SEQ ID NO: 13. In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises the 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 functional fragment thereof comprises the modification C125A relative to the sequence of mature IL-2 having SEQ ID NO:13.

[0024] In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises the 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 functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 14.

[0025] In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises a modification that reduces or eliminates binding to CD25.

[0026] In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises a modification that enhances binding to CD25.

[0027] In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises a modification that reduces or eliminates binding to CD122.

[0028] In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises a modification that enhances binding to CD122.

[0029] 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 compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at position D22 compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at position E46 compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at position E53 compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at position N71 compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at position N79 compared to the amino acid sequence of SEQ ID NO:93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at position N112 compared to the amino acid sequence of SEQ ID NO:93.

[0030] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22, E46, or E53 compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71 and N79 compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71 and N112 compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N79 and N112 compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22, E46, E53, N71, N79, or N112 compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO:94.

[0031] In some embodiments, the IL-12 polypeptide or functional fragment thereof comprises an IL-12p40 polypeptide or functional fragment thereof covalently linked to an IL-12p35 polypeptide or functional fragment thereof. In some embodiments, the IL-12p40-IL-12p35 linker is between 2 and 40 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is between 4 and 30 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is between 5 and 20 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is between 7 and 15 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is between 10 and 12 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 2 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 2 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 2 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 2 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 2 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 2 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 3 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 4 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 5 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 6 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 7 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 8 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 9 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 10 amino acids in length.In some embodiments, the IL-12p40-IL-12p35 linker is 11 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 12 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 13 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 14 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 15 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 16 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 17 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 18 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 19 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 20 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 21 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 22 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 23 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 24 amino acids in length. In some embodiments, the IL-12p40-IL-12p35 linker is 25 amino acids in length.

[0032] 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 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 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 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 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 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 compared to the amino acid sequence of SEQ ID NO: 111.

[0033] In some embodiments, the IL-12p40 polypeptide comprises at least one amino acid modification to the GAG ​​binding domain (KSKREKKDRV) compared to the amino acid sequence of SEQ ID NO:111.

[0034] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 115. In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 116.

[0035] In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having one or more cysteine ​​substitution mutations 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.

[0036] 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 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 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 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 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 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 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 compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence that has at least eight amino acid modifications compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence that has at least nine amino acid modifications compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence that has at least 10 amino acid modifications compared to the amino acid sequence of SEQ ID NO: 112.

[0037] In some embodiments, an IL-12p35 polypeptide comprises an amino acid sequence that has one amino acid modification compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, an IL-12p35 polypeptide comprises an amino acid sequence that has two amino acid modifications compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, an IL-12p35 polypeptide comprises an amino acid sequence that has three amino acid modifications compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, an IL-12p35 polypeptide comprises an amino acid sequence that has four amino acid modifications compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, an IL-12p35 polypeptide comprises an amino acid sequence that has five amino acid modifications compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, an IL-12p35 polypeptide comprises an amino acid sequence that has six amino acid modifications compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, an IL-12p35 polypeptide comprises an amino acid sequence that has seven amino acid modifications compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, an IL-12p35 polypeptide comprises an amino acid sequence that has eight amino acid modifications compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence that has nine amino acid modifications compared to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence that has ten amino acid modifications compared to the amino acid sequence of SEQ ID NO: 112. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLT FSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREK KDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 150) IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLT FSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTE RVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 151) IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLT FSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTE GRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 152) IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLT FSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKSKREK KDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 153) IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLT FSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKDNTE GRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 154)

[0038] In some embodiments, the IL-12 cytokine or functional fragment thereof comprises SEQ ID NO: 150. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises SEQ ID NO: 151. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises SEQ ID NO: 152. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises SEQ ID NO: 153. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises SEQ ID NO: 154.

[0039] In some embodiments, 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.

[0040] 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).

[0041] In some embodiments, the cleavable linker comprises between 6 and 60 amino acid residues. In some embodiments, the cleavable linker comprises between 8 and 50 amino acid residues. In some embodiments, the cleavable linker comprises between 8 and 30 amino acid residues. In some embodiments, the cleavable linker comprises between 8 and 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.

[0042] In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide. In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide flanked on its N-terminus by a spacer domain. In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide flanked on both sides by a spacer domain. In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide flanked on both sides by a spacer domain. In some embodiments, the spacer domain is between 2 and 20 amino acids in length. In some embodiments, the spacer domain is between 3 and 10 amino acids in length. In some embodiments, the spacer domain is between 3 and 6 amino acids in length. In some embodiments, the spacer domain is 2 amino acids in length. In some embodiments, the spacer domain is 3 amino acids in length. In some embodiments, the spacer domain is 4 amino acids in length. In some embodiments, the spacer domain is 5 amino acids in length. In some embodiments, the spacer domain is 6 amino acids in length. In some embodiments, the spacer domain is 7 amino acids in length. In some embodiments, the spacer domain is 8 amino acids in length. In some embodiments, the spacer domain is 9 amino acids in length. In some embodiments, the spacer domain is 10 amino acids in length. In some embodiments, the spacer domain is 11 amino acids in length. In some embodiments, the spacer domain is 12 amino acids in length. In some embodiments, the spacer domain is 13 amino acids in length. In some embodiments, the spacer domain is 14 amino acids in length. In some embodiments, the spacer domain is 15 amino acids in length.

[0043] 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 and second antigen binding domains specifically bind to the same target. In some embodiments, the first, second, and third antigen binding domains specifically bind to the same target. In some embodiments, the first, second, third, and fourth antigen binding domains specifically bind to the same target. In some embodiments, the first and second antigen binding domains specifically bind to different targets. In some embodiments, the first, second, and third antigen binding domains each specifically bind to different targets. In some embodiments, the first, second, third, and fourth antigen binding domains each specifically bind to different targets.

[0044] In some embodiments, the first and second antigen-binding domains comprise different amino acid sequences. In some embodiments, the first and second antigen-binding domains comprise the same amino acid sequence. In some embodiments, the first, second, and third antigen-binding domains comprise different amino acid sequences. In some embodiments, the first, second, and third antigen-binding domains comprise the same amino acid sequence. In some embodiments, the first, second, third, and fourth antigen-binding domains comprise different amino acid sequences. In some embodiments, the first, second, third, and fourth antigen-binding domains comprise the same amino acid sequence.

[0045] In some embodiments, the first Fc polypeptide comprises a first IgG1 Fc domain or fragment thereof. In some embodiments, the first Fc polypeptide comprises a first IgG2 Fc domain or fragment thereof. In some embodiments, the first Fc polypeptide comprises a first IgG3 Fc domain or fragment thereof. In some embodiments, the first Fc polypeptide comprises a first IgG4 Fc domain or fragment thereof. In some embodiments, the second Fc polypeptide comprises a second IgG1 Fc domain or fragment thereof. In some embodiments, the second Fc polypeptide comprises a second IgG2 Fc domain or fragment thereof. In some embodiments, the second Fc polypeptide comprises a second IgG3 Fc domain or fragment thereof. In some embodiments, the second Fc polypeptide comprises a second IgG4 Fc domain or fragment thereof.

[0046] In some embodiments, the first Fc domain contains one or more modifications that promote non-covalent association of the first and second Fc polypeptides. In some embodiments, the second Fc domain contains one or more modifications that promote non-covalent association of the first and second Fc polypeptides. In some embodiments, the first and / or second Fc domain each contains one or more modifications that promote non-covalent association of the first and second Fc polypeptides. In some embodiments, the first and second Fc domain each contain one or more modifications that promote non-covalent association of the first and second Fc polypeptides.

[0047] 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 a L368A mutation. In some embodiments, the first Fc domain comprises a Y407V mutation. In some embodiments, the first Fc domain comprises a N297A mutation. In some embodiments, the first Fc domain comprises a S354C mutation. In some embodiments, the first Fc domain comprises a T366W mutation. In some embodiments, the first Fc domain comprises a 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 a L368A mutation. In some embodiments, the second Fc domain comprises a Y407V mutation. In some embodiments, the second Fc domain comprises a N297A mutation. In some embodiments, the second Fc domain comprises a S354C mutation, a T366W mutation, or an I253A mutation.

[0048] In some embodiments, the first Fc domain comprises a Y349C mutation and a T366S mutation. In some embodiments, the first Fc domain comprises a Y349C mutation, a T366S mutation, and a L368A mutation. In some embodiments, the first Fc domain comprises a Y349C mutation and a L368A mutation. In some embodiments, the first Fc domain comprises a Y349C mutation, a T366S mutation, a L368A mutation, and a Y407V mutation. In some embodiments, the first Fc domain comprises a Y349C mutation, a T366S mutation, and a Y407V mutation. In some embodiments, the first Fc domain comprises a T366S mutation, a L368A mutation, and a Y407V mutation. In some embodiments, the first Fc domain comprises a Y349C mutation, a L368A mutation, and a Y407V mutation. In some embodiments, the first Fc domain comprises a Y349C mutation, a T366S mutation, an L368A mutation, a Y407V mutation, and an N297A mutation. In some embodiments, the first Fc domain comprises a S354C mutation and a T366W mutation. In some embodiments, the first Fc domain comprises a S354C mutation and an N297A mutation. In some embodiments, the first Fc domain comprises a T366W mutation and an N297A mutation. In some embodiments, the first Fc domain comprises a S354C mutation, a T366W mutation, and an N297A mutation. In some embodiments, the second Fc domain comprises a Y349C mutation and a T366S mutation. In some embodiments, the second Fc domain comprises a Y349C mutation, a T366S mutation, and an L368A mutation. In some embodiments, the second Fc domain comprises a Y349C mutation and an L368A mutation. In some embodiments, the second Fc domain comprises a Y349C mutation, a T366S mutation, a L368A mutation, and a Y407V mutation. In some embodiments, the second Fc domain comprises a Y349C mutation, a T366S mutation, and a Y407V mutation. In some embodiments, the second Fc domain comprises a T366S mutation, a L368A mutation, and a Y407V mutation. In some embodiments, the second Fc domain comprises a Y349C mutation, a L368A mutation, and a Y407V mutation.In some embodiments, the second Fc domain comprises a Y349C mutation, a T366S mutation, a L368A mutation, a Y407V mutation, and a N297A mutation. In some embodiments, the second Fc domain comprises a S354C mutation and a T366W mutation. In some embodiments, the second Fc domain comprises a S354C mutation and a N297A mutation. In some embodiments, the second Fc domain comprises a T366W mutation and a N297A mutation. In some embodiments, the second Fc domain comprises a S354C mutation, a T366W mutation, and a N297A mutation.

[0049] In some embodiments, the first Fc domain comprises a Y349C mutation, a T366S mutation, a L368A mutation, a Y407V mutation, and a N297A mutation, and the second Fc domain comprises a S354C mutation, a T366W mutation, and a N297A mutation. In some embodiments, the second Fc domain comprises a Y349C mutation, a T366S mutation, a L368A mutation, a Y407V mutation, and a N297A mutation, and the first Fc domain comprises a S354C mutation, a T366W mutation, and a N297A mutation.

[0050] In some embodiments, the first Fc domain comprises a Y349C mutation, a T366S mutation, a L368A mutation, a Y407V mutation, and an I253A mutation, and the second Fc domain comprises a S354C mutation, a T366W mutation, and an I253A mutation. In some embodiments, the second Fc domain comprises a Y349C mutation, a T366S mutation, a L368A mutation, a Y407V mutation, and an I253A mutation, and the first Fc domain comprises a S354C mutation, a T366W mutation, and an I253A mutation.

[0051] In some embodiments, the first Fc domain comprises a Y349C mutation, a T366S mutation, a L368A mutation, a Y407V mutation, a N297A mutation, and an I253A mutation, and the second Fc domain comprises a S354C mutation, a T366W mutation, a N297A mutation, and an I253A mutation. In some embodiments, the second Fc domain comprises a Y349C mutation, a T366S mutation, a L368A mutation, a Y407V mutation, a N297A mutation, and an I253A mutation, and the first Fc domain comprises a S354C mutation, a T366W mutation, a N297A mutation, and an I253A mutation.

[0052] In some embodiments, the first Fc polypeptide comprises a first CH3 domain comprising a modification that reduces or eliminates binding to Protein A. In some embodiments, the second Fc polypeptide comprises a second CH3 domain comprising 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.

[0053] In some embodiments, the first Fc polypeptide comprises a first CH3 domain comprising 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 comprising 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.

[0054] 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.

[0055] In some embodiments, the first CH3 domain is a human IgG1 sequence comprising an alteration at position H435 according to the Kabat numbering. In some embodiments, the first CH3 domain is a human IgG1 sequence comprising an alteration at position Y436 according to the Kabat numbering. In some embodiments, the first CH3 domain is a human IgG1 sequence comprising alterations at positions H435 and Y436 according to the Kabat numbering. In some embodiments, the first CH3 domain is a human IgG1 sequence comprising an H435R alteration according to the Kabat numbering. In some embodiments, the first CH3 domain is a human IgG1 sequence comprising a Y436F alteration. In some embodiments, the first CH3 domain is a human IgG1 sequence comprising the alterations H435R and Y436F according to the Kabat numbering.

[0056] In some embodiments, the first CH3 domain is a human IgG2 sequence comprising an alteration at position H435 according to the Kabat numbering. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising an alteration at position Y436 according to the Kabat numbering. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising alterations at positions H435 and Y436 according to the Kabat numbering. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising an H435R alteration according to the Kabat numbering. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising a Y436F alteration. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising the alterations H435R and Y436F according to the Kabat numbering.

[0057] In some embodiments, the first CH3 domain is a human IgG3 sequence comprising an alteration at position H435 according to the Kabat numbering. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising an alteration at position Y436 according to the Kabat numbering. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising alterations at positions H435 and Y436 according to the Kabat numbering. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising an H435R alteration according to the Kabat numbering. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising a Y436F alteration. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising the alterations H435R and Y436F according to the Kabat numbering.

[0058] In some embodiments, the second CH3 domain is a human IgG1 sequence comprising an alteration at position H435 according to the Kabat numbering. In some embodiments, the second CH3 domain is a human IgG1 sequence comprising an alteration at position Y436 according to the Kabat numbering. In some embodiments, the second CH3 domain is a human IgG1 sequence comprising alterations at positions H435 and Y436 according to the Kabat numbering. In some embodiments, the second CH3 domain is a human IgG1 sequence comprising an H435R alteration according to the Kabat numbering. In some embodiments, the second CH3 domain is a human IgG1 sequence comprising a Y436F alteration. In some embodiments, the second CH3 domain is a human IgG1 sequence comprising the alterations H435R and Y436F according to the Kabat numbering.

[0059] In some embodiments, the second CH3 domain is a human IgG2 sequence comprising an alteration at position H435 according to the Kabat numbering. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising an alteration at position Y436 according to the Kabat numbering. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising alterations at positions H435 and Y436 according to the Kabat numbering. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising an H435R alteration according to the Kabat numbering. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising a Y436F alteration. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising the alterations H435R and Y436F according to the Kabat numbering.

[0060] In some embodiments, the second CH3 domain is a human IgG3 sequence comprising an alteration at position H435 according to the Kabat numbering. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising an alteration at position Y436 according to the Kabat numbering. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising alterations at positions H435 and Y436 according to the Kabat numbering. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising an H435R alteration according to the Kabat numbering. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising a Y436F alteration. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising the alterations H435R and Y436F according to the Kabat numbering.

[0061] 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.

[0062] In some embodiments, the first CH3 domain comprises a human IgG3 sequence. In some embodiments, the second CH3 domain comprises a human IgG3 sequence.

[0063] 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.

[0064] 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.

[0065] In some embodiments, the targeting moiety is linked to the Fc domain via one or both of the first and second Fc polypeptides. 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 and second Fc polypeptides. In some embodiments, the targeting moiety is linked to the N-terminus of the Fc domain via one or both of the first and second Fc polypeptides. 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 and second Fc polypeptides. In some embodiments, the targeting moiety is linked to the C-terminus of the Fc domain via one or both of the first and second Fc polypeptides. 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 a second Fc polypeptide, hi some embodiments, the targeting moiety is linked to the C-terminus of the Fc domain via both the first and second Fc polypeptides.

[0066] In some embodiments, the targeting moiety comprises a variable heavy chain region (VH) and is linked to the Fc domain via one or both of a first and a second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain region (VH) and is linked to the Fc domain via a first Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain region (VH) and is linked to the Fc domain via a second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain region (VH) and is linked to the Fc domain via both a first and a second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain region (VH) and is linked to the N-terminus of the Fc domain via one or both of a first and a second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain region (VH) and is linked to the N-terminus of the Fc domain via a first Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain region (VH) and is linked to the N-terminus of the Fc domain via a second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain region (VH) and is linked to the N-terminus of the Fc domain via both the first and second Fc polypeptides. In some embodiments, the targeting moiety comprises a variable heavy chain region (VH) and is linked to the C-terminus of the Fc domain via one or both of the first and second Fc polypeptides. In some embodiments, the targeting moiety comprises a variable heavy chain region (VH) and is linked to the C-terminus of the Fc domain via the first Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain region (VH) and is linked to the C-terminus of the Fc domain via the second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy chain region (VH) and is linked to the C-terminus of the Fc domain via both the first and second Fc polypeptides.

[0067] In some embodiments, the targeting moiety comprises a Fab having an HCDR1 (GYTFTNYY) of SEQ ID NO: 4, an HCDR2 (INPSNGGT) of SEQ ID NO: 5, an HCDR3 (ARRDYRFDMGFDY) of SEQ ID NO: 6, an LCDR1 (KGVSTSGYSY) of SEQ ID NO: 9, an LCDR2 (LAS) of SEQ ID NO: 10, and an LCDR3 (QHSRDLPLT) of SEQ ID NO: 11.

[0068] 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.

[0069] In some embodiments, the cytokine is linked to the first Fc polypeptide via a non-cleavable linker. In some embodiments, the N-terminus of the cytokine is linked to the C-terminus of the first Fc polypeptide via a non-cleavable linker. In some embodiments, the C-terminus of the cytokine is linked to the N-terminus of the first Fc polypeptide via a non-cleavable 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.

[0070] In some embodiments, the masking moiety is linked to the second Fc polypeptide via a non-cleavable linker. In some embodiments, the N-terminus of the masking moiety is linked to the C-terminus of the second Fc polypeptide via a non-cleavable linker. In some embodiments, the C-terminal masking moiety is linked to the N-terminus of the second Fc polypeptide via a non-cleavable linker. In some embodiments, the masking moiety is linked to the second Fc polypeptide via a cleavable linker. In some embodiments, the N-terminus of the 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.

[0071] 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 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.

[0072] In some embodiments, the targeting moiety comprises an agent that specifically binds to the target. In some embodiments, the targeting moiety comprises a peptide that specifically binds to the target. In some embodiments, the targeting moiety comprises a polypeptide that specifically binds to the target. In some embodiments, the targeting moiety comprises a polypeptide that specifically binds to the target. In some embodiments, the targeting moiety comprises a protein that specifically binds to the target. In some embodiments, the targeting moiety comprises a nucleic acid that specifically binds to the target.

[0073] In some embodiments, the targeting moiety comprises a Fab, a single-chain Fv (scFv), a single-domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibody (bsFab), a single-domain antibody-binding Fab (s-Fab), an antibody, or a combination thereof. In some embodiments, the targeting moiety comprises a Fab. In some embodiments, the targeting moiety comprises a single-chain Fv (scFv). In some embodiments, the targeting moiety comprises a single-domain antibody (VHH). In some embodiments, the targeting moiety comprises one or more CDRs. In some embodiments, the targeting moiety comprises a variable heavy chain (VH). In some embodiments, the targeting moiety comprises a variable light chain (VL). In some embodiments, the targeting moiety comprises a Fab-like bispecific antibody (bsFab). In some embodiments, the targeting moiety comprises a single-domain antibody-binding Fab (s-Fab). In some embodiments, the targeting moiety comprises an antibody or fragment thereof.

[0074] In some embodiments, the targeting moiety comprises an antigen-binding moiety, and the antigen is expressed on an immune cell. In some embodiments, the targeting moiety specifically binds PD-1. In some embodiments, the targeting moiety specifically binds PD-L1. In some embodiments, the targeting moiety specifically binds PD-L2. In some embodiments, the targeting moiety specifically binds CTLA-4. In some embodiments, the targeting moiety specifically binds TIGIT. In some embodiments, the targeting moiety specifically binds TIM-3. In some embodiments, the targeting moiety specifically binds LAG-3. In some embodiments, the targeting moiety specifically binds CD25. In some embodiments, the targeting moiety specifically binds CD16a. In some embodiments, the targeting moiety specifically binds CD16b. In some embodiments, the targeting moiety specifically binds NKG2D. In some embodiments, the targeting moiety specifically binds NKP44. In some embodiments, the targeting moiety specifically binds NKP3. In some embodiments, the targeting moiety specifically binds CD19. In some embodiments, the targeting moiety specifically binds CD20. In some embodiments, the targeting moiety specifically binds CD30. In some embodiments, the targeting moiety specifically binds CD38. In some embodiments, the targeting moiety specifically binds BMCA. In some embodiments, the targeting moiety specifically binds HER2. In some embodiments, the targeting moiety specifically binds HER3. In some embodiments, the targeting moiety specifically binds DLL3. In some embodiments, the targeting moiety specifically binds DLL4. In some embodiments, the targeting moiety specifically binds EGFR. In some embodiments, the targeting moiety specifically binds GPC3. In some embodiments, the targeting moiety specifically binds c-MET. In some embodiments, the targeting moiety specifically binds VEGF-R1. In some embodiments, the targeting moiety specifically binds VEGF-R2. In some embodiments, the targeting moiety specifically binds OX40. In some embodiments, the targeting moiety specifically binds DR5. In some embodiments, the targeting moiety specifically binds ICOS.In some embodiments, the targeting moiety specifically binds GITR. In some embodiments, the targeting moiety specifically binds CD73. In some embodiments, the targeting moiety specifically binds CD39. In some embodiments, the targeting moiety specifically binds CD25. In some embodiments, the targeting moiety specifically binds CD16a. In some embodiments, the targeting moiety specifically binds CD8. In some embodiments, the targeting moiety specifically binds KLRC1. In some embodiments, the targeting moiety specifically binds KLRD1. In some embodiments, the targeting moiety specifically binds KLRB1. In some embodiments, the targeting moiety specifically binds CD40. In some embodiments, the targeting moiety specifically binds CD137. In some embodiments, the targeting moiety specifically binds CD28.

[0075] In some embodiments, the cleavable linker has an in vitro cleavage efficiency that yields about 1-99% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that yields about 5-80% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that yields about 8-60% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that yields about 10-40% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that yields about 10-20% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that yields at least 3% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that yields at least 5% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that yields at least 8% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that yields at least 10% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that results in an active cytokine of at least 12%. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that results in an active cytokine of at least 15%. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that results in an active cytokine of at least 20%. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that results in an active cytokine of at least 25%. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that results in an active cytokine of at least 30%. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that results in an active cytokine of at least 35%. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that results in an active cytokine of at least 40%. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that results in an active cytokine of at least 45%. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that results in an active cytokine of at least 50%.In some embodiments, the cleavable linker has an in vitro cleavage efficiency that results in an active cytokine of at least 60%. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that results in an active cytokine of at least 70%. In some embodiments, the cleavable linker has an in vitro cleavage efficiency that results in an active cytokine of at least 80%.

[0076] In one aspect, the present invention provides a targeted cytokine comprising, inter alia, a targeting moiety, a cytokine or fragment thereof, a masking moiety, and an Fc domain comprising a first Fc polypeptide linked to the cytokine or fragment thereof via a first linker and a second Fc polypeptide linked to the masking moiety via a second linker, wherein the masking moiety binds to the cytokine or fragment thereof, the first or the second linker is a cleavable linker such that the masking moiety releases the cytokine or fragment thereof upon cleavage, the cleavable linker comprising MPYDLYHP (SEQ ID NO: 34) or VPLSLYSG (SEQ ID NO: 42), and the targeting moiety is linked to the Fc domain via one or both of the first and second Fc polypeptides.

[0077] In one aspect, the invention provides a targeted cytokine comprising, inter alia, a targeting moiety, a cytokine or fragment thereof, a masking moiety, and an Fc domain comprising a first Fc polypeptide linked to the cytokine or fragment thereof via a first linker and a second Fc polypeptide linked to the masking moiety via a second linker, wherein the masking moiety binds to the cytokine or fragment thereof, the first or second linker is a tumor-specific cleavable linker such that the masking moiety releases the cytokine or fragment thereof upon cleavage by a tumor-specific protease, the tumor-specific cleavable linker comprising between 8 and 50 amino acid residues, and the targeting moiety is linked to the Fc domain via one or both of the first and second Fc polypeptides.

[0078] In one aspect, the present invention provides a targeted cytokine comprising, inter alia, a targeting moiety, a cytokine or fragment thereof, a masking moiety, and an Fc domain comprising a first Fc polypeptide linked to the cytokine or fragment thereof via a first linker and a second Fc polypeptide linked to the masking moiety via a second linker, wherein the masking moiety binds to the cytokine or fragment thereof, the first or the second linker is a cleavable linker such that the masking moiety, upon cleavage, releases the cytokine or fragment thereof to produce an active cytokine, the cleavable linker having an in vitro cleavage efficiency of at least 10% to produce an active cytokine, and the targeting moiety is linked to the Fc domain via one or both of the first and second Fc polypeptides.

[0079]

[0010] In one aspect, the invention provides a targeted cytokine comprising, inter alia, a targeting moiety, a cytokine or fragment thereof, a masking moiety comprising 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 an Fc domain comprising a first Fc polypeptide linked to the cytokine or fragment thereof via a first linker and a second Fc polypeptide linked to the masking moiety via a second linker, wherein the masking moiety binds to the cytokine or fragment thereof, and the first or second linker is a cleavable linker such that the masking moiety releases the cytokine or fragment thereof upon cleavage, and the targeting moiety is linked to the Fc domain via one or both of the first and second Fc polypeptides.

[0080] In one aspect, the invention provides a targeted cytokine comprising, inter alia, an Fc domain comprising a first Fc polypeptide comprising a CH3 domain comprising a targeting moiety, a masking moiety, the cytokine or a fragment thereof, and a modification that reduces or eliminates binding to Protein A, and a second Fc polypeptide comprising a CH3 domain that binds to Protein A.

[0081] In one aspect, the present invention provides a targeted cytokine comprising, inter alia, a targeting moiety, a cytokine or fragment thereof, a masking moiety, and an Fc domain comprising a first Fc polypeptide linked to the cytokine or fragment thereof via a first linker and a second Fc polypeptide linked to the masking moiety via a second linker, wherein the masking moiety binds to the cytokine or fragment thereof, the first or the second linker is a cleavable linker such that the masking moiety releases the cytokine or fragment thereof upon cleavage, the cleavable linker comprising MPYDLYHP (SEQ ID NO: 34) or VPLSLYSG (SEQ ID NO: 42), and the targeting moiety is linked to the Fc domain via one or both of the first and second Fc polypeptides.

[0082] In one aspect, the invention provides a targeted cytokine comprising a targeting moiety that specifically binds to PD-1, comprising, inter alia, HCDR1 (GYTFTNYY) of SEQ ID NO: 4, HCDR2 (INPSNGGT) of SEQ ID NO: 5, HCDR3 (ARRDYRFDMGFDY) of SEQ ID NO: 6, LCDR1 (KGVSTSGYSY) of SEQ ID NO: 9, LCDR2 (LAS) of SEQ ID NO: 10, LCDR3 (QHSRDLPLT) of SEQ ID NO: 11, a cytokine or fragment thereof, and an Fc domain.

[0083] In one aspect, the present invention provides, inter alia, a targeted IL-2 cytokine comprising SEQ ID NO:87.

[0084] In some embodiments, the targeted IL-2 cytokine comprises SEQ ID NO:88.

[0085] In some embodiments, the targeted IL-2 cytokine comprises SEQ ID NO:89.

[0086] In some embodiments, the targeted IL-2 cytokine comprises SEQ ID NO:90.

[0087] In some embodiments, the targeted IL-2 cytokine comprises SEQ ID NO:91.

[0088] In one aspect, the present invention provides, inter alia, a targeted IL-2 cytokine comprising SEQ ID NO:87 and SEQ ID NO:88, and a light chain comprising SEQ ID NO:8.

[0089] In one aspect, the present invention provides, inter alia, a targeted IL-2 cytokine comprising SEQ ID NO:87 and SEQ ID NO:89, and a light chain comprising SEQ ID NO:8.

[0090] In one aspect, the present invention provides, inter alia, a targeted IL-2 cytokine comprising SEQ ID NO:87 and SEQ ID NO:90, and a light chain comprising SEQ ID NO:8.

[0091] In one aspect, the present invention provides, inter alia, a targeted IL-2 cytokine comprising SEQ ID NO:87 and SEQ ID NO:91, and a light chain comprising SEQ ID NO:8.

[0092] In one aspect, the invention provides, inter alia, nucleic acids encoding the targeted cytokines of the invention. In one aspect, the invention provides, inter alia, vectors comprising nucleic acids encoding the targeted cytokines of the invention. In one aspect, the invention provides, inter alia, host cells comprising nucleic acids encoding the targeted cytokines of the invention.

[0093] In one aspect, the invention provides, inter alia, a method for producing a targeted cytokine comprising culturing a host cell comprising nucleic acid encoding a targeted cytokine of the invention under conditions for producing the targeted cytokine.

[0094] In one aspect, the present invention provides, inter alia, compositions comprising the targeted cytokines of the present invention.

[0095] In one aspect, the present invention provides, inter alia, a pharmaceutical composition comprising a targeted cytokine of the present invention and a pharmaceutically acceptable carrier.

[0096] In one aspect, the present invention provides, inter alia, kits comprising the targeted cytokines of the present invention.

[0097] In one aspect, the present invention provides, inter alia, a method of treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of a targeted cytokine of the present invention.

[0098] In one aspect, the present invention provides, inter alia, a method of treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of a composition comprising a targeted cytokine of the present invention.

[0099] In one aspect, the present invention provides, inter alia, a method of treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a targeted cytokine of the present invention.

[0100] In one aspect, the present invention provides, inter alia, a method of treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of a targeted cytokine of the present invention.

[0101] In one aspect, the present invention provides, inter alia, a method of treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of a composition comprising a targeted cytokine of the present invention.

[0102] In one aspect, the present invention provides, inter alia, a method of treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a targeted cytokine of the present invention. [Brief explanation of the drawings]

[0103] The drawings are for purposes of illustration only and not limitation.

[0104] [Figure 1] Figure 1 shows an exemplary schematic diagram of the Fc domains used in the targeted IL-2 cytokines of the present invention. The first Fc polypeptide comprises the CH2 and CH3 domains derived from IgG1. The second chain of the "chimeric Fc" comprises the CH2 domain derived from IgG1 and the CH3 domain derived from IgG3. The second chain of the "CH3 mutant" comprises the CH2 and CH3 domains derived from IgG1, with "RF mutations" (H435R and Y436F) in the CH3 domain.

[0105] [Figure 2A-1] Figure 2A shows a series of exemplary SDS-PAGE analyses of purified masked IL-2 cytokines containing either native Fc, CH3 mutant, or chimeric Fc domains. Each construct was purified by Protein A chromatography and size-exclusion chromatography (SEC) at different ratios of the first and second Fc polypeptides. Figure 2B shows a series of exemplary EC50 graphs of purified masked IL-2 cytokines containing either native Fc, CH3 mutant, or chimeric Fc domains. EC50 values ​​were determined with a cell-based reporter assay (HEK Blue IL-2 assay) that measures the percent of calculated active cytokine. [Figure 2A-2] Same as above. [Figure 2A-3] Same as above. [Figure 2B-1] Same as above. [Figure 2B-2] Same as above. [Figure 2B-3] Same as above.

[0106] [Figure 3]Figure 3 shows an exemplary schematic diagram of targeted IL-2 cytokine (TC) molecules of the present invention and their purity as determined by SEC, non-reduced capillary electrophoresis SDS (CE-SDS), and reduced CE-SDS. All TC molecules tested in this experiment contain a bivalent Fab that binds to PD-1, a first Fc polypeptide with "hole mutations" (Y349C, T366S, L368A, and Y407V), and a second Fc polypeptide with "knob mutations" (S354C and T366W) fused to an IL-2 cytokine. TC1 is "unmasked" and lacks a masking moiety that binds to IL-2. TC2, TC3, and TC4 contain CD122 as a masking moiety fused to the first Fc polypeptide. TC3 and TC4 contain cleavable linkers between the first Fc polypeptide and the masking moiety, including the MPYDLYHP (SEQ ID NO: 34) and VPLSLYSG (SEQ ID NO: 42) cleavage peptides, respectively.

[0107] [Figure 4A] Figure 4A shows an exemplary schematic diagram of different targeted IL-2 cytokines of the present invention. The targeted cytokines can be in a bivalent targeting format (format A) containing a bivalent Fab that binds to a specific antigen, or in a monovalent targeting format (formats B1, B2, and C) containing a monovalent Fab. Figure 4B is a series of exemplary EC50 graphs for the purified targeted IL-2 cytokines shown in Figure 4A. EC50 values ​​were determined in a cell-based reporter assay (HEK Blue IL-2 assay) that measures the % of calculated active cytokine. [Figure 4B] Same as above.

[0108] [Figure 5A]Figure 5A is a series of exemplary binding graphs showing the binding of PD-1 multifunctional constructs on PD-1-expressing Raji cells and on CD8+ T cells within CD3 / CD28-activated PBMCs. Figure 5B is a series of exemplary graphs showing the surface residence time of PD-1 multifunctional constructs 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-primed human PBMCs in response to stimulation with up to 1000 nM of the indicated constructs. [Figure 5B] Same as above. [Figure 5C] Same as above.

[0109] [Figure 6A-1] Figure 6A is a series of exemplary graphs showing the change in tumor weight and tumor volume over time after treatment with a targeted IL-2 cytokine of the present invention. Figure 6B is a series of exemplary graphs showing the change in CD8+ T cell quantity and CD8 proliferation in the tumor, spleen, and peripheral blood after treatment with a targeted IL-2 cytokine 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 a targeted IL-2 cytokine. Figure 6D is a series of exemplary graphs showing the change in the number of antigen-specific CD8+ T cells after treatment with a targeted IL-2 cytokine. Figure 6E is a series of exemplary graphs showing the potential increase in drug accumulation in the tumor after treatment with a targeted IL-2 cytokine. Figure 6F is a series of exemplary graphs showing the half-life, maximum plasma concentration, AUC, volume of distribution, and clearance data for a targeted IL-2 cytokine after treatment. [Figure 6A-2] Same as above. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. [Figure 6F] Same as above.

[0110] [Figure 7A] Figure 7A is an exemplary table showing binding affinity data and statistics for the targeted IL-2 cytokines shown in Figure 3. Pembrolizumab (anti-PD-1 antibody, "Pembro") and anti-human PD-L1-hFc fusion were used as controls. Figure 7B is a series of exemplary EC50 graphs of purified targeted IL-2 cytokines when performed in a cell-based reporter assay (HEK Blue IL-2 assay) to determine the calculated % active cytokine. Each graph shows binding curves in the absence and presence of various matrix metalloproteinases (MMPs). Recombinant human IL-2 (rhIL-2) and TCO (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-primed human PBMCs in response to stimulation with the indicated constructs up to 3000 nM. Figure 7D is an exemplary graph and table showing STAT5 phosphorylation on resting PBMCs using uncleaved targeted cytokines, and Figure 7E is an exemplary graph and table showing STAT5 phosphorylation on resting PBMCs using cleaved targeted cytokines. [Figure 7B-1] Same as above. [Figure 7B-2] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 7E] Same as above.

[0111] [Figure 8A] Figure 8A is a series of bar graphs showing cleavage of targeted IL-2 cytokine in tumors in vivo. Figure 8B shows cleavage of targeted IL-2 cytokine in tumors compared to plasma, demonstrating tumor-specific cleavage in vivo. [Figure 8B-1] Same as above. [Figure 8B-2] Same as above. [Figure 8B-3] Same as above.

[0112] [Figure 9A] Figure 9A is an exemplary graph showing tumor volume after treatment with targeted IL-2 cytokines 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 is a series of exemplary graphs showing the percentage of CD8+ T cells in peripheral blood and spleen cells compared to tumor cells, demonstrating that targeted cytokines of the present invention increased the percentage of CD8+ T cells in the tumor microenvironment. Figure 9C shows the % of CD8+ T cells after treatment with targeted IL-2 cytokines. Figure 9D is a series of exemplary graphs showing the % change in body weight and pulmonary edema after treatment with targeted cytokines, demonstrating minimal toxicity caused by targeted IL-2 cytokines of the present invention. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above.

[0113] [Figure 10A] Figure 10A is an exemplary table showing engineered cleavable linker sequences incorporated into targeted cytokines of the present invention, as well as the yield and purity after purification using Protein A chromatography and size-exclusion chromatography (SEC). Figure 10B is a series of exemplary tables showing the cleavage efficiency of target molecules with various engineered cleavable linker sequences by different matrix metalloproteinases (MMPs). Figure 10C is an exemplary graph showing the cleavage frequency of targeted cytokines by secreted proteases from human tumor tissue-conditioned medium. Figure 10D is an exemplary graph showing the average percentage of cleaved targeted cytokines in human plasma from lung tumor patients (n=8) as determined by Western blot. [Figure 10B-1] Same as above. [Figure 10B-2] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above.

[0114] [Figure 11A] Figure 11A is an exemplary graph and table showing EC50 values ​​as determined in a cell-based reporter assay (HEK Blue IL-2 assay) measuring the calculated % active cytokine for targeted cytokines containing an MPY sequence as a cleavable substrate. Figure 11B is an exemplary graph and table showing EC50 values ​​as determined in a cell-based reporter assay (HEK Blue IL-2 assay) measuring the calculated % active cytokine for targeted cytokines containing a VPL sequence as a cleavable substrate. Figure 11C is an exemplary graph and table showing EC50 values ​​for targeted cytokines containing an MPY sequence as a cleavable substrate after complete cleavage by MMP proteases. Figure 11D is an exemplary graph and table showing EC50 values ​​for targeted cytokines containing a VPL sequence as a cleavable substrate after complete cleavage by MMP proteases. Figure 11E is an exemplary graph and table showing EC50 values ​​for targeted cytokines containing an MPY sequence as a cleavable substrate after minimal cleavage by MMP proteases. FIG. 11F is a series of exemplary radar charts showing the cleavage efficiency of various targeted cytokines by different MMP proteases. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 11E] Same as above. [Figure 11F-1] Same as above. [Figure 11F-2] Same as above.

[0115] [Figure 12] FIG. 12 is an exemplary graph showing frequency anti-hIgG+ binding of PD-1 targeted cytokines on CD8+ T cells within CD3-activated PBMCs.

[0116] [Figure 13A]Figure 13A is a series of exemplary graphs showing the mean tumor volume and mean body weight change curves of unmasked PD-1-targeted TC29 compared to pembrolizumab. Figure 13B is a series of exemplary graphs showing the mean tumor volume and mean body weight change curves of PD-1-targeted TC3 compared to pembrolizumab. [Figure 13B] Same as above.

[0117] [Figure 14A] Figure 14A is a series of exemplary graphs showing mean tumor volume and mean body weight change curves for various targeting cytokines and pembrolizumab. Figure 14B is a series of exemplary graphs showing the amount of peripheral, splenic, and tumor-infiltrating CD8+ T cells. Figure 14C is a series of exemplary graphs showing the amount of tumor antigen-specific CD8 T cells after administration with a targeting cytokine of the present invention. Figure 14D is a series of exemplary graphs showing construct concentrations in plasma and tumor lysates. [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above.

[0118] [Figure 15] FIG. 15 is an exemplary graph showing the results of an RGA assay of various targeted masked cytokines prior to protease cleavage.

[0119] [Figure 16A] Figures 16A-16D are a series of exemplary graphs showing the results of RGA assays of various targeted masked cytokines cleaved by MMP1 (Figure 14A), MMP7 (Figure 16B), MMP9 (Figure 16C), and MMP10 (Figure 16D). [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 16D] Same as above.

[0120] [Figure 17]FIG. 17 is an exemplary graph showing the average cleavage efficiency for each cleavage site based on kinetic analysis of various constructs.

[0121] [Figure 18A] Figures 18A-18F show exemplary ELISA results demonstrating the binding affinity of various constructs to PD-1. Exemplary constructs tested include pembrolizumab scFv in VHVL format (Figure 18A), pembrolizumab scFv in VLVH format (Figure 18B), nivolumab (nivo) scFv (Figure 18C), pembrolizumab Fab (Figure 18D), or different masking moieties (Figure 18F). Figure 18E shows exemplary Kd values ​​for each construct. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 18D] Same as above. [Figure 18E] Same as above. [Figure 18F] Same as above.

[0122] [Figure 19A] Figures 19A-19F show exemplary results of PD1 / PD-1 checkpoint blockade (fold induction) demonstrated by PD1 RGA assay. Exemplary constructs tested include pembrolizumab scFv in VHVL format (Figure 19A), pembrolizumab scFv in VLVH format (Figure 19B), nivolumab (nivo) scFv (Figure 19C), pembrolizumab Fab (Figure 19D), or different masking moieties (Figure 19F). Figure 19E shows exemplary Kd values ​​for each construct. [Figure 19B] Same as above. [Figure 19C] Same as above. [Figure 19D] Same as above. [Figure 19E] Same as above. [Figure 19F] Same as above.

[0123] [Figure 20A]Figures 20A-20E show exemplary results of on-cell binding of various constructs to PD1-expressing Raji cells. Exemplary constructs tested include pembrolizumab Fab (Figure 20A), pembrolizumab scFv in VHVL format (Figure 20B), pembrolizumab scFv in VLVH format (Figure 20C), or nivolumab (nivo) scFv (Figure 20D). Figure 20E shows exemplary EC50 values ​​for each construct. [Figure 20B] Same as above. [Figure 20C] Same as above. [Figure 20D] Same as above. [Figure 20E] Same as above.

[0124] [Figure 21A] Figures 21A-21D show 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) upon administration of various targeted cytokine constructs. [Figure 21B] Same as above. [Figure 21C] Same as above. [Figure 21D] Same as above.

[0125] [Figure 22A] 22A-B show exemplary tumor growth curves (FIG. 22A) and overall survival (FIG. 22B) of subcutaneous MC38 tumors in human hPD1 transgenic mice following treatment with exemplary masked cytokines. [Figure 22B] Same as above.

[0126] [Figure 23A]Figures 23A-23F show exemplary tumor growth curves (Figure 23A, Figure 23D), overall survival (Figure 23B, Figure 23E), and body weight change (Figure 23C, Figure 23E) of subcutaneous MC38 tumors in human hPD1 transgenic mice after treatment with exemplary masked cytokines at an administration dose of 8 mg / kg (Figure 23A-Figure 23C) or 2 mg / kg (Figure 23D-Figure 23F). [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 23D] Same as above. [Figure 23E] Same as above. [Figure 23F] Same as above.

[0127] [Figure 24A] Figures 24A-24E show exemplary tumor growth curves (Figure 24A), overall survival (Figure 24B), and body weight changes (Figure 24C) of subcutaneous MC38 tumors in human hPD1 transgenic mice after treatment with exemplary masked cytokines. Schematics of exemplary test constructs are shown in Figures 24D and 24E. [Figure 24B] Same as above. [Figure 24C] Same as above. [Figure 24D] Same as above. [Figure 24E] Same as above.

[0128] [Figure 25A] Figure 25A shows exemplary results of plasma exposure of constructs over time after administration to NHPs. Figure 25B shows changes in lymphocytes after administration of exemplary constructs to NHPs. [Figure 25B] Same as above.

[0129] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for these terms and other terms are set forth throughout the specification. Publications and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are incorporated herein by reference.

[0130] It is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary, and the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0131] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an IL-2 polypeptide" optionally includes combinations of two or more such polypeptides, and the like.

[0132] As used herein, the term "about" refers to a normal range of error for the respective value, readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are inherently directed to that value or parameter.

[0133] It will be understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0134] As used herein, the term "and / or" refers to any one of the items, any combination of the items, or all of the items with which the term is associated. For example, the phrase "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A and B or C; B and A or C; C and A or B; A alone; B alone; and C alone.

[0135] The term "antibody" includes polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab'), and Fv). The term "immunoglobulin (Ig)" is used interchangeably with "antibody" herein.

[0136] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, comprising a heavy-chain variable (VH) domain connected to a light-chain variable (VL) domain in the same polypeptide chain (VH-VL).

[0137] 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, along with an additional polypeptide called the J chain, and contain 10 antigen-binding sites, while IgA antibodies consist of two to five basic four-chain units that can combine with the J chain to form multivalent aggregates. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by a single covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the a and y chains, and four CH domains for the p and s isotypes. Each L chain has a variable domain (VL) at its 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 of the heavy chain (CHI). Certain amino acid residues are thought to form an interface between the variable domains of the light and heavy chains. The pairing of a VH and a VL together forms a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0138] Light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of their heavy chain constant domains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with heavy chains designated a, 8, e, y, and p. The y and a 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 may exist in multiple polymorphic variants, called allotypes (Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7), any of which are suitable for use in the present invention. Common allotypic variants in the human population are designated by the letters a, f, n, and z.

[0139] An "isolated" antibody is an antibody that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). In some embodiments, an isolated polypeptide is free from association with all other components from its production environment. Contaminating components of its production environment, such as those resulting from recombinantly transfected cells, are materials that would typically interfere with research, diagnostic, or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide is purified to (1) greater than 95% by weight, and in some embodiments, greater than 99% by weight, of the antibody as determined, for example, by the Lowry method; (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions with Coomassie blue or silver staining, since at least one component of the antibody's natural environment will not be present. An isolated antibody includes the antibody in situ within recombinant cells. Ordinarily, however, isolated polypeptide or antibody will be prepared by at least one purification step.

[0140] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. In some embodiments, the monoclonal antibody has a C-terminal truncation in the heavy and / or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are truncated at the C-terminus of the heavy and / or light chain. In some embodiments, the C-terminal truncation removes the C-terminal lysine from the heavy chain. In some embodiments, the monoclonal antibody has an N-terminal truncation in the heavy and / or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are truncated at the N-terminus of the heavy and / or light chain. In some embodiments, truncated forms of the monoclonal antibody can be produced by recombinant techniques. In some embodiments, the monoclonal antibody is highly specific and directed against a single antigenic site. In some embodiments, monoclonal antibodies are highly specific, being directed against multiple antigenic sites (e.g., bispecific or multispecific antibodies). The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method, recombinant DNA methods, phage display technology, and techniques for producing human or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0141] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, whole antibodies include those having heavy and light chains, including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, intact antibodies may have one or more effector functions.

[0142] An "antibody fragment" comprises a portion of an intact antibody, e.g., the antigen-binding region and / or the variable region of the intact antibody, and / or the constant region of the intact antibody. Examples of antibody fragments include the Fc region of an antibody, a portion of the Fc region, or a portion of an antibody comprising the Fc region. Examples of antigen-binding antibody fragments include domain antibodies (dAbs), Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies (see U.S. Pat. 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 Fvs (scFvs), single-domain antibodies (VHHs), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like diabody (bsFab), a single-domain antibody-binding 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, isolated from camelids, sharks, libraries, or mice that have been engineered to produce single heavy chain molecules.

[0143] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments and a remaining "Fc" fragment, a name reflecting their ability to crystallize readily. The Fab fragment consists of an entire L chain, along with the variable region domain of the H chain (VH) and the first constant domain (CHI) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of antibodies produces a single large F(ab')2 fragment, roughly corresponding to two disulfide-linked Fab fragments with different antigen-binding activities and still capable of cross-linking antigen. Fab' fragments differ from Fab fragments in that they contain a few additional residues at the carboxy terminus of the CHI domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues in the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical combinations of antibody fragments are also known. The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfide bonds. The effector functions of antibodies are determined by the sequences and glycans within the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

[0144] "Amino acid sequence identity percentage (%)" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity.Alignment for determining percent amino acid sequence identity can be achieved by various methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. For example, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which may alternatively be referred to as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as perfect matches by the sequences in the alignment of A and B in the program, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A.

[0145] Antibody "effector functions" refer to the biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region) and vary depending on the antibody isotype. Examples of antibody effector functions include Clq binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0146] As used herein, "binding affinity" refers to the strength of a non-covalent interaction between a single binding site of a molecule (e.g., a cytokine) and its binding partner (e.g., a cytokine receptor). In some embodiments, the affinity of a binding protein (e.g., a cytokine) can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0147] An "isolated" nucleic acid molecule encoding a cytokine polypeptide described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced. In some embodiments, an isolated nucleic acid is free from all components associated with the environment in which it is produced. Isolated nucleic acid molecules encoding the polypeptides and cytokine polypeptides of the present invention are in a form other than the form or environment in which they are found in nature. Isolated nucleic acid molecules are therefore distinguished from nucleic acids encoding the polypeptides and cytokine polypeptides of the present invention that naturally occur in cells.

[0148] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that have unacceptable toxicity to the subject to which the formulation is administered.

[0149] As used herein, "carrier" refers to a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed to it at the dosage and concentration employed. Physiologically acceptable carriers are often aqueous pH-buffered solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0150] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include slowing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. An individual is successfully "treated," for example, 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 results in an improvement in the quality of life of the individual suffering from the disease, a reduction in the dose of other medications required to treat the disease, a reduction in the frequency of disease recurrences, a reduction in the severity of the disease, a delay in the onset or progression of the disease, and / or an extension of the individual's survival.

[0151] As used herein, "in conjunction with" or "in combination with" refers to the administration of one therapeutic modality in addition to another therapeutic modality. Thus, "in conjunction with" or "in combination with" refers to the administration of one therapeutic modality before, during, or after the administration of another therapeutic modality to an individual.

[0152] As used herein, the term "prevention" includes providing prevention with respect to the occurrence or recurrence of a disease in an individual. An individual may be predisposed to, susceptible to, or at risk of developing a disorder, but has not been diagnosed with the disorder. In some embodiments, the targeted cytokines described herein are used to delay the onset of a disorder.

[0153] As used herein, an individual "at risk" of developing a disorder may or may not have detectable disease or disease symptoms, and may or may not exhibit detectable disease or disease symptoms prior to the treatment methods described herein. "At risk," as known in the art, indicates that an individual has one or more risk factors, which are measurable parameters that correlate with the development of a disease. Individuals who have one or more of these risk factors have a higher probability of developing a disorder than individuals who do not have one or more of these risk factors.

[0154] An "effective amount" refers to at least an amount effective, at dosages and for periods of time necessary, to achieve a desired or indicated effect, including a therapeutic or prophylactic result.

[0155] An effective amount can be provided in one or more administrations. A "therapeutically effective amount" is at least the minimum concentration required to affect measurable improvement in a specific disorder. The therapeutically effective amount herein can vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the antibody to induce a desired response in an individual. A therapeutically effective amount can also be an amount in which the therapeutically beneficial effects outweigh any toxic or harmful effects of the targeted cytokine. A "prophylactically effective amount" refers to an amount effective, at the necessary dosage and for the necessary period, to achieve the desired preventative result. Typically, but not necessarily, a prophylactic dose is used in subjects before or at an early stage of disease, so the prophylactically effective amount may be less than the therapeutically effective amount.

[0156] "Chronic" administration refers to the administration of a drug continuously, as opposed to acutely, so as to predominate the initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration is treatment that is not continuous without interruption, but rather is cyclic in nature.

[0157] As used herein, an "individual" or "subject" is a mammal. For purposes of treatment, "mammals" include humans, domestic and farm animals, and zoo, sport, 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.

[0158] 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 HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid, in some embodiments, an amino acid is a d-amino acid, and in some embodiments, an amino acid is an l-amino acid. A "standard amino acid" refers to any of the 20 standard l-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or obtained from a natural source. As used herein, a "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 carboxy- and / or amino-terminal amino acids in a peptide, can be modified by methylation, amidation, acetylation, protecting groups, and / or substitutions with other chemical groups that can alter the circulating half-life of the peptide without adversely affecting its activity. Amino acids may also participate in disulfide bonds. An amino acid may include a single or post-translational modification, such as association with one or more chemical entities (e.g., a methyl group, an acetate group, an acetyl group, a phosphate group, a formyl moiety, an isoprenoid group, a sulfate group, a polyethylene glycol moiety, a lipid moiety, a carbohydrate moiety, a biotin moiety, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and can refer to a free amino acid and / or an amino acid residue of a peptide. Whether a term refers to a free amino acid or a residue of a peptide will be clear from the context in which the term is used.

[0159] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0160] Biological activity: As used herein, the phrase "biological activity" refers to the characteristic of any agent that has activity in a biological system, particularly an organism. For example, an agent that, when administered to an organism, has a biological effect on that organism is considered to be biologically active.

[0161] Delivery: As used herein, the term "delivery" encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which the mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as "local distribution" or "local delivery"), and situations in which the mRNA is delivered to a target tissue and the encoded protein is expressed, secreted into the patient's circulatory system (e.g., serum), and systemically distributed and absorbed by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[0162] Dosing interval: As used herein, a dosing interval in the context of a method for treating a disease is the frequency with which a therapeutic composition, such as an mRNA composition at an effective dose of mRNA, is administered to a subject (mammal) in need thereof so that one or more symptoms associated with the disease are reduced or one or more biomarkers associated with the disease are reduced over at least the duration of the dosing interval. Dosing frequency and dosing interval may be used interchangeably in this disclosure.

[0163] 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 an intact protein (e.g., an enzyme), and / or the post-translational modification of a polypeptide or fully assembled protein (e.g., an enzyme). In this application, the terms "expression" and "production," and grammatical equivalents, are used interchangeably.

[0164] Effective dose: As used herein, an effective dose is a dose of mRNA in a pharmaceutical composition that, when administered to a subject in need thereof, is effective to cause the mammalian subject to achieve the desired result in the subject, such as, for example, a reduction in symptoms associated with a disease, according to the methods of the present invention.

[0165] Improved, increased, or decreased: As used herein, the terms "improved," "increased," or "reduced," or grammatical equivalents, refer to a value relative to a baseline measurement, such as a measurement in the same individual before the initiation of a treatment described herein, or a measurement in a control subject (or control subjects) without a treatment described herein. A "control subject" is a subject suffering from the same type of disease as the subject being treated and who is about the same age as the subject being treated.

[0166] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism but within an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc.

[0167] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (e.g., as opposed to in vitro systems).

[0168] Patient: As used herein, the term "patient" or "subject" refers to any organism to which provided compositions 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. Human includes prenatal and postnatal forms.

[0169] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to a material that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0170] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbic acid, aspartic acid, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydrogen iodide, 2-hydroxy-ethanesulfonate, Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N(C1-4 alkyl) salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed where appropriate with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Further pharmaceutically acceptable salts include salts formed from the quaternization of amines with suitable electrophiles, e.g., alkyl halides, to form quaternized alkylated amino salts.

[0171] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, a subject is a human. A subject can be a patient, where a patient refers to a person who presents to a health care provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be suffering from or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0172] Substantially: As used herein, the term "substantially" refers to the qualitative condition indicating the extent or degree of the sum or near sum of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the possible lack of perfection inherent in many biological and chemical phenomena.

[0173] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by the disease being treated. In some embodiments, the target tissue includes tissue that exhibits disease-related pathology, symptoms, or characteristics.

[0174] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount sufficient to treat, diagnose, prevent, and / or delay the onset of symptoms of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. Those skilled in the art will understand that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0175] Treatment: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not exhibit signs of disease and / or who show only early signs of disease, with the intent of reducing the risk of developing pathologies associated with the disease.

[0176] Various aspects of the invention are described in detail in the following sections. The use of the sections is not intended to limit the invention. Each section may be applicable to any aspect of the invention. In this application, the use of "or" means "and / or" unless stated otherwise. DETAILED DESCRIPTION OF THE INVENTION

[0177] Targeted Cytokines The present invention provides, inter alia, a targeted cytokine comprising a targeting moiety, a cytokine or a variant thereof, a masking moiety, and an Fc domain, which is active at the disease site and can specifically target target cells for effective cancer treatment without causing undesirable side effects.

[0178] 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. Examples of targeting cytokines of the present invention are shown in Figures 3 and 4A. 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 variant thereof is linked to the Fc polypeptide via a cleavable linker. In some embodiments, the cytokine or variant thereof is linked to the Fc polypeptide via a non-cleavable linker.

[0179] A targeted IL-2 cytokine according to the present disclosure can combine an IL-2 cytokine or functional fragment thereof described anywhere herein, a masking moiety described anywhere herein, a first and second Fc domain described anywhere herein, a cleavable and non-cleavable linker described anywhere herein, and a targeting moiety described anywhere herein.

[0180] Targeted Cytokine Format In some embodiments, the targeted cytokine is a bivalent targeting format comprising: 1) a first chain comprising a variable heavy chain region and a heavy chain constant region of IgG1 or IgG4 with "hole mutations" (Y349C, T366S, L368A, and Y407V); 2) a second chain comprising a variable heavy chain region and a heavy chain constant region with "knob mutations" (S354C and T366W) fused to a cytokine or a variant thereof, wherein the CH1 and CH2 domains are from IgG1 or IgG4 and the CH3 domain is from IgG3; and 3) a third chain comprising a variable light chain region and an immunoglobulin kappa or lambda constant region.

[0181] In some embodiments, the targeted cytokine is a bivalent targeting format comprising: 1) a first chain comprising a variable heavy chain region and a heavy chain constant region of IgG1 or IgG4 with "hole mutations" (Y349C, T366S, L368A, and Y407V), 2) a second chain comprising a variable heavy chain region and a heavy chain constant region from IgG1 or IgG4 with "knob mutations" (S354C and T366W) and "RF mutations" (H435R and Y436F) fused to the cytokine or a variant thereof, and 3) a third chain comprising a variable light chain region and an immunoglobulin kappa or lambda constant region.

[0182] In some embodiments, the targeted cytokine is a bivalent targeting format comprising: 1) a first chain comprising a variable heavy chain region and an IgG1 or IgG4 heavy chain constant region with "hole mutations" (Y349C, T366S, L368A, and Y407V) fused to the cytokine or a variant thereof; 2) a second chain comprising a variable heavy chain region and a heavy chain constant region with "knob mutations" (S354C and T366W) 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 chain region and an immunoglobulin kappa or lambda constant region.

[0183] In some embodiments, the targeted cytokine is a bivalent targeting format comprising: 1) a first chain comprising a variable heavy chain region and a heavy chain constant region of IgG1 or IgG4 with "hole mutations" (Y349C, T366S, L368A, and Y407V) fused to the cytokine or a variant thereof; 2) a second chain comprising a variable heavy chain region and a heavy chain constant region from IgG1 or IgG4 with "knob mutations" (S354C and T366W) and "RF mutations" (H435R and Y436F) fused to a masking moiety; and 3) a third chain comprising a variable light chain region and an immunoglobulin kappa or lambda constant region.

[0184] In some embodiments, the targeted cytokine is a bivalent targeting format comprising: 1) a first chain comprising a variable heavy chain region and an IgG1 or IgG4 heavy chain constant region with "hole mutations" (Y349C, T366S, L368A, and Y407V) fused to a masking moiety; 2) a second chain comprising a variable heavy chain region and a heavy chain constant region with "knob mutations" (S354C and T366W) fused to a cytokine or a variant thereof, wherein the CH1 and CH2 domains are from IgG1 or IgG4 and the CH3 domain is from IgG3; and 3) a third chain comprising a variable light chain region and an immunoglobulin kappa or lambda constant region.

[0185] In some embodiments, the targeted cytokine is a bivalent targeting format comprising: 1) a first chain comprising a variable heavy chain region and a heavy chain constant region of IgG1 or IgG4 with "hole mutations" (Y349C, T366S, L368A, and Y407V) fused to a masking moiety; 2) a second chain comprising a variable heavy chain region and a heavy chain constant region from IgG1 or IgG4 with "knob mutations" (S354C and T366W) and "RF mutations" (H435R and Y436F) fused to the cytokine or a variant thereof; and 3) a third chain comprising a variable light chain region and an immunoglobulin kappa or lambda constant region.

[0186] In some embodiments, the targeted cytokine is a monovalent targeting format comprising: 1) a first chain comprising a Fab fused to a first Fc polypeptide chain derived from IgG1 or IgG4 with "hole mutations" (Y349C, T366S, L368A, and Y407V), and 2) a second chain comprising a second Fc polypeptide chain derived from IgG1 or IgG4 with "knob mutations" (S354C and T366W) and "RF mutations" (H435R and Y436F).

[0187] In some embodiments, the targeted cytokine is a monovalent targeting format comprising: 1) a first chain comprising a Fab fused to a first Fc polypeptide chain derived from IgG1 or IgG4 with "hole mutations" (Y349C, T366S, L368A, and Y407V); and 2) a second chain comprising a second Fc polypeptide chain with "knob mutations" (S354C and T366W), wherein the CH1 and CH2 domains are derived from IgG1 or IgG4 and the CH3 domain is derived from IgG3.

[0188] In some embodiments, the targeted cytokine is a monovalent targeting format comprising: 1) a first chain comprising a first Fc polypeptide chain derived from IgG1 or IgG4 with "hole mutations" (Y349C, T366S, L368A, and Y407V); and 2) a second chain comprising a Fab fused to a second Fc polypeptide chain derived from IgG1 or IgG4 with "knob mutations" (S354C and T366W) and "RF mutations" (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, a masking moiety is fused to the first Fc polypeptide chain. In some embodiments, a masking moiety is fused to the second Fc polypeptide chain.

[0189] In some embodiments, the targeted cytokine is a monovalent targeting format comprising: 1) a first chain comprising a first Fc polypeptide chain derived from IgG1 or IgG4 with "hole mutations" (Y349C, T366S, L368A, and Y407V); and 2) a second chain comprising a Fab fused to a second Fc polypeptide chain with "knob mutations" (S354C and T366W), wherein the CH1 and CH2 domains are derived from IgG1 or IgG4 and the CH3 domain is derived from IgG3. In some embodiments, the cytokine is fused to the second Fc polypeptide chain. In some embodiments, a masking moiety is fused to the first Fc polypeptide chain. In some embodiments, a masking moiety is fused to the second Fc polypeptide chain.

[0190] In some embodiments, the targeted cytokine is a monovalent targeting format comprising: 1) a first chain comprising a variable light chain region and an IgG kappa or lambda constant region fused to an IgG1 or IgG4 heavy chain constant region with "hole mutations" (Y349C, T366S, L368A, and Y407V); and 2) a second chain comprising a variable heavy chain region and a heavy chain constant region from IgG1 or IgG4 with "knob mutations" (S354C and T366W) and "RF mutations" (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 a second Fc polypeptide chain. In some embodiments, a masking moiety is fused to the first Fc polypeptide chain. In some embodiments, a masking moiety is fused to the second Fc polypeptide chain.

[0191] In some embodiments, the targeted cytokine is a monovalent targeting format comprising: 1) a first chain comprising a variable heavy chain region and a heavy chain constant region of IgG1 or IgG4 with "hole mutations" (Y349C, T366S, L368A, and Y407V); and 2) a second chain comprising a variable heavy chain region fused to a heavy chain constant region with "knob mutations" (S354C and T366W) fused to a cytokine or a variant thereof, wherein the CH1 and CH2 domains are from IgG1 or IgG4 and the CH3 domain is from IgG3.

[0192] In some embodiments, the targeted cytokine is a monovalent targeting format comprising: 1) a first chain comprising a variable heavy chain region fused to a heavy chain constant region of IgG1 or IgG4 with "hole mutations" (Y349C, T366S, L368A, and Y407V); and 2) a second chain comprising a variable light chain region and a constant region of IgG kappa or lambda fused to a heavy chain constant region from IgG1 or IgG4 with "knob mutations" (S354C and T366W) and "RF mutations" (H435R and Y436F), wherein the C-terminus of the CH3 of the second chain is fused to the cytokine or a variant thereof.

[0193] In some embodiments, the targeted cytokine is a monovalent targeting format comprising: 1) a first chain comprising a variable heavy chain region fused to a heavy chain constant region of IgG1 or IgG4 with "hole mutations" (Y349C, T366S, L638A, and Y407V); and 2) a second chain comprising a variable light chain region and a constant region of IgG kappa or lambda fused to a heavy chain constant region with "knob mutations" (S354C and T366W) fused to a cytokine or a variant thereof, wherein the CH1 and CH2 domains are from IgG1 or IgG4 and the CH3 domain is from IgG3.

[0194] targeting part Provided herein are targeted cytokines 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.

[0195] In some embodiments, the targeting moiety comprises an antigen-binding moiety, and the antigen is expressed on an immune cell. In some embodiments, the targeting moiety comprises an antigen-binding moiety, and 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.

[0196] In some embodiments, the targeting moiety is selected from the group consisting of 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), prostate six-transmembrane epithelial antigen 1 (STEAPl), 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), and Claudin Specifically binds 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.

[0197] In some embodiments, the targeting moiety binds a tumor-associated antigen. In some embodiments, the targeting moiety is an antibody or antigen-binding fragment that binds a tumor-associated antigen. In some embodiments, the targeting moiety is a bispecific antibody or antigen-binding fragment that binds a tumor-associated antigen. In some embodiments, the targeting moiety is an anti-alpha-fetoprotein (AFP) antibody or fragment thereof. In some embodiments, the targeting moiety is an anti-B2M antibody or fragment thereof. In some embodiments, the targeting moiety is an anti-beta-human chorionic gonadotropin (beta-hCG) antibody or fragment thereof. In some embodiments, the targeting moiety is an anti-CD117 antibody or fragment thereof. In some embodiments, the targeting moiety is an anti-CD19 antibody or fragment thereof. In some embodiments, the targeting moiety is an anti-CD20 antibody or fragment thereof. In some embodiments, the targeting moiety is an anti-CD22 antibody or fragment thereof. In some embodiments, the targeting moiety is an anti-CD25 antibody or fragment thereof. In some embodiments, the targeting moiety is an anti-CD30 antibody or fragment thereof. In some embodiments, the targeting moiety is an anti-CD33 antibody or fragment thereof. In some embodiments, the targeting moiety is an anti-CD151 antibody or fragment thereof. In some embodiments, the targeting moiety is an anti-MUC-1 antibody or fragment thereof.

[0198] In some embodiments, the targeting moiety specifically binds PD-1. In some embodiments, the targeting moiety is an anti-PD1 Fab. In some embodiments, the targeting moiety is an anti-PD1 scFv.

[0199] In some embodiments, the targeting moiety is derived from an anti-PD1 antibody, hi some embodiments, the targeting moiety is derived from pembrolizumab or nivolumab.

[0200] In some embodiments, the targeting moiety binds PD-L1.

[0201] In some embodiments, the targeting moiety comprises a drug, peptide, or polypeptide that specifically binds to a target.

[0202] In some embodiments, the targeting moiety comprises a Fab, a single-chain Fv (scFv), a single-domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibody (bsFab), a single-domain antibody-binding Fab (s-Fab), an antibody, or a combination thereof. In some embodiments, the targeting moiety comprises a Fab. In some embodiments, the targeting moiety comprises a single-chain Fv (scFv). In some embodiments, the targeting moiety comprises a single-domain antibody (VHH). In some embodiments, the targeting moiety comprises one or more CDRs. In some embodiments, the targeting moiety comprises a variable heavy chain (VH). In some embodiments, the targeting moiety comprises a variable light chain (VL). In some embodiments, the targeting moiety comprises a Fab-like bispecific antibody (bsFab). In some embodiments, the targeting moiety comprises a single-domain antibody-binding Fab (s-Fab). In some embodiments, the targeting moiety comprises an antibody or fragment thereof.

[0203] In some embodiments, the targeting moiety is It contains the heavy chain variable region of QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 1).

[0204] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:1. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:1. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:1. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO:1. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:1. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO:1. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:1. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:1. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:1.

[0205] In some embodiments, the targeting moiety is QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 2).

[0206] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:2. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:2.

[0207] In some embodiments, the targeting moiety is QVQLVQSVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTT VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 3).

[0208] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:3. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:3.

[0209] In some embodiments, the targeting moiety comprises the heavy chain CDR1 sequence GYTFTNYY (SEQ ID NO: 4). In some embodiments, the targeting moiety comprises the heavy chain CDR2 sequence INPSNGGT (SEQ ID NO: 5). In some embodiments, the targeting moiety comprises the heavy chain CDR3 sequence ARRDYRFDMGFDY (SEQ ID NO: 6). In some embodiments, the targeting moiety comprises an HCDR1 of SEQ ID NO: 4, an HCDR2 of SEQ ID NO: 5, and an HCDR3 of SEQ ID NO: 6.

[0210] In some embodiments, the targeting moiety is It contains the light chain variable region of EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKTSENLYFQ (SEQ ID NO: 7).

[0211] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:7. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:7.

[0212] In some embodiments, the targeting moiety is EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 8).

[0213] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:8. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:8.

[0214] In some embodiments, the targeting moiety comprises the light chain CDR1 sequence KGVSTSGYSY (SEQ ID NO: 9). In some embodiments, the targeting moiety comprises the light chain CDR2 sequence LAS (SEQ ID NO: 10). In some embodiments, the targeting moiety comprises the light chain CDR3 sequence QHSRDLPLT (SEQ ID NO: 11). In some embodiments, the targeting moiety comprises an LCDR1 of SEQ ID NO: 9, an LCDR2 of SEQ ID NO: 10, and an LCDR3 of SEQ ID NO: 11.

[0215] In some embodiments, the targeting moiety comprises an HCDR1 of SEQ ID NO:4, an HCDR2 of SEQ ID NO:5, an HCDR3 of SEQ ID NO:6, an LCDR1 of SEQ ID NO:9, an LCDR2 of SEQ ID NO:10, and an LCDR3 of SEQ ID NO:11.

[0216] In some embodiments, the targeting moiety comprises the heavy chain variable region or the light chain variable region of nivolumab.

[0217] In some embodiments, the targeting moiety is QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS (SEQ ID NO: 159).

[0218] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 159. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 159.

[0219] In some embodiments, the targeting moiety is QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK (SEQ ID NO: 157).

[0220] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 157. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 157.

[0221] In some embodiments, the targeting moiety comprises the heavy chain CDR1 sequence GITFSNSG (SEQ ID NO: 161). In some embodiments, the targeting moiety comprises the heavy chain CDR2 sequence VIWYDGSKRYYADSVKG (SEQ ID NO: 162). In some embodiments, the targeting moiety comprises the heavy chain CDR3 sequence ATNDDY (SEQ ID NO: 163). In some embodiments, the targeting moiety comprises HCDR1 GITFSNSG (SEQ ID NO: 161), HCDR2 VIWYDGSKRYYADSVKG (SEQ ID NO: 162), and HCDR3 ATNDDY (SEQ ID NO: 163).

[0222] In some embodiments, the targeting moiety comprises a heavy chain variable region and a CH1 domain comprising SEQ ID NO:264. QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 264)

[0223] In some embodiments, the targeting moiety is It contains the light chain variable region of EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK (SEQ ID NO: 160).

[0224] In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 160. In some embodiments, the targeting moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 160.

[0225] In some embodiments, the targeting moiety is EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGE (SEQ ID NO: 158) light chain.

[0226] 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.

[0227] In some embodiments, the targeting moiety comprises the light chain CDR1 sequence QSVSSY (SEQ ID NO: 164). In some embodiments, the targeting moiety comprises the light chain CDR2 sequence DAS (SEQ ID NO: 165). In some embodiments, the targeting moiety comprises the light chain CDR3 sequence QQSSNWPRT (SEQ ID NO: 166). In some embodiments, the targeting moiety comprises an LCDR1 of SEQ ID NO: 164, an LCDR2 of SEQ ID NO: 165, and an LCDR3 of SEQ ID NO: 166.

[0228] In some embodiments, the targeting moiety comprises an HCDR1 of SEQ ID NO: 161, an HCDR2 of SEQ ID NO: 162, an HCDR3 of SEQ ID NO: 163, an LCDR1 of SEQ ID NO: 164, an LCDR2 of SEQ ID NO: 165, and an LCDR3 of SEQ ID NO: 166.

[0229] 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 a CH1 domain comprise the amino acid sequence (SEQ ID NO: 264). QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 264)

[0230] 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 a Fab is fused to an Fc polypeptide. In some embodiments, the C-terminus of the heavy chain of a Fab is fused to the N-terminus of the Fc polypeptide. In some embodiments, the Fc polypeptide comprises a cleavage site.

[0231] 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.

[0232] In some embodiments, the targeting moiety comprises a CH1 domain comprising 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)

[0233] cytokines The immune system is a highly skilled communicator designed to respond rapidly, specifically, and comprehensively to protect the organism from foreign invaders and disease. The cytokine superfamily of proteins is an integral part of the cell-cell signaling network and is essential for the generation and regulation of the immune system. These interacting biological signals have remarkable capabilities, including influencing growth and development, hematopoiesis, lymphocyte recruitment, differentiation of T-cell subsets, and inflammation.

[0234] Cytokines can be part of a larger immune program, such as T cell subset differentiation. Mature CD4 and CD8 T cells leave the thymus with a naive phenotype and produce a variety of cytokines. In the periphery, these T cells encounter antigen-presenting cells (APCs) that display either major histocompatibility complex (MHC) class I molecules (presenting cytosolic peptides to CD8 T cells) or MHC class II molecules (presenting degraded peptides in intracellular vesicles to CD4 T cells). After activation, CD4 T helper (Th) cells can be classified into two major subpopulations in mice and humans based on their distinct cytokine and chemokine secretion profiles. 3-7 Th1 cells primarily secrete IL-2, interferon-γ (IFN-γ), and tumor necrosis factor-β (TNF-β), whereas Th2 cells primarily secrete IL-4, IL-5, IL-6, IL-10, and IL-13. Th1 cells support cell-mediated immunity, resulting in inflammation, cytotoxicity, and delayed-type hypersensitivity (DTH). Th2 cells support humoral immunity and downregulate the inflammatory effects of Th1 cells. This paradigm is an excellent example of an integrated biological network and is extremely useful for simplifying our understanding of typical immune responses and the immune responses that alter pathogenesis. For example, failure to communicate "self" can lead to a loss of tolerance to our own antigens, promoting destructive immune responses against self-tissues and autoimmune disease. Autoimmunity, the primary focus of this paper, is the underlying mechanism for a range of conditions, including type 1 diabetes, multiple sclerosis, and rheumatoid arthritis. Autoimmune diseases can be caused, in part, by cytokine- and chemokine-mediated dysregulation of Th cell subset differentiation. Aside from situations in which antigen and costimulatory signals are presented, the primary factors influencing Th subset development are cytokines and chemokines in the stimulatory environment. A better understanding of the properties and interactions of individual cytokines and chemokines that play a role in Th cell activation may lead to more advanced treatments for autoimmune diseases.

[0235] Targeted cytokines of the present invention can include any cytokine known in the art or variants thereof. See, e.g., Cameron MJ, Kelvin DJ. Cytokines, Chemokines and Their Receptors. In: Madame Curie Bioscience Database. Austin (TX): Landes Bioscience; 2000-2013, the contents of which are incorporated in their entirety. For example, the cytokine incorporated into the targeted cytokine can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-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.

[0236] 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 is 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.

[0237] Interleukin 2 (IL-2) Provided herein is an IL-2 cytokine or functional fragment thereof for use in targeting cytokines or cleavage products thereof. Cytokines play a role in cell signaling, particularly in cells of the immune system. IL-2 is an interleukin, a type of immune system cytokine signaling molecule that regulates the activity of white blood cells. A suitable IL-2 cytokine for use in the present invention can be any IL-2 or functional fragment thereof. In some embodiments, the IL-2 is native IL-2, IL-2 containing one or more substitutions (e.g., an IL-2 mutein or IL-2 variant), or truncated IL-2. In some embodiments, the IL-2 is a polypeptide that retains at least one characteristic of IL-2 biological activity.

[0238] In some embodiments, the IL-2 is native IL-2. In some embodiments, the IL-2 comprises a C125A substitution of mature IL-2 (SEQ ID NO: 13).

[0239] In some embodiments, the amino acid substitution reduces the affinity of the IL-2 polypeptide or functional fragment thereof for CD25 (IL-2Rα).

[0240] In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence resulting from the introduction of one or more amino acid substitutions into the amino acid sequence of the IL-2 polypeptide or functional fragment thereof that increase the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rb or IL-2Rγ. In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions, compared to the amino acid sequence of wild-type IL-2, that enhance the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Rb (CD122). In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions, compared to the amino acid sequence of wild-type IL-2, that reduce the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2Ra (CD25) and having one or more amino acid substitutions, compared to the amino acid sequence of wild-type IL-2, that enhance the affinity of the IL-2 polypeptide or functional fragment thereof for IL-2R (CD122).

[0241] In some embodiments, IL-2 binds to IL-2Ra with an affinity equal to or greater than that of wild-type IL-2. In some embodiments, IL-2 preferentially binds to CD25 (e.g., alpha-biased). In some embodiments, IL-2 has reduced affinity for CD122 and / or CD132. In some embodiments, IL-2 comprises N88D and C125A relative to SEQ ID NO: 13. In some embodiments, IL-2 is SEQ ID NO: 167. APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 167)

[0242] In eukaryotic cells, natural IL-2 is synthesized as a 153 amino acid precursor polypeptide having SEQ ID NO:12. MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 12)

[0243] This is then processed to mature IL-2 by removing amino acid residues 1 to 20. This results in the mature form of IL-2 consisting of 133 amino acids (amino acid residues 21 to 153) having SEQ ID NO: 13. APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 13)

[0244] A "functional fragment" of an IL-2 cytokine includes a portion of a full-length cytokine protein that retains or has altered cytokine receptor binding ability (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity compared to the full-length cytokine protein). Cytokine receptor binding ability can be demonstrated, for example, by the ability of the cytokine to bind to its cognate receptor or a component thereof (e.g., one or more chains of a heterotrimeric receptor complex).

[0245] In some embodiments, the IL-2 cytokine or functional fragment thereof is any naturally occurring interleukin-2 (IL-2) protein or modified variant thereof capable of binding to the interleukin-2 receptor, particularly the IL-2Rα chain. In the context of IL-2 cytokine binding, the target protein can be IL-2R (including the IL-2Rα chain, IL-2Rβ chain, and IL-2Rγ chain), the IL-2Rα chain, the IL-2Rβ chain, or the IL-2Rα / β dimeric complex. In some embodiments, the IL-2 cytokine or 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 functional fragment thereof comprises SEQ ID NO: 12, the amino acid sequence of mature IL-2.

[0246] In some embodiments, the IL-2 cytokine or 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 of the at least one amino acid modification can be any amino acid modification, such as a substitution, insertion, or deletion. In some embodiments, the IL-2 cytokine or 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 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.

[0247] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 13.

[0248] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions compared to the amino acid sequence of wild-type IL-2 of SEQ ID NO: 13 that decrease the affinity of the IL-2 peptide or functional fragment thereof for IL-2Rα (CD25). In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 13 such that one or more amino acid residues 38, 42, 45, and 62 are alanine (A). In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 13 such that one or more amino acid residues 38, 42, 45, and 62 are alanine (A).

[0249] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises the amino acid sequence substitution C125A compared to the amino acid sequence of SEQ ID NO:13.

[0250] In some embodiments, the IL-2 cytokine or 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 functional fragment thereof comprises an amino acid sequence having amino acid residues R38, F42, Y45, and E62 substituted with alanine (A) in the amino acid sequence of SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or 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.

[0251] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO:14. APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 14)

[0252] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 92% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 14.

[0253] In some embodiments, the IL-2 cytokine or functional fragment thereof has one or more amino acid residues, e.g., residues 1-3, removed compared to the amino acid sequence of mature IL-2 of SEQ ID NO: 12 to remove an O-glycosylation site. In some embodiments, the IL-2 cytokine or functional fragment thereof has one or more amino acid residues substituted compared to the amino acid sequence of mature IL-2 of SEQ ID NO: 12 to remove an O-glycosylation site. In some embodiments, the IL-2 cytokine or functional fragment thereof has one or more amino acid residues inserted compared to the amino acid sequence of mature IL-2 of SEQ ID NO: 12 to remove an O-glycosylation site, e.g., in the region of residues 1-3. In some embodiments, the IL-2 cytokine or functional fragment thereof does not have an O-glycosylation site within residues 1-3.

[0254] Interleukin 15 (IL-15) The present invention provides an IL-15 cytokine or a functional fragment thereof for use in targeting cytokines or their cleavage products.Cytokines play a role in cell signaling, particularly in cells of the immune system.IL-15 is an interleukin, a type of cytokine signaling molecule of the immune system that regulates the activity of white blood cells.

[0255] In eukaryotic cells, IL-15 is synthesized as a 162 amino acid precursor polypeptide (SEQ ID NO: 92) that is then processed to mature IL-15 by removal of amino acid residues 1 to 48. This results in a mature form of IL-15 consisting of 114 amino acids (amino acid residues 49 to 162) that is secreted in its mature, active form (see SEQ ID NO: 93). IL-15 precursor polypeptide (SEQ ID NO: 92): MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS IL-15 mature polypeptide (SEQ ID NO: 93): NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0256] 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 naturally occurring 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), as well as any form of IL-15 resulting from intracellular processing (e.g., the mature form of IL-15 consisting of amino acid residues 49-162). Thus, the term encompasses the protein encoded by the amino acid sequence of SEQ ID NO:93, as well as sequence variants thereof. The term also encompasses naturally occurring variants of IL-15. The term also encompasses non-naturally occurring variants of IL-15, such as truncations, deletions, forms in which IL-15 is linked to another molecule, and variants caused by at least one amino acid change to the amino acid sequence (e.g., by substitution, addition, or deletion). In some embodiments, a variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50-, 100-, or 114-contiguous amino acid portion) 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. 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, and includes variants thereof, such as variants made by one or more amino acid substitutions to the amino acid sequence of SEQ ID NO: 92 or 93.

[0257] A "functional fragment" of an IL-15 cytokine includes a portion of a full-length cytokine protein that retains or has altered cytokine receptor binding ability (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity compared to the full-length cytokine protein). Cytokine receptor binding ability can be demonstrated, for example, by the ability of the cytokine to bind to its cognate receptor or a component thereof (e.g., one or more chains of a heterotrimeric receptor complex).

[0258] In some embodiments, the IL-15 cytokine or functional fragment thereof is any naturally occurring interleukin-2 (IL-15) protein or modified variant thereof capable of binding to the interleukin-2 receptor, particularly the IL-15Rα chain.

[0259] In some embodiments, the IL-15 cytokine or fragment thereof comprises SEQ ID NO: 93 or a functional fragment thereof.

[0260] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO:93.

[0261] In some embodiments, the IL-15 cytokine or 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: 93. Each of the at least one amino acid modification can be any amino acid modification, such as a substitution, insertion, or deletion. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least five amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 93.

[0262] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 93.

[0263] In some embodiments, the IL-15 cytokine or 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:93.

[0264] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22, E46, E53 compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22, E46, E53, N71, N79, and N112 compared to the amino acid sequence of SEQ ID NO: 93.

[0265] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position D22 compared to the amino acid sequence of SEQ ID NO:93.

[0266] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position E46 compared to the amino acid sequence of SEQ ID NO:93.

[0267] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position E53 compared to the amino acid sequence of SEQ ID NO:93.

[0268] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N71 compared to the amino acid sequence of SEQ ID NO:93.

[0269] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N79 compared to the amino acid sequence of SEQ ID NO:93.

[0270] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N112 compared to the amino acid sequence of SEQ ID NO:93.

[0271] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions E46 and E53 compared to the amino acid sequence of SEQ ID NO:93.

[0272] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71 and N79 compared to the amino acid sequence of SEQ ID NO:93.

[0273] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71 and N112 compared to the amino acid sequence of SEQ ID NO:93.

[0274] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N79 and N112 compared to the amino acid sequence of SEQ ID NO:93.

[0275] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71, N79, and N112 compared to the amino acid sequence of SEQ ID NO:93.

[0276] In some embodiments, the amino acid substitution at position D22 is D22A.

[0277] In some embodiments, the amino acid substitution at position E46 is E46A.

[0278] In some embodiments, the amino acid substitution at position E46 is E46R.

[0279] In some embodiments, the amino acid substitution at position E46 is E46S.

[0280] In some embodiments, the amino acid substitution at position E53 is E53A.

[0281] In some embodiments, the amino acid substitution at position E53 is E53R.

[0282] In some embodiments, the amino acid substitution at position E53 is E53S.

[0283] In some embodiments, the amino acid substitution at position N71 is N71Q.

[0284] In some embodiments, the amino acid substitution at position N79 is N79Q.

[0285] In some embodiments, the amino acid substitution at position N112 is N112Q.

[0286] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having the amino acid substitution D22A compared to the amino acid sequence of SEQ ID NO:93.

[0287] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO:94. NWVNVISDLKKIEDLIQSMHIAATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 94)

[0288] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:94.

[0289] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having the amino acid substitution E46A compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 95. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLALQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 95)

[0290] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 95.

[0291] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46A and E53A compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 96. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLALQVISLASGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 96)

[0292] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46R and E53R compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 97. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:6. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLRLQVISLRSGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 97)

[0293] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46S and E53S compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 98. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLSLQVISLSSGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 98)

[0294] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having the amino acid substitution E53A compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 99. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLASGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 99)

[0295] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 100 and the amino acid sequence of SEQ ID NO: 101. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 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. NWVNVISDLKKIEDLIQS (SEQ ID NO: 100) KVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 101)

[0296] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having the amino acid substitution N71Q compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 102. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAQNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 102)

[0297] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having the amino acid substitution N79Q compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 103. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 103)

[0298] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having the amino acid substitution N112Q compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 104. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 104. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS (SEQ ID NO: 104)

[0299] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions N71Q and N79Q compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 105. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 105. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAQNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 105)

[0300] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions N71Q and N112Q compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 106. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 106. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAQNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS (SEQ ID NO: 106)

[0301] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions N79Q and N112Q compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 107. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 107. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS (SEQ ID NO: 107)

[0302] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions N71Q, N79Q, and N112Q compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 108. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 108. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAQNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS (SEQ ID NO: 108)

[0303] In some embodiments, an additional mutation may be included in any of the above sequences at position N71. In some embodiments, the mutation is N71A, N71R, N71W, N71F, N71P, N71M, N71L, N71T, N71S, or N71Y.

[0304] In some embodiments, an additional mutation may be included in any of the above sequences at position S73. In some embodiments, the mutation is S73A, S73W, S73V, or S73M.

[0305] In some embodiments, additional mutations may be included in any of the above sequences at one or more amino acid positions N72, N79, V80, T81, and N112. In some embodiments, one or more additional mutations selected from N72A, N79A, V80A, T81A, and N112R may be included in any of the above sequences.

[0306] In some embodiments, additional mutations may be included in any of the above sequences at one or more amino acid positions N72, S73, N79, V80, T81, and N112. In some embodiments, one or more additional mutations N72A, S73A, N79A, V80A, T81A, and N112 may be included in any of the above sequences.

[0307] In some embodiments, the IL-15 cytokine or functional fragment thereof has one or more amino acid residues, e.g., residues 1-3, deleted compared to the amino acid sequence of mature IL-15 of SEQ ID NO: 93 to remove an O-glycosylation site. In some embodiments, the IL-15 cytokine or functional fragment thereof has one or more amino acid residues substituted compared to the amino acid sequence of mature IL-15 of SEQ ID NO: 93 to remove an O-glycosylation site. In some embodiments, the IL-15 cytokine or functional fragment thereof has one or more amino acid residues inserted compared to the amino acid sequence of mature IL-15 of SEQ ID NO: 93 to remove an O-glycosylation site, e.g., in the region of residues 1-3. In some embodiments, the IL-15 cytokine or functional fragment thereof does not have an O-glycosylation site within residues 1-3.

[0308] Interleukin-12 (IL-12) The present invention provides IL-12 cytokine or its functional fragment for use in targeting cytokine or its cleavage product.Cytokines play a role in cell signaling, particularly in cells of the immune system.IL-12 is an interleukin, a type of cytokine signaling molecule of the immune system that regulates the activity of white blood cells.

[0309] Endogenous IL-12 exists as two distinct molecules, IL-12 p40 and IL-12 p35, which dimerize intracellularly during biosynthesis.

[0310] The full sequences of IL-12 p40 and IL-12 p35 are as follows (the propeptide cleaved during biosynthesis is shown in bold): TIFF2025508882000002.tif50170

[0311] The mature forms are as follows: IL-12 p40 subunit: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 111) IL-12 p35 subunit: RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 112)

[0312] They are expressed as two chains that covalently dimerize during biosynthesis via a disulfide bond between the two subunits: cysteine ​​C199 of the p40 subunit associates with cysteine ​​C96 of the p35 subunit.

[0313] A "functional fragment" of an IL-12 cytokine includes a portion of a full-length cytokine protein that retains or has altered cytokine receptor binding ability (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity compared to the full-length cytokine protein). Cytokine receptor binding ability can be demonstrated, for example, by the ability of the cytokine to bind to its cognate receptor or a component thereof.

[0314] In some embodiments, the IL-12 cytokine or functional fragment thereof is any naturally occurring interleukin-2 (IL-12) protein or modified variant thereof capable of binding to the interleukin-12 receptor.

[0315] In some embodiments, the IL-12 polypeptide or functional fragment thereof comprises an IL-12p40 polypeptide or functional fragment thereof covalently linked to an IL-12p35 polypeptide or functional fragment thereof.

[0316] The IL-12p40 polypeptide or functional fragment thereof may have a first polypeptide chain of the formula: N'HL1-L1-MM C' Including, the second polypeptide chain N'HL2-L2-[IL-12p40-linker-IL-12p35] C' and may be added to the first half-life extending domain to comprise: wherein "IL-12p40" is an IL-12p40 polypeptide or a functional fragment thereof, and "IL-12p35" is an IL-12p35 polypeptide or a functional fragment thereof.

[0317] 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 compared to the amino acid sequence of SEQ ID NO: 111. Each of the at least one amino acid modification can be any amino acid modification, such as a substitution, insertion, or deletion. In some embodiments, the IL-12 cytokine or 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: 111. In some embodiments, the IL-12 cytokine or 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: 111.

[0318] The IL-12p40 polypeptide contains a glycosaminoglycan (GAG)-binding domain. GAGs, such as heparin and heparan sulfate, have been shown to bind to numerous growth factors and cytokines, including IL-12. The physiological significance of this binding is twofold. First, GAGs can serve as coreceptors on the cell surface, maintaining high local concentrations of cytokines. Second, GAGs can regulate the biological activity of growth factors and cytokines through multiple mechanisms, including dimerization and protection from proteolysis.

[0319] The GAG-binding domain of the mature form of the IL-12 p40 subunit is shown below in bold. TIFF2025508882000003.tif20170

[0320] Modifications to the GAG-binding domain KSKREKKDRV (SEQ ID NO: 114) are shown herein to increase the PK profile of constructs containing the IL-12 cytokine with the mutated GAG-binding domain, without any reduction in cytokine activity. Thus, in some embodiments, the IL-12p40 polypeptide comprises at least one amino acid modification to the GAG-binding domain. In some embodiments, the modification to the GAG-binding domain is a deletion mutation. In some embodiments, the modification to the GAG-binding domain is a deletion mutation and at least one substitution mutation.

[0321] In some embodiments, the GAG ​​binding domain comprises the amino acid sequence KDNTERV. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO:115. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTERVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 115)

[0322] 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. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 116)

[0323] 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.

[0324] In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having one or more cysteine ​​substitutions compared to the amino acid sequence of SEQ ID NO: 111. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having an amino acid substitution at position C252 compared to the amino acid sequence of SEQ ID NO: 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. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEF GDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTC WWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAA EESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTW STPHSYFSLTFSVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEW ASVPCS (SEQ ID NO: 117)

[0325] In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having one or more cysteine ​​substitutions compared to the amino acid sequence of SEQ ID NO: 111 and at least one amino acid modification to the GAG-binding domain. In some embodiments, the IL-12p40 polypeptide comprises an amino acid substitution at position C252S compared to the amino acid sequence of SEQ ID NO: 111, and the GAG-binding domain comprises the amino acid sequence KDNTERV. In some embodiments, the IL-12p40 polypeptide comprises an amino acid substitution at position C252S compared to the amino acid sequence of SEQ ID NO: 111, 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. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 118)

[0326] 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 compared to the amino acid sequence of SEQ ID NO: 112. Each of the at least one amino acid modification can be any amino acid modification, such as a substitution, insertion, or deletion. In some embodiments, the IL-12 cytokine or 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: 112. In some embodiments, the IL-12 cytokine or 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: 112.

[0327] In some embodiments, the IL-12p40-IL-12p35 linker is between 5 and 20 amino acids in length.

[0328] In some embodiments, the IL-12p40-IL-12p35 linker is rich in G and S amino acid residues.

[0329] In some embodiments, the IL-12p40-IL-12p35 linker comprises only amino acid residue types selected from the group consisting of G and S.

[0330] In some embodiments, the IL-12p40-IL-12p35 linker comprises [(G)nS], where n=4 or 5.

[0331] In some embodiments, the IL-12p40-IL-12p35 linker comprises (GGGGS) repeats.

[0332] In some embodiments, the IL-12p40-IL-12p35 linker comprises SEQ ID NO: 119. (GGGGSGGGGSGGGGS)

[0333] In some embodiments, the IL-12 cytokine or functional fragment thereof comprises SEQ ID NO: 120. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises an amino acid sequence having at least one amino acid modification compared to the amino acid sequence of SEQ ID NOs: 111 and 112. Each of the at least one amino acid modification can be any amino acid modification, such as a substitution, insertion, or deletion. In some embodiments, the IL-12 cytokine or 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 NOs: 111 and 112. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises an amino acid sequence having at least 5 amino acid substitutions compared to the amino acid sequence of SEQ ID NOs: 111 and 112. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKT FLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGG SGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMA (SEQ ID NO: 120)

[0334] In some embodiments, the IL-12 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 120. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 120.

[0335] In some embodiments, the IL-12 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 121. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 121. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTIST DLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQG KDNTERVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 121)

[0336] In some embodiments, the IL-12 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 122. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 122. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTD LTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGK DNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 122)

[0337] In some embodiments, the IL-12 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 123. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 123. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTD LTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKS KREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 123)

[0338] In some embodiments, the IL-12 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 124. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 124. IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKT FLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGKDNTEGRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSG GGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALC (SEQ ID NO: 124)

[0339] Masking part Provided herein is a masking moiety for use in targeting cytokines. It will be understood that the masking moiety is cleaved from the targeting cytokine to form its cleavage product. The masking moiety binds to the cytokine moiety and inhibits the biological activity of the cytokine. Upon cleavage, the masking moiety is released from the cytokine and activates the function of the cytokine in the target of interest.

[0340] In some embodiments, the masking moiety comprises an agent, peptide, or polypeptide that binds to a cytokine.

[0341] In some embodiments, the masking moiety comprises a Fab, a single chain Fv (scFv), a single domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibody (bsFab), a single domain antibody-binding Fab (s-Fab), an antibody, or a combination thereof. In some embodiments, the masking moiety comprises a Fab that binds to a cytokine. In some embodiments, the masking moiety comprises a single chain Fv (scFv) that binds to a cytokine. In some embodiments, the masking moiety comprises a single domain antibody (VHH) that binds to a cytokine. In some embodiments, the masking moiety comprises one or more CDRs that bind to a cytokine. In some embodiments, the masking moiety comprises a variable heavy chain (VH) that binds to a cytokine. In some embodiments, the masking moiety comprises a variable light chain (VL) that binds to a cytokine. In some embodiments, the masking moiety comprises a Fab-like bispecific antibody (bsFab) that binds to a cytokine. In some embodiments, the masking moiety comprises a single domain antibody-binding Fab (s-Fab) that binds to a cytokine. In some embodiments, the masking moiety comprises an antibody or fragment thereof that binds to a cytokine. In some embodiments, the masking moiety comprises an antibody to a cytokine, or a binding fragment of that antibody.

[0342] In some embodiments, the masking moiety is a cytokine receptor. In some embodiments, the masking moiety is a fragment of a cytokine receptor. In some embodiments, the masking moiety comprises the extracellular domain (ECD) of a cytokine receptor.

[0343] In some embodiments, the cytokine is IL-1α or IL-1β and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to IL-1α or IL-1β. In some embodiments, the cytokine is IL-1α or IL-1β and the masking moiety is CD121a, CDw121b, or a fragment thereof.

[0344] In some embodiments, the cytokine is IL-2 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to IL-2. In some embodiments, the cytokine is IL-2 and the masking moiety is IL-2Rα, IL-2Rβ, CD25, CD122, CD132, or a fragment thereof.

[0345] In some embodiments, the cytokine is IL-18 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to IL-18. In some embodiments, the cytokine is IL-18 and the masking moiety is IL-18Rα, IL-18Rβ, or a fragment thereof.

[0346] In some embodiments, the cytokine is IL-4 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to IL-4. In some embodiments, the cytokine is IL-4 and the masking moiety is CD124, CD213a13, CD132, or a fragment thereof.

[0347] In some embodiments, the cytokine is IL-7 and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to IL-7. In some embodiments, the cytokine is IL-7 and the masking moiety is CD127, CD132, or a fragment thereof.

[0348] In some embodiments, the cytokine is IL-9 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to IL-9. In some embodiments, the cytokine is IL-9 and the masking moiety is IL-9R, CD132, or a fragment thereof.

[0349] In some embodiments, the cytokine is IL-13 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to IL-13. In some embodiments, the cytokine is IL-13 and the masking moiety is CD213a1, CD213a2, or a fragment thereof.

[0350] In some embodiments, the cytokine is IL-15 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to IL-15. In some embodiments, the cytokine is IL-15 and the masking moiety is IL-15Ra, CD122, CD132, or a fragment thereof.

[0351] In some embodiments, the cytokine is IL-3 and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to IL-3. In some embodiments, the cytokine is IL-3 and the masking moiety is CD123, CDw131, or a fragment thereof.

[0352] In some embodiments, the cytokine is IL-5 and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to IL-5. In some embodiments, the cytokine is IL-5 and the masking moiety is CDw125, CD131, or a fragment thereof.

[0353] In some embodiments, the cytokine is GM-CSF and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to GM-CSF, hi some embodiments, the cytokine is GM-CSF and the masking moiety is CD116, CDw131, or a fragment thereof.

[0354] In some embodiments, the cytokine is IL-6 and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to IL-6. In some embodiments, the cytokine is IL-6 and the masking moiety is CD126, CD130, or a fragment thereof.

[0355] In some embodiments, the cytokine is IL-11 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to IL-11. In some embodiments, the cytokine is IL-11 and the masking moiety is IL-11Ra, CD130, or a fragment thereof.

[0356] In some embodiments, the cytokine is G-CSF and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to G-CSF. In some embodiments, the cytokine is G-CSF and the masking moiety is CD114 or a fragment thereof.

[0357] In some embodiments, the cytokine is IL-12 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to IL-12. In some embodiments, the cytokine is IL-12 and the masking moiety is CD212 or a fragment thereof.

[0358] In some embodiments, the cytokine is LIF and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to LIF. In some embodiments, the cytokine is LIF and the masking moiety is LIFR, CD130, or a fragment thereof.

[0359] In some embodiments, the cytokine is OSM and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to OSM, hi some embodiments, the cytokine is OSM and the masking moiety is OSMR, CD130, or a fragment thereof.

[0360] In some embodiments, the cytokine is IL-10 and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to IL- 10. In some embodiments, the cytokine is IL-10 and the masking moiety is CDw210 or a fragment thereof.

[0361] In some embodiments, the cytokine is IL-20 and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to IL-20. In some embodiments, the cytokine is IL-20 and the masking moiety is IL-20Rα, IL-20Rβ, or a fragment thereof.

[0362] In some embodiments, the cytokine is IL-14 and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to IL-14. In some embodiments, the cytokine is IL-14 and the masking moiety is IL-14R or a fragment thereof.

[0363] In some embodiments, the cytokine is IL-16 and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to IL-16. In some embodiments, the cytokine is IL-16 and the masking moiety is CD4 or a fragment thereof.

[0364] In some embodiments, the cytokine is IL-17 and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to IL-17. In some embodiments, the cytokine is IL-17 and the masking moiety is CDw217 or a fragment thereof.

[0365] In some embodiments, the cytokine is IFN-α and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to IFN-α. In some embodiments, the cytokine is IFN-α and the masking moiety is CD118 or a fragment thereof.

[0366] In some embodiments, the cytokine is IFN-β and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to IFN-β. In some embodiments, the cytokine is IFN-β and the masking moiety is CD118 or a fragment thereof.

[0367] In some embodiments, the cytokine is IFN-γ and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to IFN-γ. In some embodiments, the cytokine is IFN-γ and the masking moiety is CDw119 or a fragment thereof.

[0368] In some embodiments, the cytokine is CD154 and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to CD154. In some embodiments, the cytokine is CD154 and the masking moiety is CD40 or a fragment thereof.

[0369] In some embodiments, the cytokine is LT-β and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to LT-β. In some embodiments, the cytokine is LT-β and the masking moiety is LT-βR or a fragment thereof.

[0370] In some embodiments, the cytokine is TNF-α and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to TNF-α, hi some embodiments, the cytokine is TNF-α and the masking moiety is CD120a, CD120b, or a fragment thereof.

[0371] In some embodiments, the cytokine is TNF-β and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to TNF-β, hi some embodiments, the cytokine is TNF-β and the masking moiety is CD120a, CD120b, or a fragment thereof.

[0372] In some embodiments, the cytokine is 4-1BBL and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to 4-1BBL. In some embodiments, the cytokine is 4-1BBL and the masking moiety is CDw137, 4-1BB, or a fragment thereof.

[0373] In some embodiments, the cytokine is APRIL and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to APRIL. In some embodiments, the cytokine is APRIL and the masking moiety is BCMA, TACI, or a fragment thereof.

[0374] In some embodiments, the cytokine is CD70 and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to CD70. In some embodiments, the cytokine is CD70 and the masking moiety is CD27 or a fragment thereof.

[0375] In some embodiments, the cytokine is CD153 and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to CD153. In some embodiments, the cytokine is CD153 and the masking moiety is CD30 or a fragment thereof.

[0376] In some embodiments, the cytokine is CD178 and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to CD178. In some embodiments, the cytokine is CD178 and the masking moiety is CD95 or a fragment thereof.

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

[0378] In some embodiments, the cytokine is LIGHT and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to LIGHT. In some embodiments, the cytokine is LIGHT and the masking moiety is LTVβR, HVEM, or a fragment thereof.

[0379] In some embodiments, the cytokine is OX40 and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to OX40. In some embodiments, the cytokine is OX40 and the masking moiety is OX40 or a fragment thereof.

[0380] In some embodiments, the cytokine is TALL-1 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to TALL-1. In some embodiments, the cytokine is TALL-1 and the masking moiety is BCMA, TACI, or a fragment thereof.

[0381] In some embodiments, the cytokine is TRAIL and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to TRAIL. In some embodiments, the cytokine is TRAIL and the masking moiety is TRAILR1-4 or a fragment thereof.

[0382] In some embodiments, the cytokine is TWEAK and the masking moiety is a Fab, single chain Fv (scFv), or single domain antibody to TWEAK. In some embodiments, the cytokine is TWEAK and the masking moiety is Apo3 or a fragment thereof.

[0383] In some embodiments, the cytokine is TRANCE and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to TRANCE, hi some embodiments, the cytokine is TRANCE and the masking moiety is RANK, OPG, or a fragment thereof.

[0384] In some embodiments, the cytokine is TGF-β1 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to TGF-β1. In some embodiments, the cytokine is TGF-β1 and the masking moiety is TGF-βR1 or a fragment thereof.

[0385] In some embodiments, the cytokine is TGF-β2 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to TGF-β2. In some embodiments, the cytokine is TGF-β2 and the masking moiety is TGF-βR2 or a fragment thereof.

[0386] In some embodiments, the cytokine is TGF-β3 and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to TGF-β3. In some embodiments, the cytokine is TGF-β3 and the masking moiety is TGF-βR3 or a fragment thereof.

[0387] In some embodiments, the cytokine is Epo and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to Epo. In some embodiments, the cytokine is Epo and the masking moiety is EpoR or a fragment thereof.

[0388] In some embodiments, the cytokine is Tpo and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to Tpo. In some embodiments, the cytokine is Tpo and the masking moiety is TpoR or a fragment thereof.

[0389] In some embodiments, the cytokine is Flt-3L and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to Flt-3L. In some embodiments, the cytokine is Flt-3L and the masking moiety is Flt-3 or a fragment thereof.

[0390] In some embodiments, the cytokine is SCF and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to SCF. In some embodiments, the cytokine is SCF and the masking moiety is CD117 or a fragment thereof.

[0391] In some embodiments, the cytokine is M-CSF and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to M-CSF, hi some embodiments, the cytokine is M-CSF and the masking moiety is CD115 or a fragment thereof.

[0392] In some embodiments, the cytokine is MSP and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to MSP. In some embodiments, the cytokine is MSP and the masking moiety is CDw136 or a fragment thereof.

[0393] In some embodiments, the cytokine is an IL-15 agonist polypeptide and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody to IL-15. In some embodiments, the cytokine moiety comprises an IL-15 agonist polypeptide and the chimeric molecule further comprises the sushi domain of IL-15 receptor α (IL-15Ra sushi domain).

[0394] In some embodiments, the cytokine is an IL-2 agonist polypeptide and the masking moiety is the extracellular domain of IL-21 receptor a (IL-21Ra ECD) or a functional analog thereof. In some embodiments, the cytokine is an IL-2 agonist polypeptide or an IL-15 agonist polypeptide and the masking moiety is the extracellular domain of IL-2 receptor b (IL-2R β ECD). In some embodiments, the cytokine is an IL-21 agonist polypeptide and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-21.

[0395] In some embodiments, the cytokine is an IL-2 agonist polypeptide and the masking moiety is the extracellular domain of IL-21 receptor a (IL-21Ra ECD) or a functional analog thereof. In some embodiments, the cytokine is an IL-2 agonist polypeptide or an IL-15 agonist polypeptide and the masking moiety is the extracellular domain of IL-2 receptor b (IL-2R β ECD). In some embodiments, the cytokine is an IL-21 agonist polypeptide and the masking moiety is a Fab, single-chain Fv (scFv), or single-domain antibody against IL-21.

[0396] CD122 (masking moiety of IL-2 and IL-15) The masking moiety masks the cytokine or functional fragment thereof in the targeted cytokine, thereby reducing or preventing binding of the cytokine or functional fragment thereof to its cognate receptor. In some embodiments, the masking moiety reduces or prevents binding of the IL-2 cytokine or functional fragment thereof to IL-2Rα (CD25). In some embodiments, the masking moiety provided herein refers to a moiety capable of binding to or otherwise exhibiting affinity for the IL-2 cytokine or functional fragment thereof, such as an anti-IL-2 antibody or an IL-2 cognate receptor protein. In some embodiments, the masking moiety reduces or prevents binding of the IL-15 cytokine or functional fragment thereof to IL-15Rα. In some embodiments, the masking moiety provided herein refers to a moiety capable of binding to or otherwise exhibiting affinity for the IL-15 cytokine or functional fragment thereof, 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.

[0397] In some embodiments, the masking moiety comprises an IL-2 cytokine receptor, or a subunit or functional fragment thereof.

[0398] In some embodiments, the masking moiety comprises CD122 (also known as IL-2Rβ) or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-2 and IL-15.

[0399] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:15. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 15)

[0400] 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: 15. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 15 with 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: 15 with one or two amino acid substitutions.

[0401] In some embodiments, CD122 or a fragment, portion, or variant thereof has a mutation at amino acid position C122 compared to CD122 of SEQ ID NO:15.

[0402] In some embodiments, CD122 or a fragment, portion, or variant thereof has a mutation C122S at amino acid position 122 compared to CD122 of SEQ ID NO:15.

[0403] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 15 with a C122 mutation.

[0404] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 15 with a C122S mutation.

[0405] In some embodiments, CD122 or a fragment, portion, or variant thereof has a mutation at amino acid position C168 compared to CD122 of SEQ ID NO:15.

[0406] In some embodiments, CD122 or a fragment, portion, or variant thereof has a mutation at amino acid position 168, C168S, compared to CD122 of SEQ ID NO:15.

[0407] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 15 with a C168 mutation.

[0408] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 15 with a C168S mutation.

[0409] In some embodiments, CD122 or a fragment, portion, or variant thereof has mutations at amino acid positions C122 and C168 compared to CD122 of SEQ ID NO:15.

[0410] In some embodiments, CD122 or a fragment, portion, or variant thereof has the mutations C122S and C168S compared to CD122 of SEQ ID NO:15.

[0411] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:16. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRSNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWISLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 16)

[0412] In some embodiments, the masking moiety comprises an amino acid sequence having at least about 80% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 91% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 16.

[0413] 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 a mutation F8C compared to CD122 of SEQ ID NO: 15.

[0414] 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 a mutation A94C compared to CD122 of SEQ ID NO: 15.

[0415] 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 a mutation L106C compared to CD122 of SEQ ID NO: 15.

[0416] 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 a mutation V117C compared to CD122 of SEQ ID NO: 15.

[0417] 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 a mutation C122S compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety has a mutation C122V ​​compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety has a mutation C122A compared to CD122 of SEQ ID NO: 15.

[0418] 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 a mutation N123C compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety has a mutation N123Q compared to CD122 of SEQ ID NO: 15.

[0419] In some embodiments, the masking moiety has a mutation at amino acid position C168 compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety has a mutation C168S compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety has a mutation C168V compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety has a mutation C168A compared to CD122 of SEQ ID NO: 15.

[0420] In some embodiments, the masking moiety has a mutation at amino acid position L169 compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety has a mutation L169C compared to CD122 of SEQ ID NO: 15.

[0421] In some embodiments, the masking moiety has a mutation at amino acid position Q177 compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety has a mutation Q177C compared to CD122 of SEQ ID NO: 15.

[0422] In some embodiments, the masking moiety has a mutation at amino acid position V184 compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety has a mutation V184C compared to CD122 of SEQ ID NO: 15.

[0423] In some embodiments, the masking moiety has a mutation at amino acid position S195 compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety has a mutation S195C compared to CD122 of SEQ ID NO: 15.

[0424] In some embodiments, the masking moiety has a mutation at amino acid position R204 compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety has a mutation R204C compared to CD122 of SEQ ID NO: 15.

[0425] In some embodiments, the masking moiety has the mutations C122V / C168V compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 17. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 17)

[0426] In some embodiments, the masking moiety has the mutations C122A / C168V compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 18. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 18)

[0427] In some embodiments, the masking moiety has the mutation C168V compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 19. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 19)

[0428] 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. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 20)

[0429] 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. (AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRACISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 21)

[0430] 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 moiety comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 21. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 21.

[0431] In some embodiments, the masking moiety has the mutations C122V / N123C compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:22. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRVCISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 22)

[0432] 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. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 23)

[0433] 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. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHCETHRCQISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 24)

[0434] 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. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCQISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIACETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 25)

[0435] In some embodiments, the masking moiety has the mutations L106C / C122A / C168A / S195C compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:26. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRVKPLQGEFTTWCPWSQPLAFRTKPAALGKD (SEQ ID NO: 26)

[0436] In some embodiments, the masking moiety has the mutations L106C / C122A / C168A / V184C compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:27. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRCKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 27)

[0437] In some embodiments, the masking moiety has the mutations C122A / C168A / V184C / S195C compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:28. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTQYEFQVRCKPLQGEFTTWCPWSQPLAFRTKPAALGKD (SEQ ID NO: 28)

[0438] In some embodiments, the masking moiety has the mutations C122A / C168A / Q177C / R204C compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:29. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIALETLTPDTCYEFQVRVKPLQGEFTTWSPWSQPLAFCTKPAALGKD (SEQ ID NO: 29)

[0439] In some embodiments, the masking moiety has the mutations L106C / C122V / C168V / S195C compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:30. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMAPISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWCPWSQPLAFRTKPAALGKD (SEQ ID NO: 30)

[0440] In some embodiments, the masking moiety has the mutations F8C / A94C / C122V / C168V compared to CD122 of SEQ ID NO: 15. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:31. AVNGTSQCTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMCIQDFKPFENLRLMAPISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 31)

[0441] IL-12 receptor The masking moiety masks the IL-12 cytokine or functional fragment thereof in the targeted cytokine, thereby reducing or preventing binding of the IL-cytokine or functional fragment thereof to its cognate receptor.

[0442] IL-12 receptor, beta 1, or IL-12Rβ1, is a subunit of the IL-12 receptor complex. IL-12Rβ1 is also known as CD212. This protein binds interleukin-12 (IL-12) with low affinity. The protein forms disulfide-linked oligomers, which are required for IL-12 binding activity. IL-12 receptor, beta 2, or IL-12Rβ2, is a subunit of the IL-12 receptor complex. Coexpression of IL-12Rβ1 and IL-12Rβ2 proteins has been shown to lead to the formation of a high-affinity IL-12 binding site.

[0443] In some embodiments, the masking moiety comprises the extracellular domain of the IL-12 cytokine receptor, or a subunit or functional fragment thereof.

[0444] The interleukin-12 receptor subunit beta-1, also known as CD212, has the following sequence: TIFF2025508882000004.tif45170

[0445] The interleukin-12 receptor subunit beta-2 has the following sequence: TIFF2025508882000005.tif55170

[0446] Bold indicates the propeptide, underlined italics indicates the extracellular domain, italics indicates the transmembrane domain, and underlined bold indicates the cytoplasmic domain.

[0447] 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.

[0448] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of human IL-12Rβ2 with one to four amino acid substitutions (SEQ ID NO: 125). 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.

[0449] In some embodiments, the masking moiety comprises residues 24 to 237 of human IL-12Rβ1, i.e., the sequence having SEQ ID NO: 126, or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-12. CRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRQLGSQGSSWSKWSSPVCVPPENP (SEQ ID NO: 126)

[0450] In some embodiments, the masking portion comprises an IL-12Rβ1 having SEQ ID NO: 126. 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: 126. In some embodiments, the masking portion comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 126. In some embodiments, the masking portion comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 126. In some embodiments, the masking portion comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 126. In some embodiments, the masking portion comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 126. In some embodiments, the masking portion comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 126. In some embodiments, the masking portion comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 126. In some embodiments, the masking portion comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 126. In some embodiments, the masking portion comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 126. In some embodiments, the masking portion comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 126. In some embodiments, the masking portion comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 126.

[0451] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 126 with 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 with one or two amino acid substitutions.

[0452] In some embodiments, the masking moiety comprises residues 24 to 545 of human IL-12Rβ1, i.e., the sequence having SEQ ID NO: 127, or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-12. CRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAG QLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRQLGSQGSSWSKWSSPVCVPPENPPQPQVRFSVEQLGQDGRRRLTLKEQPTQLELPEGCQGLAPGTEVTYRL QLHMLSCPCKAKATRTLHLGKMPYLSGAAYNVAVISSNQFGPGLNQTWHIPADTHTEPVALNISVGTNGTTMYWPARAQSMTYCIEWQPVGQDGGLATCSLTAPQDPDPAGMATYSWSRESGAMGQEKCYYITIFASAHPEKLTLWSTVLSTYHFGGNASAAGTPHHVSVKNHSLDSVSVDWAPSLLSTCPGVLKEYVVRCRDEDSKQVSEHPVQPTETQVTLSGLRAGVAYTVQVRADTAWLRGVWSQPQRFSIEVQVSD (SEQ ID NO: 127)

[0453] In some embodiments, the masking portion comprises an IL-12Rβ1 having SEQ ID NO: 127. 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: 127. In some embodiments, the masking portion comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 127. In some embodiments, the masking portion comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 127. In some embodiments, the masking portion comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 127. In some embodiments, the masking portion comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 127. In some embodiments, the masking portion comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 127. In some embodiments, the masking portion comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 127. In some embodiments, the masking portion comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 127. In some embodiments, the masking portion comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 127. In some embodiments, the masking portion comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 127. In some embodiments, the masking portion comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 127.

[0454] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 127 with 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 with one or two amino acid substitutions.

[0455] 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) 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-12Rβ2 with one or two amino acid substitutions. MAHTFRGCSLAFMFFIITWLLIKAKIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACIN SDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPST FTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTG MLDVWYMKRHIDYSRQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANS EGMDNILVTWQPPRKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSSILGNSKHKAPLSGPHINAITEEKGSIL ISWNSIPVQEQMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNSHPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKANWMAFVAPSICIAIIMVGIFSTHYF QQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML (SEQ ID NO: 155)

[0456] In some embodiments, the masking moiety comprises residues 24-212 of human IL-12Rβ2, ie, the sequence having SEQ ID NO:128. KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSL (SEQ ID NO: 128)

[0457] In some embodiments, the masking portion comprises an IL-12Rβ1 having SEQ ID NO: 128. 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 any one of the amino acid sequences of SEQ ID NO: 128. In some embodiments, the masking portion comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 128. In some embodiments, the masking portion comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 128. In some embodiments, the masking portion comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 128. In some embodiments, the masking portion comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 128. In some embodiments, the masking portion comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 128. In some embodiments, the masking portion comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 128. In some embodiments, the masking portion comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 128. In some embodiments, the masking portion comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 128. In some embodiments, the masking portion comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 128. In some embodiments, the masking portion comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 128.

[0458] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 128 with 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 with one or two amino acid substitutions.

[0459] In some embodiments, the masking moiety comprises residues 24-222 of human IL-12Rβ2, ie, the sequence having SEQ ID NO:129. KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIV (SEQ ID NO: 129)

[0460] In some embodiments, the masking portion comprises an IL-12Rβ1 having SEQ ID NO: 129. 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 any one of the amino acid sequences of SEQ ID NO: 129. In some embodiments, the masking portion comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 129. In some embodiments, the masking portion comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 129. In some embodiments, the masking portion comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 129. In some embodiments, the masking portion comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 129. In some embodiments, the masking portion comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 129. In some embodiments, the masking portion comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 129. In some embodiments, the masking portion comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 129. In some embodiments, the masking portion comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 129. In some embodiments, the masking portion comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 129. In some embodiments, the masking portion comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 129.

[0461] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 129 with 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 with one or two amino acid substitutions.

[0462] In some embodiments, the masking moiety comprises residues 24-319 of human IL-12Rβ2, ie, the sequence having SEQ ID NO:130. KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEE (SEQ ID NO: 130)

[0463] In some embodiments, the masking portion comprises an IL-12Rβ1 having SEQ ID NO: 130. 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 any one of the amino acid sequences of SEQ ID NO: 130. In some embodiments, the masking portion comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 130. In some embodiments, the masking portion comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 130. In some embodiments, the masking portion comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 130. In some embodiments, the masking portion comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 130. In some embodiments, the masking portion comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 130. In some embodiments, the masking portion comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 130. In some embodiments, the masking portion comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 130. In some embodiments, the masking portion comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 130. In some embodiments, the masking portion comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 130. In some embodiments, the masking portion comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 130.

[0464] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 130 with 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 with one or two amino acid substitutions.

[0465] In some embodiments, the masking moiety comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 130, with one or more cysteine ​​substitutions. In some embodiments, the masking moiety comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 130, with 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. KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRSTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEE (SEQ ID NO: 131)

[0466] 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: 131. In some embodiments, the masking portion consists of the amino acid sequence of SEQ ID NO: 131.

[0467] In some embodiments, the masking moiety comprises residues 24 to 622 of human IL-12Rβ2, ie, the sequence having SEQ ID NO:132. KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYT LQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQ ISSKLHLYKGSWSDWSESLRAQTPEEEPTGMLDVWYMKRHIDYSRQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGMDNILVTWQPPRKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSSILGNSKHKAPLSGPHINAITEEKGSILISWNSIPVQEQMGCLLHYRIYWKER (SEQ ID NO: 132)

[0468] In some embodiments, the masking portion comprises an IL-12Rβ1 having SEQ ID NO: 132. 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 SEQ ID NO: 132. In some embodiments, the masking portion comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 132. In some embodiments, the masking portion comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 132. In some embodiments, the masking portion comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 132. In some embodiments, the masking portion comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 132. In some embodiments, the masking portion comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 132. In some embodiments, the masking portion comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 132. In some embodiments, the masking portion comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 132. In some embodiments, the masking portion comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 132. In some embodiments, the masking portion comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 132. In some embodiments, the masking portion comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 132.

[0469] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 132 with 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 with one or two amino acid substitutions.

[0470] In some embodiments, the masking moiety comprises residues 24-227 of human IL-12Rβ2, ie, the sequence having SEQ ID NO:133. KIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPP (SEQ ID NO: 133)

[0471] In some embodiments, the masking portion comprises an IL-12Rβ1 having SEQ ID NO: 133. 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: 133. In some embodiments, the masking portion comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 133. In some embodiments, the masking portion comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 133. In some embodiments, the masking portion comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 133. In some embodiments, the masking portion comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 133. In some embodiments, the masking portion comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 133. In some embodiments, the masking portion comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 133. In some embodiments, the masking portion comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 133. In some embodiments, the masking portion comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 133. In some embodiments, the masking portion comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 133. In some embodiments, the masking portion comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 133.

[0472] In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 133 with 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 with one or two amino acid substitutions.

[0473] Antibody-based masking moiety The masking moiety that masks the cytokine or its functional fragment in the targeting cytokine can be an antibody or its antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment binds to the cytokine. The cytokine and anti-cytokine antibody or binding fragment complex in the targeting cytokine thereby reduces or prevents the cytokine or its functional fragment from binding to its cognate receptor or ligand (e.g., masks the activity of the cytokine).

[0474] The cytokine-specific antibody can be an intact antibody or a binding fragment thereof. In some embodiments, binding fragments derived from antibodies include, but are 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.

[0475] Single-chain variable fragment (scFv) 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.

[0476] 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. [Table 1]

[0477] 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 GGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 182).

[0478] In some embodiments, the scFv masking moiety is linked to the Fc domain by a cleavable linker. In some embodiments, the scFv masking moiety is linked to the Fc domain by a cleavable linker described herein (e.g., a linker comprising a cleavable peptide described in Table 1).

[0479] In some embodiments, the masking portion comprises an scFv having an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences of SEQ ID NO: 180. In some embodiments, the masking portion comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 180. In some embodiments, the masking portion comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 180. In some embodiments, the masking portion comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 180. In some embodiments, the masking portion comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 180. In some embodiments, the masking portion comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 180. In some embodiments, the masking portion comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 180. In some embodiments, the masking portion comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 180. In some embodiments, the masking portion comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 180. In some embodiments, the masking portion comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 180. In some embodiments, the masking portion comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 180.

[0480] VHH antibody VHH antibodies (or nanobodies) are antigen-binding fragments of heavy chain-only antibodies. In some embodiments, the masking moiety is a heavy chain-only antibody (VHH). In some embodiments, the masking moiety is a heavy chain-only antibody (VHH) that binds to a cytokine. VHHs can be derived from organisms that produce VHH antibodies, such as camelids, and sharks. In some cases, VHHs can be recombinant VHHs. VHH technology is based on fully functional antibodies from camelids that lack light chains. These heavy chain antibodies contain a single variable domain (VH A VHH comprises a single polypeptide chain having three CDRs and four framework regions (FR1-FR4). As used herein, "framework region" or "FR" refers to the region in the variable domain located between the CDRs. As used herein, "complementarity-determining region" or "CDR" refers to the variable region in a VHH that contains an amino acid sequence capable of specifically binding to an antigenic target (e.g., a cytokine).

[0481] In some embodiments, a VHH comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at the N- or C-terminus to comprise only partial FR1 and / or FR4, or to lack one or both of these framework regions, so long as the VHH substantially maintains cytokine binding and specificity.

[0482] VHHs are not limited to a particular biological source or a particular preparation method. For example, VHHs generally consist of (1) the VH of a natural heavy chain antibody; H (2) By isolating the H domain, the natural V H (3) expression of a nucleotide sequence encoding the H domain to produce a naturally occurring V H By "humanizing" the H domain or by such a humanized V H(4) by "camelization" of a naturally occurring VH domain from any animal species, e.g., from a mammalian species such as human, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by "camelization" of a "domain antibody" or "Dab" as described in the art, or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides, or other amino acid sequences known in the art; (7) by preparing a nucleic acid encoding a VHH using techniques for nucleic acid synthesis known in the art, followed by expression of the nucleic acid thus obtained; and / or (8) by any combination of one or more of the foregoing.

[0483] In some embodiments, the IL-2 masking moiety is an anti-IL-2 VHH antibody. The IL-2 masking moiety is a VHH of a natural heavy chain antibody directed against IL-2. H The VHH corresponding to the H domain of such an anti-IL-2 VHH is H The H sequence can generally be generated or obtained by appropriate immunization of a camelid species with an IL-2 molecule. In some embodiments, a V sequence directed against an IL-2 molecule is H The H sequence is included by appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies.

[0484] 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 an hCDR1 of SEQ ID NO: 169, an hCDR2 of SEQ ID NO: 170, and an hCDR3 of SEQ ID NO: 171.

[0485] [Table 2]

[0486] In some embodiments, the masking moiety comprises a VHH having an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences of 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 portion comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 168. In some embodiments, the masking portion comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 168. In some embodiments, the masking portion comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 168. In some embodiments, the masking portion comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 168. In some embodiments, the masking portion comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 168.

[0487] In some embodiments, the VHH masking moiety is linked to the Fc domain by a cleavable linker. In some embodiments, the VHH masking moiety is linked to the Fc domain by a cleavable linker described herein (e.g., a linker comprising a cleavable peptide described in Table 1).

[0488] Linker Provided herein are linkers for use in targeting cytokines or cleavage products thereof. The linkers provided herein refer to peptides of two or more amino acids that are used to link together two functional components in the targeting cytokines described herein.

[0489] The targeted cytokine comprises a first linker and a second linker, wherein at least the first linker or the second linker comprises a proteolytically cleavable peptide.

[0490] In some embodiments, the first Fc polypeptide is linked to the cytokine or its variant 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 its variant 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 its variant 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 its variant 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 its variant 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 variant thereof via a non-cleavable linker and the second Fc polypeptide is linked to the masking moiety via a non-cleavable linker.

[0491] Non-cleavable linkers In some embodiments, the non-cleavable linker is between 2 and 25 amino acids in length. In some embodiments, the non-cleavable linker is between 3 and 21 amino acids in length. In some embodiments, the non-cleavable linker is between 3 and 18 amino acids in length. In some embodiments, the non-cleavable linker is between 5 and 18 amino acids in length. In some embodiments, the non-cleavable linker is between 3 and 8 amino acids in length. In some embodiments, the non-cleavable linker is between 4 and 6 amino acids in length.

[0492] In some embodiments, the non-cleavable linker is 15 amino acids in length. In some embodiments, the non-cleavable linker is 16 amino acids in length. In some embodiments, the non-cleavable linker is 17 amino acids in length. In some embodiments, the non-cleavable linker is 18 amino acids in length. In some embodiments, the non-cleavable linker is 19 amino acids in length. In some embodiments, the non-cleavable linker is 20 amino acids in length.

[0493] In some embodiments, the non-cleavable linker is rich in the amino acid residues G, S, and P. In some embodiments, the non-cleavable linker comprises only amino acid residue types selected from the group consisting of G, S, and P. In some embodiments, the non-cleavable linker comprises "GS" repeats. In some embodiments, the non-cleavable linker comprises an N'-terminal "P" residue.

[0494] In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 32 (PGSGS).

[0495] In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 33 (GGSSPPGGGSSGGGSGP).

[0496] In some embodiments, the non-cleavable linker comprises the amino acid sequence GGS.

[0497] In some embodiments, the non-cleavable linker is [(G) n S], where n=4 or 5.

[0498] In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 134 (GGGGS).

[0499] In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 135 (GGGGSGGGGS).

[0500] In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 136 (GGSGGGSGGGGGS).

[0501] In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 137 (GGSGGSGGSGGSGGSSGP).

[0502] In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 138 (PGGSGP).

[0503] In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO:56 (GGSPG).

[0504] In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 181 (GGSSGSGGSGGGSGSGGG).

[0505] In some embodiments, the second linker comprises a proteolytically cleavable peptide such that the second linker is a proteolytically cleavable linker, the first linker does not comprise a proteolytically cleavable peptide such that the first linker is a non-proteolytically cleavable linker, and the non-cleavable linker is between 3 and 18 amino acids in length. In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 134 (GGGGS). In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 135 (GGGGSGGGGS). In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 136 (GGSGGGSGGGGGS). In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 137 (GGSGGSGGSGGSGGSSGP). In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 138 (PGGSGP). In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 139 (GGSPG).

[0506] In some embodiments, the first and second polypeptide chains are desirably the same or similar length to facilitate association of the first half-life prolonging domain with the second half-life prolonging domain and masking of the cytokine or functional fragment thereof in the assembled construct. Thus, if the masking moiety is a shorter amino acid sequence than the cytokine or functional fragment thereof, the difference in length can be fully or partially compensated for by using a longer linker L1.

[0507] Cleavable Linker In some embodiments, the cleavable linker is between 2 and 25 amino acids in length. In some embodiments, the cleavable linker is between 3 and 21 amino acids in length. In some embodiments, the cleavable linker is between 3 and 18 amino acids in length. In some embodiments, the cleavable linker is between 5 and 18 amino acids in length. In some embodiments, the cleavable linker is between 3 and 8 amino acids in length. In some embodiments, the cleavable linker is between 4 and 6 amino acids in length.

[0508] In some embodiments, the cleavable linker is 15 amino acids in length. In some embodiments, the cleavable linker is 16 amino acids in length. In some embodiments, the cleavable linker is 17 amino acids in length. In some embodiments, the cleavable linker is 18 amino acids in length. In some embodiments, the cleavable linker is 19 amino acids in length. In some embodiments, the cleavable linker is 20 amino acids in length.

[0509] In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide (CP) flanked by spacer domains (SD) as shown in the following formula: SD-CP-SD

[0510] Cleavable peptides The cleavable linker comprises a cleavable peptide.

[0511] A cleavable peptide is a polypeptide that contains a protease cleavage site so that the cleavable peptide can be proteolytically cleaved. A protease is an enzyme that cleaves and hydrolyzes a peptide bond between two specific amino acid residues in a target substrate protein. As used herein, "cleavage site" refers to a recognizable site for cleavage of a portion of a cleavable peptide found in any of the linkers comprising the cleavable peptides described herein. Thus, the cleavage site can be found in the sequence of the cleavable peptides described herein. In some embodiments, the cleavage site is an amino acid sequence that is recognized and cleaved by a cleaving agent.

[0512] 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, the expression of which is specific to or upregulated in tumor cells or the tumor cell environment.

[0513] The tumor cell environment is complex and may contain multiple different proteases. Therefore, the exact site at which a given cleavable peptide is cleaved in the tumor cell environment may vary between tumor types, between patients with the same tumor type, and even between cleavage products formed within the same tumor, depending on the specific tumor cell environment. Furthermore, even after cleavage, further modification of the initial cleavage product, for example, by removal of one or two terminal amino acids, may occur due to further action of proteases in the tumor cell environment. Therefore, the distribution of cleavage products can be expected to be formed in the tumor cell environment of a patient after administration of a single-structure targeting cytokine as described herein.

[0514] As referred to herein, a cleavage site is understood to refer to a site between two specific amino acid residues within a cleavable peptide that is a target of a protease known to be associated with the tumor cell environment. In this sense, two or more cleavage sites may be present within a cleavable peptide described herein, and different proteases may cleave the cleavable peptide at different cleavage sites. Also, two or more proteases may act on the same cleavage site within a cleavable peptide. Discussion of protease cleavage sites can be found in the art.

[0515] Thus, the cleavable peptides disclosed herein may be cleaved by one or more proteases. In some embodiments, the protease cleavage site is a tumor-associated protease cleavage site. The tumor-associated protease cleavage site can be recognized by a tumor-associated protease. As a non-limiting example, the tumor-associated protease is a matrix metalloproteinase (MMP) selected from the group consisting of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP27, and MMP28. In one embodiment, the tumor-associated protease is MMP2. In another embodiment, the tumor-associated protease is MMP3. In some embodiments, the tumor-associated protease is MMP7. In yet other embodiments, the tumor-associated protease is MMP9. In yet other embodiments, the tumor-associated protease is MMP10. Other disease-associated and tissue-selective proteases include cathepsins (cathepsin B (catB), cathepsin D, cathepsin F, cathepsin K, cathepsin L, cathepsin V, cathepsin S, cathepsin W), ADAMs, ADAMTS, kallikreins 1-15, HTRA1-2-3, HGFAc, PRSS, TMPRSS, elastase, PR-3, granzymes (granzymes A, B, M, H, and K), fibroblast activation protein (FAP), plasmin, urokinase plasminogen activator (uPA), tryptase, caspase, thrombin, legumain, chymase, collagenase, napsin A, and matriptase 1-2. In some embodiments, the disease-associated and / or tissue-selective protease is cathepsin (cathepsin B (catB)).

[0516] In some embodiments, the cleavable peptide is a substrate for a protease that is co-localized in a region or tissue that expresses a cytokine receptor.

[0517] In some embodiments, the cleavable peptide is a 5-mer (i.e., a peptide 5 amino acids in length), a 6-mer (i.e., a peptide 6 amino acids in length), a 7-mer (i.e., a peptide 7 amino acids in length), an 8-mer (i.e., a peptide 8 amino acids in length), a 9-mer (i.e., a peptide 9 amino acids in length), a 10-mer (i.e., a peptide 10 amino acids in length), an 11-mer (i.e., a peptide 11 amino acids in length), a 12-mer (i.e., a peptide 12 amino acids in length), a 13-mer (i.e., a peptide 13 amino acids in length), a 14-mer (i.e., a peptide 14 amino acids in length), a 15-mer (i.e., a peptide 15 amino acids in length), a 16-mer (i.e., a peptide 16 amino acids in length), a 17-mer (i.e., a peptide 17 amino acids in length), or an 18-mer (i.e., a peptide 18 amino acids in length).

[0518] In some embodiments, the cleavable peptide is between 5 and 18 amino acids in length. In some embodiments, the cleavable peptide is between 6 and 10 amino acids in length.

[0519] In some embodiments, the cleavable peptide in the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 35, 36, 37, 38, 39, and 40. In some embodiments, the cleavable peptide in the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 35, 36, 37, 38, 39, and 40.

[0520] In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 34. In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 35. In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 36. In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 37. In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 38. In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 39. In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 40. In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 183 (PANLVAPDP). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 184 (IHVTLKSL). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 185 (NSYTIKGL). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 186 (SNESLSLS). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 187 (SQESLSLS). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 188 (QVSSSLSP). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 189 (PTSTSLSP). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 190 (ESLSLSEE). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 191 (ASLSLAPV). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 192 (PLGL). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 193 (RPLALWRS). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 194 (TQKPLGLS). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 195 (APAGLIVPYN).In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 196 (PVSLRSGS). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 197 (GMPKDLYHAS). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 198 (RSKYLATA). In some embodiments, the cleavable peptide in the cleavable linker comprises SEQ ID NO: 199 (IVGRPRHQGV). [Table 3] TIFF2025508882000009.tif235170

[0521] Purely by way of example, in the table above, * indicates a known or observed protease cleavage site within the cleavable peptide.

[0522] In some embodiments, the cleavable peptide comprises an amino acid sequence selected from Table 1.

[0523] In some embodiments, the cleavable peptide comprises the amino acid sequence 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).

[0524] In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 41 (MPYDLYHPS). In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 42 (VPLSLYSG). In som...

Claims

1. a) targeting moiety; b) a cytokine or a fragment thereof; c) a masking portion, and d) an Fc domain comprising a first Fc polypeptide linked to said cytokine or fragment thereof via a first linker and a second Fc polypeptide linked to said masking moiety via a second linker; A targeted cytokine, comprising: the masking moiety binds to the cytokine or a fragment thereof; the first or second linker is a cleavable linker, such that the masking moiety, upon cleavage, releases the cytokine or fragment thereof; the cleavable linker (i) MPYDLYHP (SEQ ID NO: 34) or VPLSLYSG (SEQ ID NO: 42); (ii) an amino acid sequence selected from Table 1; (iii) 8 to 50 amino acid residues, and / or (iv) has an in vitro cleavage efficiency that yields at least 10% active cytokine; and the targeting moiety is linked to the Fc domain via one or both of the first and second Fc polypeptides; Targeted cytokines.

2. a) targeting moiety; b) a cytokine or a fragment thereof; c) a masking moiety comprising 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 comprising a first Fc polypeptide linked to said cytokine or fragment thereof via a first linker and a second Fc polypeptide linked to said masking moiety via a second linker; A targeted cytokine, comprising: the masking moiety binds to the cytokine or a fragment thereof; the first or second linker is a cleavable linker, such that the masking moiety, upon cleavage, releases the cytokine or fragment thereof; the targeting moiety is linked to the Fc domain via one or both of the first and second Fc polypeptides, and optionally CD122 or a fragment thereof is (i) C122S and C168S, (ii) C122V ​​and C168V; (iii) C122A and C168V; (iv) C168V, (v) C122V ​​and C168A; (vi) C122A and N123C, (vii) C122V ​​and N123C; (viii) C122A and C168A, (ix) V117C, N123Q and C168A, (x) N123Q, C168A and L169C, (xi) L106C, C122A, C168A and S195C (xii) L106C, C122A, C168A and V184C (xiii) C122A, C168A, V184C, and S195C, (xiv) C122A, C168A, Q177C, and R204C, (xv) L106C, C122V, C168V, and S195C, or (xvi) F8C, A94C, C122V ​​and C168V, comprising a mutation selected from Targeted cytokines.

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

4. The targeting moiety may be PD-1, PD-L1, PD-L2, CTLA-4, TIGIT, TIM-3, LAG-3, CD25, CD16a, CD16b, NKG2D, NKP44, NKP30, CD19, CD20, CD30, CD38, BCMA, human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epidermal growth factor receptor (EGFR), glypican-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGF R1), vascular endothelial growth factor receptor 2 (VEGF R2), or a combination thereof. R2), Nectin-4, Liv-1, glycoprotein NMB (GPNMB), prostate-specific membrane antigen (PSMA), Trop-2, carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), prostate six-transmembrane epithelial antigen 1 (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 4. The targeted cytokine of any one of claims 1 to 3, which specifically binds to 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.

5. a) a targeting moiety that specifically binds to PD-1; b) a cytokine or a fragment thereof; c) a masking portion, and d) an Fc domain comprising a first Fc polypeptide linked to said cytokine or fragment thereof via a first linker and a second Fc polypeptide linked to said masking moiety via a second linker; A targeted cytokine, comprising: the masking moiety binds to the cytokine or a fragment thereof; the first or second linker is a cleavable linker, such that the masking moiety, upon cleavage, releases the cytokine or fragment thereof; the cleavable linker (i) MPYDLYHP (SEQ ID NO: 34) or VPLSLYSG (SEQ ID NO: 42), and / or (ii) an amino acid sequence selected from Table 1; and A targeted cytokine, wherein the targeting moiety is linked to the Fc domain via one or both of the first and second Fc polypeptides.

6. a) (i) HCDR1 (GYTFTNYY) of SEQ ID NO: 4, HCDR2 (INPSNGGT) of SEQ ID NO: 5, HCDR3 (ARRDYRFDMGFDY) of SEQ ID NO: 6, LCDR1 (KGVSTSGYSY) of SEQ ID NO: 9, LCDR2 (LAS) of SEQ ID NO: 10, LCDR3 (QHSRDLPLT) of SEQ ID NO: 11; or (ii) HCDR1 of GITFSNSG (SEQ ID NO: 161), HCDR2 of VIWYDGSKRYYADSVKG (SEQ ID NO: 162), HCDR3 of ATNDDY (SEQ ID NO: 163), LCDR1 of QSVSSY (SEQ ID NO: 164), LDR2 of DAS (SEQ ID NO: 165), and LCDR3 of QQSSNWPRT (SEQ ID NO: 166); a targeting moiety that specifically binds to PD-1, comprising: b) a cytokine or a fragment thereof, and c) an Fc domain, optionally comprising (i) a first Fc polypeptide comprising a CH3 domain comprising a modification that reduces or eliminates binding to Protein A; and (ii) a second Fc polypeptide comprising a CH3 domain that binds to Protein A; 2. A targeted cytokine comprising:

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

8. The targeted cytokine of claim 6, wherein the targeted cytokine further comprises a masking moiety, and wherein the first Fc polypeptide is linked to the cytokine or a fragment thereof via a first linker, the second Fc polypeptide is linked to the masking moiety via a second linker, and optionally the targeting moiety is linked to the Fc domain via one or both of the first and second Fc polypeptides.

9. 9. The targeted cytokine of claim 8, wherein the masking moiety binds to the cytokine or a fragment thereof and / or the first or second linker is a cleavable linker, such that the masking moiety releases the cytokine or a fragment thereof upon cleavage.

10. 10. The targeted cytokine of any one of claims 7-9, 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).

11. 8. The targeted cytokine of any one of claims 1-3 and 5-7, wherein the cleavable linker comprises a proteolytically cleavable peptide flanked on both sides by spacer domains, optionally the spacer domains being between 3 and 10 amino acids, or between 3 and 6 amino acids in length.

12. 8. The targeted cytokine of any one of claims 1 to 3 and 5 to 7, wherein the targeting moiety and / or masking moiety comprises one or more antigen-binding domains, peptides, polypeptides, proteins, ligands, or drugs that specifically bind to the cytokine or a fragment thereof, or wherein the targeting moiety comprises an antigen-binding domain selected from the group consisting of a Fab, a single-chain Fv (scFv), a single-domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibody (bsFab), a single-domain antibody-binding Fab (s-Fab), an antibody, and combinations thereof.

13. the targeted cytokine comprises a first antigen-binding domain and a second antigen-binding domain, and optionally (i) the first and second antigen-binding domains specifically bind to the same target; (ii) the first and second antigen-binding domains comprise the same amino acid sequence; (iii) the first and second antigen-binding domains specifically bind to different targets; or (iv) The targeted cytokine of any one of claims 1 to 3 and 5 to 7, wherein the first and second antigen-binding domains comprise different amino acid sequences.

14. the C-terminus of the first antigen-binding domain is linked to the N-terminus of the first Fc polypeptide; the C-terminus of the first Fc polypeptide is linked to the N-terminus of the cytokine or fragment thereof; the C-terminus of the second antigen-binding domain is linked to the N-terminus of the second Fc polypeptide; and 14. The targeted cytokine of claim 13, wherein the C-terminus of the second Fc polypeptide is linked to the N-terminus of the masking moiety.

15. 8. The targeted cytokine of any one of claims 1 to 3 and 5 to 7, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises a first IgG1, IgG2, or IgG4 Fc domain or a fragment thereof.

16. The first and / or second Fc domains each contain one or more modifications that promote non-covalent association of said first and said second Fc polypeptides, and optionally: a) the first Fc domain comprises a Y349C mutation, a T366S mutation, a L368A mutation, a Y407V mutation, and a N297A mutation, and the second Fc domain comprises a S354C mutation, a T366W mutation, and a N297A mutation; or b) the second Fc domain comprises a Y349C mutation, a T366S mutation, a L368A mutation, a Y407V mutation, and a N297A mutation, and the first Fc domain comprises a S354C mutation, a T366W mutation, and a N297A mutation; or c) the first Fc domain comprises a Y349C mutation, a T366S mutation, a L368A mutation, a Y407V mutation, a N297A mutation, and a I253A mutation, and the second Fc domain comprises a S354C mutation, a T366W mutation, a N297A mutation, and a I253A mutation; or d) the second Fc domain comprises a Y349C mutation, a T366S mutation, a L368A mutation, a Y407V mutation, a N297A mutation, and a I253A mutation, and the first Fc domain comprises a S354C mutation, a T366W mutation, a N297A mutation, and a I253A mutation; or e) the first Fc polypeptide comprises a first CH3 domain comprising 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; The targeted cytokine of any one of claims 1 to 3 and 5 to 7.

17. 17. The targeted cytokine of claim 16, wherein the CH3 domain that binds Protein A is a human IgGl, IgG2, or IgG4 sequence, and optionally the second CH3 domain is a human IgGl, IgG2, or IgG4 sequence comprising an alteration at positions H435 and / or Y436 according to the Kabat numbering. (i) the first or second Fc domain comprises SEQ ID NO:85 or SEQ ID NO:80; or (ii) The targeted cytokine of any one of claims 1-3 and 5-7, wherein the first or second Fc domain comprises SEQ ID NO:84 or SEQ ID NO:

80.

19. The cytokine or 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, 8. The targeted cytokine of any one of claims 1-3 and 5-7, which is IFN-β, IFN-γ, CD154, LT-β, ​​4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β, M-CSF, or MSP, or a fragment thereof.

20. The masking moiety is a receptor for the cytokine or a fragment thereof, and optionally is selected from the group consisting of 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.

21. 8. The targeted cytokine of any one of claims 1 to 3 and 5 to 7, wherein the cytokine is an IL-2 cytokine or a functional fragment thereof that is modified compared to the sequence of mature IL-2 having SEQ ID NO:

13.

22. The modified IL-2 cytokine or functional fragment thereof comprises: (i) reducing or eliminating binding to CD25; (ii) enhances binding to CD25; (iii) reduces or eliminates binding to CD122, or (iv) comprises a modification that enhances binding to CD122; Optionally, the modified IL-2 cytokine or functional fragment thereof comprises: (i) the modifications R38A, F42A, Y45A, and E62A relative to the sequence of mature IL-2 having SEQ ID NO: 13; (ii) the 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; 22. The targeted cytokine of claim 21.

23. The cytokine comprises an 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 compared to the amino acid sequence of SEQ ID NO:93, and optionally, the modification is: (i) one or more amino acid substitutions at positions D22, E46, or E53 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 compared to the amino acid sequence of SEQ ID NO: 93; (iii) amino acid substitutions at positions N71 and N79 compared to the amino acid sequence of SEQ ID NO: 93; (iv) amino acid substitutions at positions N71 and N112 compared to the amino acid sequence of SEQ ID NO: 93; or (v) comprising amino acid substitutions at positions N79 and N112 compared to the amino acid sequence of SEQ ID NO: 93; The targeted cytokine of any one of claims 1-3 and 5-6.

24. The cytokine of claim 24, wherein the cytokine comprises an 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, and optionally: (a) the IL-12p40 polypeptide comprises SEQ ID NO:111 or an amino acid sequence having at least one amino acid modification compared to the amino acid sequence of SEQ ID NO:111; (b) the IL-12p40 polypeptide comprises at least one amino acid modification to the GAG ​​binding domain (KSKREKKDRV) of SEQ ID NO: 114, compared to the amino acid sequence of SEQ ID NO: 111; (c) the IL-12p40 polypeptide comprises SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, or SEQ ID NO:118; (d) the IL-12p35 polypeptide comprises an amino acid sequence comprising SEQ ID NO: 112 or having at least one amino acid modification compared to the amino acid sequence of SEQ ID NO: 112; and / or (e) the IL-12 polypeptide or 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; The targeted cytokine of any one of claims 1-3 and 5-6.

25. the IL-12p40-IL-12p35 linker is between 5 and 20 amino acids in length, and optionally the IL-12p40-IL-12p35 linker is (i) is rich in the amino acid residues G and S; (ii) comprises SEQ ID NO: 119 (GGGGSGGGGSGGGGGS); 25. The targeted cytokine of claim 24.

26. 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, and optionally, the masking moiety comprises: (i) 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: 132; or (ii) comprising the extracellular domain of human IL-12Rβ2, or a fragment, portion, or variant thereof that retains or otherwise exhibits affinity for IL-12; 25. The targeted cytokine of claim 24.

27. A nucleic acid or vector encoding a targeted cytokine according to any one of claims 1 to 3 and 5 to 7.

28. 28. A host cell comprising the nucleic acid or vector of claim 27.

29. A method for producing a targeted cytokine, comprising culturing a host cell according to claim 28 under conditions for producing the targeted cytokine.

30. A pharmaceutical composition comprising a targeted cytokine according to any one of claims 1 to 3 and 5 to 7 and a pharmaceutically acceptable carrier.

31. A kit comprising a targeted cytokine according to any one of claims 1 to 3 and 5 to 7.

32. a) For treating neoplastic diseases; b) for treating or preventing inflammatory diseases; or c) for treating or preventing autoimmune diseases; A medicament comprising a targeted cytokine according to any one of claims 1 to 3 and 5 to 7.

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