Immunocytokines and uses thereof

Abstract

The present application relates to immunocytokines, methods for making and uses thereof, including cytokines or variants thereof located in the hinge region of a heavy chain of an antibody (e.g., a full-length antibody) or in the hinge region between an antigen-binding fragment (e.g., a ligand, receptor, or antibody fragment) and an Fc domain subunit or portion thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 130,339, filed December 23, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] Submission of sequence listing as an ASCII text file The contents of the following ASCII text file submission are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 754392000640SEQLIST.TXT, Recorded: December 23, 2021, Size: 1,037,246 bytes).

[0003] The present invention relates to immunocytokines, methods for making and uses thereof, including cytokines or variants thereof located in the hinge region of the heavy chain of an antibody (e.g., a full-length antibody) or in the hinge region between an antigen-binding fragment (e.g., a ligand, receptor, or antibody fragment) and an Fc domain subunit or portion thereof. [Background technology]

[0004] Cytokines are key regulators of the innate and adaptive immune systems, enabling immune cells to communicate with each other. Cytokine therapy to stimulate the immune system in cancer patients has always been a major area of ​​interest in clinical cancer research. A key challenge with cytokine monotherapy is achieving effective antitumor responses without causing treatment-limiting toxicities. The low response rates and notorious toxicities of IL-2 and IL-12 therapy exemplify this dilemma. High doses of IL-2 have been shown to induce blood leak syndrome (VLS), tumor tolerance caused by activation-induced cell death (AICD), and immunosuppression caused by the activation of regulatory T cells (Tregs). These severe side effects often limit optimal IL-2 dosing and therefore limit the number of patients who successfully respond to this therapy. IL-12 has demonstrated modest antitumor responses in clinical trials, but is often accompanied by significant toxicity issues (Lasek et al., Cancer Immunol Immunother., 2014). IL-12 treatment has been found to be associated with systemic flu-like symptoms (e.g., fever, chills, fatigue, erythromelalgia, and headache) and toxic effects on the bone marrow and liver. Dose-finding studies have shown that patients can only tolerate IL-12 at doses below 1 μg / kg, far below the therapeutically effective dose. IL-12, whether used as monotherapy or in combination with other drugs, has failed to demonstrate strong and sustained therapeutic efficacy in clinical trials (Lasek et al., 2014).

[0005] Several approaches have been taken to overcome the problems associated with cytokine monotherapy. Recently, NKTR-214, a recombinant human IL-2 conjugated with polyethylene glycol (PEG) ("IL-2-PEG"), has shown promising results in animal models. IL-2-PEG offers two benefits. First, steric hindrance of PEG masks the region on IL-2 that interacts with the IL-2 receptor alpha (IL-2Rα) subunit, which is involved in the activation of immunosuppressive Tregs, thereby biasing activity toward tumor-killing CD8+ T cells (Charych et al., Clin Cancer Res., 2016). Second, PEG conjugation significantly improves plasma half-life and proteolytic stability, reducing immunogenicity and hepatic uptake (Chaffee et al., J Clin Invest., 1992; Pyatak et al., Res Commun Chem Pathol Pharmacol., 1980). Targeted delivery of cytokines (e.g., IL-12) to tumor sites by local injection or by using immunocytokines (cytokines fused to antibodies, antibody fragments, or ligand / receptor-Fc fusion proteins) has also been developed to overcome the side effects of cytokine therapy. Immunocytokines can target cytokines to cells or tissues of interest, such as tumor cells or immune effector cells (Klein et al., Oncoimmunology, 2017; King et al., J Clin Oncol., 2004).

[0006] The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entireties. Summary of the Invention [Means for solving the problem]

[0007] One aspect of the present application provides an immunocytokine comprising: a) an antigen-binding protein (e.g., an antibody, or a ligand / receptor-Fc fusion protein) that specifically recognizes a target antigen; and b) a cytokine or variant thereof, wherein the antigen-binding protein comprises, from N' to C': an antigen-binding polypeptide (e.g., a heavy chain, or a ligand / receptor-Fc fusion polypeptide) comprising an antigen-binding fragment (e.g., an antibody fragment, a ligand, or a receptor), a hinge region, and an Fc domain subunit or portion thereof, and the cytokine or variant thereof is located in the hinge region (e.g., at the N-terminus, the C-terminus, or within the hinge region).

[0008] In some embodiments of any one of the immunocytokines described above, in the presence of an antigen binding protein (e.g., an antibody, or a ligand / receptor-Fc fusion protein) binding to a target antigen, the activity of the cytokine or variant thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antigen binding protein to the target antigen.

[0009] In some embodiments of any one of the immunocytokines described above, in the absence of binding of an antigen binding protein (e.g., an antibody or a ligand / receptor-Fc fusion protein) to a target antigen, the activity of the cytokine or variant thereof located in the hinge region is about 70% or less (such as about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding cytokine or variant thereof in the free state.

[0010] In some embodiments of any one of the immunocytokines described above, the cytokine or variant thereof is a cytokine variant, and the activity of the cytokine variant in its free state is about 80% or less (e.g., about 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% or less) of the activity of the corresponding wild-type cytokine in its free state.

[0011] In some embodiments of any one of the immunocytokines described above, the antigen binding protein (e.g., an antibody or a ligand / receptor-Fc fusion protein) comprises two antigen binding polypeptides (e.g., heavy chains or ligand / receptor-Fc fusion polypeptides) each comprising a hinge region, and only one of the antigen binding polypeptides comprises the cytokine or variant thereof located in the hinge region. In some embodiments, the antigen binding protein comprises two antigen binding polypeptides each comprising a hinge region, and each antigen binding polypeptide comprises the cytokine or variant thereof located in the hinge region.

[0012] In some embodiments of any one of the immunocytokines described above, the immunocytokine comprises two or more (e.g., two, three, four, or more) cytokines or variants thereof, wherein the two or more cytokines or variants thereof are located in tandem in the hinge region of the antigen-binding polypeptide (e.g., a heavy chain, or a ligand / receptor-Fc fusion polypeptide).

[0013] In some embodiments of any one of the immunocytokines described above, the cytokine or variant thereof is a monomeric cytokine or variant thereof (e.g., IL-2, IFN-α such as IFN-α2b). In some embodiments, the cytokine or variant thereof is a dimeric cytokine or variant thereof (e.g., IFN-γ, IL-10, IL-12, or IL-23). ​​In some embodiments, both subunits of the dimeric cytokine or variant thereof are located in tandem in the hinge region of an antigen-binding polypeptide (e.g., a heavy chain, or a ligand / receptor-Fc fusion polypeptide). In some embodiments, the antigen-binding protein comprises two antigen-binding polypeptides each comprising a hinge region, wherein one subunit of the dimeric cytokine or variant thereof is located in the hinge region of one antigen-binding polypeptide and the other subunit of the dimeric cytokine or variant thereof is located in the hinge region of the other antigen-binding polypeptide.

[0014] In some embodiments of any one of the immunocytokines described above, two or more (e.g., two, three, four, or more) cytokines or variants thereof are the same. In some embodiments, two or more (e.g., two, three, four, or more) cytokines or variants thereof are different.

[0015] In some embodiments of any one of the immunocytokines described above, the antigen binding protein is a monospecific antigen binding protein (e.g., a monospecific antibody or a ligand / receptor-Fc fusion protein). In some embodiments, the antigen binding protein is a multispecific antigen binding protein (e.g., a multispecific antibody or a ligand / receptor-Fc fusion protein).

[0016] In some embodiments of any one of the immunocytokines described above, the target antigen is selected from the group consisting of TIGIT, PD-1, PD-L1, PD-L2, CTLA-4, CD3, CD4, CD8, CD123, CD25, and HER2.

[0017] In some embodiments of any one of the immunocytokines described above, the cytokine or variant thereof is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IL-35, IFN-α, IFN-β, IFN-γ, TNF-α, TGF-β, VEGF, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, and GM-CSF. In some embodiments, the cytokine or variant thereof is selected from the group consisting of IL-2, IL-10, IL-12, IL-23, IFN-α, and IFN-γ. In some embodiments, the cytokine or variant thereof is IL-2 or a variant thereof. In some embodiments, the IL-2 variants comprise one or more mutations at positions selected from the group consisting of L18, Q22, F24, K35, R38, F42, K43, E61, P65, Q126, and S130 relative to wild-type IL-2 comprising the sequence of SEQ ID NO: 1. In some embodiments, the IL-2 variants comprise one or more mutations selected from the group consisting of L18R, Q22E, F24A, R38D, K43E, E61R, P65L, Q126T, and S130R relative to wild-type IL-2 comprising the sequence of SEQ ID NO: 1. In some embodiments, the IL-2 variant comprises an R38D / K43E / E61R mutation, an L18R / Q22E / R38D / K43E / E61R mutation, an R38D / K43E / E61R / Q126T mutation, an L18R / Q22E / R38D / K43E / E61R / Q126T mutation, or an L18R / Q22E / R38D / K43E / E61R / Q126T / S130R mutation relative to wild-type IL-2 comprising the sequence of SEQ ID NO: 1. In some embodiments, the IL-2 variant comprises the sequence of any one of SEQ ID NOs: 2 and 251-254. In some embodiments, the cytokine or variant thereof is IFN-α or a variant thereof.In some embodiments, the IFN-α variant comprises one or more mutations at positions selected from the group consisting of R22, L26, F27, L30, K31, D32, R33, H34, D35, F36, S68, T79, K83, Y85, Y89, R120, K121, Y122, Q124, Y129, K131, E132, R144, and E146 compared to an IFN-α comprising the sequence of SEQ ID NO: 3. In some embodiments, the IFN-α variant comprises one or more mutations selected from the group consisting of L30A, K31A, D32A, R33A, H34A, and D35A compared to an IFN-α comprising the sequence of SEQ ID NO: 3. In some embodiments, the IFN-α variant comprises the sequence of any of SEQ ID NOs: 4-9. In some embodiments, the IFN-α variant comprises an L30A mutation compared to an IFN-α comprising the sequence of SEQ ID NO: 3. In some embodiments, the IFN-α variant comprises the sequence of SEQ ID NO: 4. In some embodiments, the cytokine or variant thereof is IFN-γ or a variant thereof. In some embodiments, the IFN-γ variant comprises one or more mutations within one or both IFN-γ subunits at positions selected from the group consisting of V5, S20, D21, V22, A23, D24, N25, G26, H111, and Q115 relative to a wild-type IFN-γ subunit comprising the sequence of SEQ ID NO: 10. In some embodiments, the IFN-γ variant comprises one or more mutations within one or both IFN-γ subunits at positions selected from the group consisting of S20A, D21A, D21K, V22A, A23S, A23E, A23Q, A23V, D24A, D24E, N25A, N25K, and H111D relative to a wild-type IFN-γ subunit comprising the sequence of SEQ ID NO: 10. In some embodiments, the IFN-γ variant comprises, within one or both IFN-γ subunits, one or more mutations selected from the group consisting of S20A / D21A, D21K, V22A / A23S, D24A / N25A, A23E / D24E / N25K, A23Q, and A23V relative to a wild-type IFN-γ subunit comprising the sequence of SEQ ID NO: 10. In some embodiments, one or both subunits of the IFN-γ variant comprise the sequence of any of SEQ ID NOs: 11-17.In some embodiments, the IFN-γ variant comprises an A23V mutation within one or both IFN-γ subunits relative to a wild-type IFN-γ subunit comprising the sequence of SEQ ID NO: 10. In some embodiments, one or both subunits of the IFN-γ variant comprise the sequence of SEQ ID NO: 13. In some embodiments, the two subunits of the IFN-γ or variant thereof are connected by a linker. In some embodiments, the IFN-γ variant comprises the sequence of SEQ ID NO: 19. In some embodiments, the cytokine or variant thereof is IL-10 or a variant thereof. In some embodiments, the IL-10 variant comprises one or more mutations within one or both IL-10 subunits at a position selected from the group consisting of N21, M22, R24, D25, L26, R27, D28, A29, F30, S31, R32, H90, and S93 relative to a wild-type IL-10 subunit comprising the sequence of SEQ ID NO: 20. In some embodiments, the IL-10 variant comprises, within one or both IL-10 subunits, one or more mutations selected from the group consisting of R24A, D25A, L26A, R27A, D28A, A29S, F30A, S31A, and R32A relative to a wild-type IL-10 subunit comprising the sequence of SEQ ID NO: 20. In some embodiments, the IL-10 variant comprises, within one or both IL-10 subunits, one or more mutations selected from the group consisting of R24A, D25A / L26A, R27A, D28A / A29S, F30A / S31A, and R32A relative to a wild-type IL-10 subunit comprising the sequence of SEQ ID NO: 20. In some embodiments, one or both subunits of the IL-10 variant comprise the sequence of any of SEQ ID NOs: 21-26. In some embodiments, the IL-10 variant comprises an R27A mutation within one or both IL-10 subunits compared to a wild-type IL-10 subunit comprising the sequence of SEQ ID NO: 20. In some embodiments, one or both subunits of the IL-10 variant comprise the sequence of SEQ ID NO: 23. In some embodiments, the two subunits of IL-10 or a variant thereof are connected by a linker. In some embodiments, the IL-10 variant comprises the sequence of SEQ ID NO: 28.In some embodiments, the cytokine or variant thereof is IL-12 or a variant thereof. In some embodiments, the IL-12 variant comprises one or more mutations within the p40 subunit at positions selected from the group consisting of E45, Q56, V57, K58, E59, F60, G61, D62, A63, G64, Q65, and C177 relative to a wild-type p40 subunit comprising the sequence of SEQ ID NO: 30. In some embodiments, the IL-12 variant comprises one or more mutations within the p40 subunit selected from the group consisting of Q56A, V57A, K58A, E59A, F60A, G61A, D62A, A63S, G64A, and Q65A relative to a wild-type p40 subunit comprising the sequence of SEQ ID NO: 30. In some embodiments, the p40 subunit of the IL-12 variant comprises the sequence of any of SEQ ID NOs: 31-34. In some embodiments, the IL-12 variant comprises an E59A / F60A mutation within the p40 subunit compared to a wild-type p40 subunit comprising the sequence of SEQ ID NO: 30. In some embodiments, the p40 subunit of the IL-12 variant comprises the sequence of SEQ ID NO: 31. In some embodiments, the IL-12 variant comprises an F60A mutation within the p40 subunit compared to a wild-type p40 subunit comprising the sequence of SEQ ID NO: 30. In some embodiments, the p40 subunit of the IL-12 variant comprises the sequence of SEQ ID NO: 33. In some embodiments, the p40 subunit and the p35 subunit of the IL-12 or variant thereof are connected by a linker. In some embodiments, the IL-12 variant comprises the sequence of SEQ ID NO: 36, 275, 331, or 332. In some embodiments, the cytokine or variant thereof is IL-23 or a variant thereof. In some embodiments, the IL-23 variant comprises one or more mutations within the p40 subunit at positions selected from the group consisting of E45, Q56, V57, K58, E59, F60, G61, D62, A63, G64, Q65, and C177 relative to a wild-type p40 subunit comprising the sequence of SEQ ID NO: 30.In some embodiments, the IL-23 variant comprises one or more mutations within the p40 subunit selected from the group consisting of Q56A, V57A, K58A, E59A, F60A, G61A, D62A, A63S, G64A, and Q65A relative to a wild-type p40 subunit comprising the sequence of SEQ ID NO: 30. In some embodiments, the p40 subunit of the IL-23 variant comprises the sequence of any of SEQ ID NOs: 31-34. In some embodiments, the IL-23 variant comprises an E59A / F60A mutation within the p40 subunit relative to a wild-type p40 subunit comprising the sequence of SEQ ID NO: 30. In some embodiments, the p40 subunit of the IL-23 variant comprises the sequence of SEQ ID NO: 31. In some embodiments, the p40 subunit and p19 subunit of IL-23 or its variants are connected by a linker. In some embodiments, the IL-23 variant comprises the sequence of SEQ ID NO: 39. In some embodiments, the linker comprises the sequence of any of SEQ ID NOs: 194-242, such as any of SEQ ID NOs: 227-229.

[0018] In some embodiments of any one of the immunocytokines described above, the cytokine or variant thereof is located within the hinge region of the antigen-binding polypeptide (e.g., the heavy chain, or the ligand / receptor-Fc fusion polypeptide).

[0019] In some embodiments of any one of the immunocytokines described above, the Fc domain subunit, or portion thereof, comprises a knob-into-hole (KIH) mutation.

[0020] In some embodiments of any one of the immunocytokines described above, the antigen-binding protein is an antibody that specifically recognizes a target antigen, the antigen-binding polypeptide comprising the hinge region is a heavy chain of the antibody, and the cytokine or variant thereof is located in the hinge region of the heavy chain. In some embodiments, the antibody is an anti-HER2 antibody comprising: i) a VH-CDR1 comprising the sequence of SEQ ID NO: 188; ii) a VH-CDR2 comprising the sequence of SEQ ID NO: 189; iii) a VH-CDR3 comprising the sequence of SEQ ID NO: 190; iv) a VL-CDR1 comprising the sequence of SEQ ID NO: 191; v) a VL-CDR2 comprising the sequence of SEQ ID NO: 192; and vi) a VL-CDR3 comprising the sequence of SEQ ID NO: 193. In some embodiments, the antibody comprises a VH comprising the sequence of SEQ ID NO: 150 and a VL comprising the sequence of SEQ ID NO: 151. In some embodiments, the antibody comprises a light chain comprising the sequence of SEQ ID NO: 154, and (i) the cytokine or variant thereof is an IL-2 variant comprising the sequence of SEQ ID NO: 2, and the heavy chain comprising the IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 156; (ii) the cytokine or variant thereof is an IFN-α variant comprising the sequence of SEQ ID NO: 4, and the heavy chain comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 157; (iii) the cytokine or variant thereof is an IFN-γ variant comprising the sequence of SEQ ID NO: 19, and the heavy chain comprising the IFN-γ variant located in the hinge region comprises the sequence of SEQ ID NO: 158. (iv) the cytokine or variant thereof is an IL-10 variant comprising the sequence of SEQ ID NO: 28, and the heavy chain comprising the IL-10 variant located in the hinge region comprises the sequence of SEQ ID NO: 159; (v) the cytokine or variant thereof is an IL-12 variant comprising the sequence of SEQ ID NO: 36, and the heavy chain comprising the IL-12 variant located in the hinge region comprises the sequence of SEQ ID NO: 160; or (vi) the cytokine or variant thereof is an IL-23 variant comprising the sequence of SEQ ID NO: 39, and the heavy chain comprising the IL-23 variant located in the hinge region comprises the sequence of SEQ ID NO: 161. In some embodiments, the antibody comprises a second heavy chain that does not comprise a cytokine or variant thereof located in the hinge region, and this second heavy chain comprises the sequence of SEQ ID NO: 155.In some embodiments, the antibody is an anti-CD3 antibody comprising i) a VH-CDR1 comprising the sequence of SEQ ID NO: 85; ii) a VH-CDR2 comprising the sequence of SEQ ID NO: 86; iii) a VH-CDR3 comprising the sequence of SEQ ID NO: 87; iv) a VL-CDR1 comprising the sequence of SEQ ID NO: 88; v) a VL-CDR2 comprising the sequence of SEQ ID NO: 89; and vi) a VL-CDR3 comprising the sequence of SEQ ID NO: 90. In some embodiments, the antibody comprises a VH comprising the sequence of SEQ ID NO: 91 and a VL comprising the sequence of SEQ ID NO: 92. In some embodiments, the antibody comprises a light chain comprising the sequence of SEQ ID NO: 94, and (i) the cytokine or variant thereof is an IL-2 variant comprising the sequence of SEQ ID NO: 2, and the heavy chain comprising the IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 96; (ii) the cytokine or variant thereof is an IFN-α variant comprising the sequence of SEQ ID NO: 4, and the heavy chain comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 97; (iii) the cytokine or variant thereof is an IFN-γ variant comprising the sequence of SEQ ID NO: 19, and the heavy chain comprising the IFN-γ variant located in the hinge region comprises the sequence of SEQ ID NO: , comprising the sequence of SEQ ID NO: 98; (iv) the cytokine or variant thereof is an IL-10 variant comprising the sequence of SEQ ID NO: 28, and the heavy chain comprising the IL-10 variant located in the hinge region comprises the sequence of SEQ ID NO: 99; (v) the cytokine or variant thereof is an IL-12 variant comprising the sequence of SEQ ID NO: 36, and the heavy chain comprising the IL-12 variant located in the hinge region comprises the sequence of SEQ ID NO: 100; or (vi) the cytokine or variant thereof is an IL-23 variant comprising the sequence of SEQ ID NO: 39, and the heavy chain comprising the IL-23 variant located in the hinge region comprises the sequence of SEQ ID NO: 101. In some embodiments, the antibody comprises a second heavy chain that does not comprise a cytokine or variant thereof located in the hinge region, and this second heavy chain comprises the sequence of SEQ ID NO: 95. In some embodiments, the antibody is an anti-PD-1 antibody comprising a VH-CDR1, VH-CDR2, and VH-CDR3 comprising the sequence of SEQ ID NO: 102, and a VL-CDR1, VL-CDR2, and VL-CDR3 comprising the sequence of SEQ ID NO: 103. In some embodiments, the antibody comprises a VH comprising the sequence of SEQ ID NO: 102 and a VL comprising the sequence of SEQ ID NO: 103.In some embodiments, the antibody comprises a light chain comprising the sequence of SEQ ID NO: 106, and (i) the cytokine or variant thereof is an IL-2 variant comprising the sequence of SEQ ID NO: 2, and the heavy chain comprising the IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 108; (ii) the cytokine or variant thereof is an IFN-α variant comprising the sequence of SEQ ID NO: 4, and the heavy chain comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 109; (iii) the cytokine or variant thereof is an IFN-γ variant comprising the sequence of SEQ ID NO: 19, and the heavy chain comprising the IFN-γ variant located in the hinge region comprises the sequence of SEQ ID NO: (iv) the cytokine or variant thereof is an IL-10 variant comprising the sequence of SEQ ID NO: 28, and the heavy chain comprising the IL-10 variant located in the hinge region comprises the sequence of SEQ ID NO: 111; (v) the cytokine or variant thereof is an IL-12 variant comprising the sequence of SEQ ID NO: 36, and the heavy chain comprising the IL-12 variant located in the hinge region comprises the sequence of SEQ ID NO: 112; or (vi) the cytokine or variant thereof is an IL-23 variant comprising the sequence of SEQ ID NO: 39, and the heavy chain comprising the IL-23 variant located in the hinge region comprises the sequence of SEQ ID NO: 113. In some embodiments, the antibody comprises a second heavy chain that does not comprise a cytokine or variant thereof located in the hinge region, and this second heavy chain comprises the sequence of SEQ ID NO: 107. In some embodiments, the antibody is an anti-CD4 antibody comprising i) a VH-CDR1 comprising the sequence of SEQ ID NO: 67; ii) a VH-CDR2 comprising the sequence of SEQ ID NO: 68; iii) a VH-CDR3 comprising the sequence of SEQ ID NO: 69; iv) a VL-CDR1 comprising the sequence of SEQ ID NO: 70; v) a VL-CDR2 comprising the sequence of SEQ ID NO: 71; and vi) a VL-CDR3 comprising the sequence of SEQ ID NO: 72. In some embodiments, the antibody comprises a VH comprising the sequence of SEQ ID NO: 73 and a VL comprising the sequence of SEQ ID NO: 74.In some embodiments, the antibody comprises a light chain comprising the sequence of SEQ ID NO: 77, and (i) the cytokine or variant thereof is an IL-2 variant comprising the sequence of SEQ ID NO: 2, and the heavy chain comprising the IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 79; (ii) the cytokine or variant thereof is an IFN-α variant comprising the sequence of SEQ ID NO: 4, and the heavy chain comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 80; (iii) the cytokine or variant thereof is an IFN-γ variant comprising the sequence of SEQ ID NO: 19, and the heavy chain comprising the IFN-γ variant located in the hinge region comprises the sequence of SEQ ID NO: 80. comprises the sequence of SEQ ID NO: 81; (iv) the cytokine or variant thereof is an IL-10 variant comprising the sequence of SEQ ID NO: 28, and the heavy chain comprising the IL-10 variant located in the hinge region comprises the sequence of SEQ ID NO: 82; (v) the cytokine or variant thereof is an IL-12 variant comprising the sequence of SEQ ID NO: 36, and the heavy chain comprising the IL-12 variant located in the hinge region comprises the sequence of SEQ ID NO: 83; or (vi) the cytokine or variant thereof is an IL-23 variant comprising the sequence of SEQ ID NO: 39, and the heavy chain comprising the IL-23 variant located in the hinge region comprises the sequence of SEQ ID NO: 84. In some embodiments, the antibody comprises a second heavy chain that does not comprise a cytokine or variant thereof located in the hinge region, and this second heavy chain comprises the sequence of SEQ ID NO: 78. In some embodiments, the antibody is an anti-CD8 antibody comprising VH-CDR1, VH-CDR2, and VH-CDR3 of a VH comprising the sequence of SEQ ID NO: 114, and VL-CDR1, VL-CDR2, and VL-CDR3 of a VL comprising the sequence of SEQ ID NO: 115. In some embodiments, the antibody comprises a VH comprising the sequence of SEQ ID NO: 114 and a VL comprising the sequence of SEQ ID NO: 115.In some embodiments, the antibody comprises a light chain comprising the sequence of SEQ ID NO: 117, and (i) the cytokine or variant thereof is an IL-2 variant comprising the sequence of SEQ ID NO: 2, and the heavy chain comprising the IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 119; (ii) the cytokine or variant thereof is an IFN-α variant comprising the sequence of SEQ ID NO: 4, and the heavy chain comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 120; (iii) the cytokine or variant thereof is an IFN-γ variant comprising the sequence of SEQ ID NO: 19, and the heavy chain comprising the IFN-γ variant located in the hinge region comprises the sequence of SEQ ID NO: (iv) the cytokine or variant thereof is an IL-10 variant comprising the sequence of SEQ ID NO: 28, and the heavy chain comprising the IL-10 variant located in the hinge region comprises the sequence of SEQ ID NO: 122; (v) the cytokine or variant thereof is an IL-12 variant comprising the sequence of SEQ ID NO: 36, and the heavy chain comprising the IL-12 variant located in the hinge region comprises the sequence of SEQ ID NO: 123; or (vi) the cytokine or variant thereof is an IL-23 variant comprising the sequence of SEQ ID NO: 39, and the heavy chain comprising the IL-23 variant located in the hinge region comprises the sequence of SEQ ID NO: 124. In some embodiments, the antibody comprises a second heavy chain that does not comprise a cytokine or variant thereof located in the hinge region, and this second heavy chain comprises the sequence of SEQ ID NO: 118. In some embodiments, the antibody is an anti-CTLA-4 antibody comprising VH-CDR1, VH-CDR2, and VH-CDR3 of a VH comprising the sequence of SEQ ID NO: 125, and VL-CDR1, VL-CDR2, and VL-CDR3 of a VL comprising the sequence of SEQ ID NO: 126. In some embodiments, the antibody comprises a VH comprising the sequence of SEQ ID NO: 125 and a VL comprising the sequence of SEQ ID NO: 126.In some embodiments, the antibody comprises a light chain comprising the sequence of SEQ ID NO: 129, and (i) the cytokine or variant thereof is an IL-2 variant comprising the sequence of SEQ ID NO: 2, and the heavy chain comprising the IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 131; (ii) the cytokine or variant thereof is an IFN-α variant comprising the sequence of SEQ ID NO: 4, and the heavy chain comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 132; (iii) the cytokine or variant thereof is an IFN-γ variant comprising the sequence of SEQ ID NO: 19, and the heavy chain comprising the IFN-γ variant located in the hinge region comprises the sequence of SEQ ID NO: (iv) the cytokine or variant thereof is an IL-10 variant comprising the sequence of SEQ ID NO: 28, and the heavy chain comprising the IL-10 variant located in the hinge region comprises the sequence of SEQ ID NO: 134; (v) the cytokine or variant thereof is an IL-12 variant comprising the sequence of SEQ ID NO: 36, and the heavy chain comprising the IL-12 variant located in the hinge region comprises the sequence of SEQ ID NO: 135; or (vi) the cytokine or variant thereof is an IL-23 variant comprising the sequence of SEQ ID NO: 39, and the heavy chain comprising the IL-23 variant located in the hinge region comprises the sequence of SEQ ID NO: 136. In some embodiments, the antibody comprises a second heavy chain that does not comprise a cytokine or variant thereof located in the hinge region, and this second heavy chain comprises the sequence of SEQ ID NO: 130. In some embodiments, the antibody comprises i) a VH-CDR1 comprising the sequence of SEQ ID NO: 243; ii) a VH-CDR2 comprising the sequence of SEQ ID NO: 244; iii) a VH-CDR3 comprising the sequence of SEQ ID NO: 245; iv) a VL-CDR1 comprising the sequence of SEQ ID NO: 246; v) a VL-CDR2 comprising the sequence of SEQ ID NO: 247; and vi) a VL-CDR3 comprising the sequence of SEQ ID NO: 248. In some embodiments, the antibody comprises a VH comprising the sequence of SEQ ID NO: 137 and a VL comprising the sequence of SEQ ID NO: 138. In some embodiments, the antibody comprises a light chain comprising the sequence of SEQ ID NO: 140, and (i) the cytokine or variant thereof is an IL-2 variant comprising the sequence of SEQ ID NO: 2, and the heavy chain comprising the IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 144; (ii) the cytokine or variant thereof is an IFN-α variant comprising the sequence of SEQ ID NO: 4, and the heavy chain comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 145; (iii) the cytokine or variant thereof is an IFN-γ variant comprising the sequence of SEQ ID NO: 19, and the heavy chain comprising the IFN-γ variant located in the hinge region comprises the sequence of SEQ ID NO: 146. (iv) the cytokine or variant thereof is an IL-10 variant comprising the sequence of SEQ ID NO: 28, and the heavy chain comprising the IL-10 variant located in the hinge region comprises the sequence of SEQ ID NO: 147; (v) the cytokine or variant thereof is an IL-12 variant comprising the sequence of SEQ ID NO: 36, and the heavy chain comprising the IL-12 variant located in the hinge region comprises the sequence of SEQ ID NO: 148; or (vi) the cytokine or variant thereof is an IL-23 variant comprising the sequence of SEQ ID NO: 39, and the heavy chain comprising the IL-23 variant located in the hinge region comprises the sequence of SEQ ID NO: 149. In some embodiments, the antibody comprises a second heavy chain that does not comprise a cytokine or variant thereof located in the hinge region, and this second heavy chain comprises the sequence of SEQ ID NO: 143. In some embodiments, the antibody is an anti-CD25 antibody comprising VH-CDR1, VH-CDR2, and VH-CDR3 of a VH comprising the sequence of SEQ ID NO: 162, and VL-CDR1, VL-CDR2, and VL-CDR3 of a VL comprising the sequence of SEQ ID NO: 163. In some embodiments, the antibody comprises a VH comprising the sequence of SEQ ID NO: 162 and a VL comprising the sequence of SEQ ID NO: 163.In some embodiments, the antibody comprises a light chain comprising the sequence of SEQ ID NO: 165, and (i) the cytokine or variant thereof is an IL-2 variant comprising the sequence of SEQ ID NO: 2, and the heavy chain comprising the IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 169; (ii) the cytokine or variant thereof is an IFN-α variant comprising the sequence of SEQ ID NO: 4, and the heavy chain comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 170; (iii) the cytokine or variant thereof is an IFN-γ variant comprising the sequence of SEQ ID NO: 19, and the heavy chain comprising the IFN-γ variant located in the hinge region comprises the sequence of SEQ ID NO: (iv) the cytokine or variant thereof is an IL-10 variant comprising the sequence of SEQ ID NO: 28, and the heavy chain comprising the IL-10 variant located in the hinge region comprises the sequence of SEQ ID NO: 172; (v) the cytokine or variant thereof is an IL-12 variant comprising the sequence of SEQ ID NO: 36, and the heavy chain comprising the IL-12 variant located in the hinge region comprises the sequence of SEQ ID NO: 173; or (vi) the cytokine or variant thereof is an IL-23 variant comprising the sequence of SEQ ID NO: 39, and the heavy chain comprising the IL-23 variant located in the hinge region comprises the sequence of SEQ ID NO: 174. In some embodiments, the antibody comprises a second heavy chain that does not comprise a cytokine or variant thereof located in the hinge region, and this second heavy chain comprises the sequence of SEQ ID NO: 168. In some embodiments, the antibody is an anti-PD-1 antibody that has reduced binding affinity to PD-1 relative to nivolumab (at least about 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 50-, 100-, 1000-, or more fold reduced). In some embodiments, the antibody is an anti-PD-1 antibody comprising VH-CDR1, VH-CDR2, and VH-CDR3 of a VH comprising the sequence of SEQ ID NO: 102, and VL-CDR1, VL-CDR2, and VL-CDR3 of a VL comprising the sequence of SEQ ID NO: 103, wherein VH-CDR3 comprises any one of the following mutations relative to SEQ ID NO: 102: D100N, D100G, D100R, N99G, N99A, or N99M.In some embodiments, the antibody comprises a light chain comprising the sequence of SEQ ID NO: 106, wherein the cytokine or variant thereof is an IL-12 variant comprising the sequence of SEQ ID NO: 36, and wherein the heavy chain comprising the IL-12 variant located in the hinge region comprises the sequence of any one of SEQ ID NOs: 268-273.

[0021] In some embodiments of any one of the immunocytokines described above, (i) the antigen-binding fragment is a ligand and the target antigen is a receptor specifically recognized by the ligand; or (ii) the antigen-binding fragment is a receptor and the target antigen is a ligand specifically recognized by the receptor. In some embodiments, the ligand or receptor is selected from the group consisting of IL-2, IL-2Rα (CD25), PD-1, PD-L1, PD-L2, NKG2A, NKG2C, NKG2F, NKG2D, BCMA, APRIL, BAFF, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the ligand is PD-L2. In some embodiments, the ligand comprises the sequence of SEQ ID NO: 176.In some embodiments, (i) the cytokine or variant thereof is an IL-2 variant comprising the sequence of SEQ ID NO: 2, and the antigen-binding polypeptide comprising the IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 180; (ii) the cytokine or variant thereof is an IFN-α variant comprising the sequence of SEQ ID NO: 4, and the antigen-binding polypeptide comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 182; (iii) the cytokine or variant thereof is an IFN-γ variant comprising the sequence of SEQ ID NO: 19, and the antigen-binding polypeptide comprising the IFN-γ variant located in the hinge region comprises the sequence of SEQ ID NO: 183; (iv) the cytokine or variant thereof is an IL-10 variant comprising the sequence of SEQ ID NO: 28, and the antigen-binding polypeptide comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 184. (v) the cytokine or variant thereof is an IL-12 variant comprising the sequence of SEQ ID NO: 36, and the antigen-binding polypeptide comprising the IL-12 variant located in the hinge region comprises the sequence of SEQ ID NO: 186; (vi) wherein the cytokine or variant thereof is an IL-12 variant comprising the sequence of SEQ ID NO: 275, and the antigen-binding polypeptide comprising the IL-12 variant located in the hinge region comprises the sequence of SEQ ID NO: 274; or (vii) the cytokine or variant thereof is an IL-23 variant comprising the sequence of SEQ ID NO: 39, and the antigen-binding polypeptide comprising the IL-23 variant located in the hinge region comprises the sequence of SEQ ID NO: 187. In some embodiments, the antigen-binding protein comprises a second antigen-binding polypeptide that does not comprise a cytokine or variant thereof located in the hinge region, and this second antigen-binding polypeptide comprises the sequence of any of SEQ ID NOs: 178, 179, 181, and 185. In some embodiments, the ligand is or is derived from the extracellular domain of PD-L1. In some embodiments, the PD-L1 ligand exhibits increased binding to PD-1 compared to wild-type PD-L1 (e.g., an increase of at least about any of a 2-, 3-, 4-, 5-, 6-, 10-, 20-, 50-, 100-, 100-fold, or more).In some embodiments, the PD-L1 ligand comprises any or a combination of the following mutations relative to SEQ ID NO: 249: I54Q, E58M, R113T, M115L, S117A, and G119K. In some embodiments, the PD-L1 ligand comprises any of the following mutations relative to SEQ ID NO: 249: E58M / R113T / M115L / S117A / G119K, I54Q / E58M / R113T / M115L / S117A / G119K, I54Q / R113T / M115L / S117A / G119K, I54Q / E58M / M115L / S117A / G119K, I54Q / E58M / R113T / S117A / G119K, I54Q / E58M / R113T / M115L / G119K, or I54Q / E58M / R113T / M115L / S117A. In some embodiments, the PD-L1 ligand comprises the sequence of any one of SEQ ID NOs: 255-261. In some embodiments, the cytokine or variant thereof is an IL-12 variant comprising the sequence of SEQ ID NO: 36, and the antigen-binding polypeptide comprising the IL-12 variant located in the hinge region comprises the sequence of SEQ ID NO: 277. In some embodiments, the antigen-binding protein comprises a second antigen-binding polypeptide comprising a second cytokine or variant thereof located in the hinge region or C' of the Fc fragment. In some embodiments, the second cytokine or variant is any cytokine or variant described herein, such as IL-2 or IFN-γ or variants thereof. In some embodiments, the antigen-binding protein comprises a second antigen-binding polypeptide comprising a second cytokine or variant thereof located in the hinge region, and the second antigen-binding polypeptide comprises the sequence of any of SEQ ID NOs: 285-288 and 327. In some embodiments, the ligand is or is derived from the extracellular domain of PD-L2. In some embodiments, the PD-L2 ligand exhibits increased binding to PD-1 compared to wild-type PD-L2 (e.g., an increase of at least about any of 2, 3, 4, 5, 6, 10, 20, 50, 100, 100-fold, or more).In some embodiments, the PD-L2 ligand comprises any mutation or combination thereof compared to SEQ ID NO: 176: T56V, S58V, Q60L, or T56V / S58V / Q60L. In some embodiments, the PD-L2 ligand comprises the sequence of any one of SEQ ID NOs: 262-265. In some embodiments, the cytokine or variant thereof is an IL-12 variant comprising the sequence of SEQ ID NO: 36, wherein the antigen-binding polypeptide comprising the IL-12 variant located in the hinge region comprises the sequence of SEQ ID NO: 293. In some embodiments, the antigen-binding protein comprises a second antigen-binding polypeptide comprising a second cytokine or variant thereof located in the hinge region or C' of the Fc fragment. In some embodiments, the second cytokine or variant is any cytokine or variant described herein, such as IL-2 or IFN-γ or variants thereof. In some embodiments, the ligand is or is derived from the extracellular domain of CD155. In some embodiments, the CD155 ligand comprises the sequence of SEQ ID NO: 267.

[0022] The present application also provides any of the anti-PD-1 antibodies described herein, including anti-PD-1 antibodies having the HC-CDR3 mutations described herein.

[0023] The present application also provides any of the immunocytokines described herein, such as any of the constructs described in the Examples, including single-chain fusions of cytokines at the hinge region, dual-chain fusions of cytokines (same or different) at the hinge region, or fusions in which one cytokine is fused at the hinge region of one chain and the other is fused to the C' of the Fc of the other chain. In some embodiments, the cytokine is any of the cytokines or variants described herein, such as IL-2, IL-12, IFN-γ, etc.

[0024] Also provided herein is an immunocytokine comprising a cytokine or variant thereof fused to the C' of an antigen-binding protein that specifically recognizes a target antigen (e.g., to the C' of an Fc fragment). In some embodiments, the immunocytokine comprises: a) an antigen-binding protein that specifically recognizes a target antigen; and b) a cytokine or variant thereof (e.g., any described herein), where the antigen-binding protein comprises, from N' to C': an antigen-binding polypeptide comprising an antigen-binding fragment (e.g., anti-PD-1, PD-L1, or PD-L2), a hinge region, and an Fc domain subunit or portion thereof, and where the cytokine or variant thereof is located at the C' of the Fc domain subunit or portion thereof.

[0025] Also provided herein is an immunocytokine comprising: a) an antigen-binding protein that specifically recognizes a target antigen; b) a first cytokine (e.g., IL-2 or IFN-γ) or a variant thereof; and c) a second cytokine (e.g., IL-12) or a variant thereof, wherein the antigen-binding protein comprises, from N' to C': a first antigen-binding polypeptide comprising an antigen-binding fragment, a hinge region, and an Fc domain subunit or a portion thereof, wherein the first cytokine or variant thereof is located in the hinge region; and from N' to C': a second antigen-binding polypeptide comprising an antigen-binding fragment, a hinge region, and an Fc domain subunit or a portion thereof, wherein the second cytokine or variant thereof is located in the hinge region.

[0026] Also provided herein is an immunocytokine comprising: a) an antigen-binding protein that specifically recognizes a target antigen; b) a first cytokine (e.g., IL-2 or IFN-γ) or a variant thereof; and c) a second cytokine (e.g., IL-12) or a variant thereof, wherein the antigen-binding protein comprises, from N' to C': a first antigen-binding polypeptide comprising an antigen-binding fragment, a hinge region, and an Fc domain subunit or a portion thereof, wherein the first cytokine or a variant thereof is located in the hinge region; and from N' to C': a second antigen-binding polypeptide comprising an antigen-binding fragment, a hinge region, and an Fc domain subunit or a portion thereof, wherein the second cytokine or a variant thereof is located in C' of the Fc domain.

[0027] Another aspect of the present application provides a method for selectively activating the activity of a cytokine or a variant thereof against cells expressing a target antigen in an individual, the method comprising administering to the individual an effective amount of an immunocytokine, wherein the immunocytokine comprises: a) an antigen-binding protein (e.g., an antibody, or a ligand / receptor-Fc fusion protein) that specifically recognizes the target antigen; and b) a cytokine or a variant thereof, wherein the antigen-binding protein comprises, from N' to C': an antigen-binding polypeptide (e.g., a heavy chain, or a ligand / receptor-Fc fusion polypeptide) comprising an antigen-binding fragment (e.g., an antibody fragment, ligand, or receptor), a hinge region, and an Fc domain subunit or a portion thereof, and the cytokine or variant thereof is located in the hinge region (e.g., at the N-terminus, the C-terminus, or within a range thereof), and wherein binding of the antigen-binding protein to the target antigen selectively activates the activity of the cytokine or variant thereof. In some embodiments, the present application provides a method of selectively activating the activity of a cytokine or variant thereof against cells expressing a target antigen in an individual, the method comprising administering to the individual an effective amount of any one of the immunocytokines described above, and wherein upon binding of the antigen binding protein to the target antigen, the activity of the cytokine or variant thereof is selectively activated. In some embodiments, in the presence of binding of the antigen binding protein to the target antigen, the activity of the cytokine or variant thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antigen binding protein to the target antigen. In some embodiments, in the absence of binding of the antigen binding protein to a target antigen, the activity of a cytokine or variant thereof located in the hinge region is about 70% or less (such as about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0% or less) of the activity of the corresponding cytokine or variant thereof in the free state.In some embodiments, the cytokine or variant thereof is a cytokine variant, and the activity of the cytokine variant in the free state is about 80% or less (e.g., about 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% or less) of the activity of the corresponding wild-type cytokine in the free state. In some embodiments, the antigen-binding protein is an antibody that specifically recognizes a target antigen, the antigen-binding polypeptide comprising a hinge region is a heavy chain of the antibody, and the cytokine or variant thereof is located in the hinge region of the heavy chain. In some embodiments, (i) the antigen-binding fragment is a ligand, and the target antigen is a receptor specifically recognized by the ligand; or (ii) the antigen-binding fragment is a receptor, and the target antigen is a ligand specifically recognized by the receptor.

[0028] Additionally provided are isolated nucleic acids encoding any one of the immunocytokines described herein, vectors (e.g., lentiviral vectors) comprising such nucleic acids, host cells (e.g., CHO cells) comprising such nucleic acids or vectors, and methods of making any one of the immunocytokines described herein.

[0029] Also provided are compositions (e.g., pharmaceutical compositions), kits, and articles of manufacture comprising any of the immunocytokines described herein. Methods of treating a disease or disorder in an individual (e.g., cancer, an infectious disease, an autoimmune disease, an allergy, transplant rejection, or graft-versus-host disease (GvHD)) using an effective amount of any of the immunocytokines or compositions (e.g., pharmaceutical compositions) described herein are also provided. [Brief explanation of the drawings]

[0030] [Figure 1]Figure 1A shows a full-length antibody structure. Figure 1B shows an exemplary immunocytokine structure comprising a cytokine or variant thereof fused to the N-terminus of a subunit of the Fc fragment of a parent full-length antibody. Figure 1C shows an exemplary immunocytokine structure comprising a cytokine or variant thereof fused to the C-terminus of the heavy chain of a parent full-length antibody. Figure 1D shows an exemplary immunocytokine structure comprising a cytokine or variant thereof fused to the N-terminus of the heavy chain variable domain (VH) of a parent full-length antibody. Figure 1E shows an exemplary immunocytokine structure comprising a cytokine or variant thereof fused to the C-terminus of the light chain constant region (CL) of a parent full-length antibody. The light gray filled area indicates the parent antibody scaffold. [Figure 2] Figures 2A-2D show exemplary immunocytokine structures of the present invention in which one or more cytokines or variants thereof (or subunits thereof) are located in the hinge region of one or both heavy chains of a parent full-length antibody. Figure 2A shows a monomeric cytokine or variant thereof (e.g., IL-2 or IFN-α) located in the hinge region of one heavy chain of a parent full-length antibody. Figure 2B shows a dimeric (homodimeric or heterodimeric) cytokine or variant thereof (e.g., IFN-γ, IL-10, IL-12, or IL-23) expressed as a single chain and located in the hinge region of one heavy chain of a parent full-length antibody. Figure 2C shows two cytokines or variants thereof located in tandem in the hinge region of one heavy chain of a parent full-length antibody. Figure 2D shows two cytokines or variants thereof, each located in the hinge region of one heavy chain of the parent full-length antibody, or a dimeric (homodimeric or heterodimeric) cytokine or variant thereof, where each subunit is located in the hinge region of one heavy chain of the parent full-length antibody. The light gray filled area indicates the parent antibody scaffold. [Figure 3] Figures 3A-3C show alternative exemplary immunocytokine structures of the invention in which the cytokine or variants thereof are located between the VH (e.g., within the scope of a Fab or scFv) and the Fc fragment subunits. The light grey region indicates the parent antibody scaffold. [Figure 4]Figures 4A-4C show exemplary immunocytokine structures of the invention in which one or more cytokines or variants thereof (or subunits thereof) are located in the hinge region of one or both polypeptides of a parent ligand / receptor-hinge-Fc fusion protein. Figure 4A shows a monomeric cytokine or variant thereof (e.g., IL-2 or IFN-α) located in the hinge region of one polypeptide of the parent ligand / receptor-hinge-Fc fusion protein. Figure 4B shows two cytokines or variants thereof, each located in the hinge region of one polypeptide of the parent ligand / receptor-hinge-Fc fusion protein, or a dimeric (homodimeric or heterodimeric) cytokine or variant thereof, in which each subunit is located in the hinge region of one polypeptide of the parent ligand / receptor-hinge-Fc fusion protein. Figure 4C shows a dimeric (homodimeric or heterodimeric) cytokine or variant thereof (e.g., IFN-γ, IL-10, IL-12, or IL-23) expressed as a single chain, or two cytokines or variants thereof fused in tandem, located in the hinge region of one polypeptide of a parent ligand / receptor-hinge-Fc fusion protein. The light gray area indicates the parent ligand / receptor-hinge-Fc fusion protein scaffold. [Figure 5] Figure 5 shows tumor volumes in mice bearing 4T1 syngeneic tumors treated with IL-12 (E59A / F60A) / anti-PD-1 immunocytokine (#48), IL-12 (E59A / F60A) / PD-L2-Fc immunocytokine (#29), IL-12 (E59A / F60A) / IL-2 (R38D / K43E / E61R) / anti-PD-1 immunocytokine (#54), or PBS (negative control). Black arrows indicate the days of injection. [Figure 6] Figure 6 shows tumor volumes in mice bearing EMT6 syngeneic tumors treated with IL-12 (E59A / F60A) / anti-PD-1 immunocytokine (#48), IL-12 (E59A / F60A) / PD-L2-Fc immunocytokine (#29), IL-2 (R38D / K43E / E61R) / PD-L2-Fc immunocytokine (#11), or PBS (negative control). Black arrows indicate the days of injection. [Figure 7]Figures 7A and 7B show tumor volumes in mice bearing CT26 syngeneic tumors treated with IL-12(F60A) / PD-L2-Fc immunocytokine (#30), PD-L2-Fc / IL-12(F60A) immunocytokine (#34), or PBS (negative control). Black arrows indicate the day of injection. Figure 7A shows the mean tumor volume for all mice receiving the indicated IL-12 immunocytokine or control. The mean tumor size (±STD) for each group at the time of initial treatment administration is shown in parentheses. Figure 7B shows tumor volumes for individual mice receiving the indicated IL-12 immunocytokine. [Figure 8] Figure 8 shows tumor volumes in mice pre-treated with IL-12(F60A) / PD-L2-Fc immunocytokine (#30) or PD-L2-Fc / IL-12(F60A) immunocytokine (#34) against CT26 tumors and then re-challenged with CT26 mouse colon cancer cells in the right flank and EMT6 mouse breast cancer cells (as a control) in the left flank. [Figure 9] Figure 9 shows tumor volumes in mice with late-stage CT26 syngeneic tumors treated with IL-12 (E59A / F60A) / PD-L2 Fc immunocytokine (#29) or IL-12 (F60A) / PD-L2-Fc immunocytokine (#30). Black arrows indicate the day of injection. The tumor size for each mouse at the time of administration of the first treatment is shown in parentheses. [Figure 10] Figure 10 shows regression of tumor size in a late-stage CT26 syngeneic tumor mouse model treated with IL-12(F60A) / PD-L2-Fc immunocytokine (construct #30). [Figure 11A]Figure 11A shows tumor volumes in mice bearing EMT6 syngeneic breast tumors treated with IL-12 (E59A / F60A) / PD-L2 Fc immunocytokine (#29), IL-12 (F60A) / PD-L2-Fc immunocytokine (#30), IL-12 (E59A / F60A) / anti-PD-1 immunocytokine (#48), or PBS (negative control). Black arrows indicate the day of injection. Figure 11A shows the mean tumor volumes for all mouse groups, and the mean tumor size (±STD) at the time of administration of the first treatment is shown in parentheses. Figure 11B shows tumor volumes for individual mice receiving the indicated IL-12 immunocytokine. [Figure 11B] Figure 11B shows tumor volumes in mice bearing EMT6 syngeneic breast tumors treated with IL-12 (E59A / F60A) / PD-L2 Fc immunocytokine (#29), IL-12 (F60A) / PD-L2-Fc immunocytokine (#30), IL-12 (E59A / F60A) / anti-PD-1 immunocytokine (#48), or PBS (negative control). Black arrows indicate the day of injection. Figure 11A shows the mean tumor volumes for all mouse groups, with the mean tumor size (±STD) at the time of administration of the first treatment indicated in parentheses. Figure 11B shows tumor volumes for individual mice receiving the indicated IL-12 immunocytokine. [Figure 12] Figure 12 shows tumor volumes in mice pre-treated with IL-12 (E59A / F60A) / PD-L2 Fc immunocytokine (#29), IL-12 (F60A) / PD-L2-Fc immunocytokine (#30), or IL-12 (E59A / F60A) / anti-PD-1 immunocytokine (#48) against EMT6 tumors and then re-challenged with EMT6 mouse breast cancer cells in the right flank and CT26 mouse colon cancer cells (as a control) in the left flank. [Figure 13] Figure 13 shows 4T1 mouse breast cancer tumors extracted from the mammary fat pads of mice treated with IL-12(E59A / F60A) / PD-L2-Fc immunocytokine (#29), IL-12(F60A) / PD-L2-Fc (#30), a combination of anti-PD-1 and anti-CTLA-4 antibodies, or PBS (negative control). [Figure 14]Figure 14 shows 4T1 mouse breast cancer cells metastasized to the lungs in mice injected with 4T1 cells into the mammary fat pad and treated with IL-12(E59A / F60A) / PD-L2-Fc immunocytokine (#29), IL-12(F60A) / PD-L2-Fc (#30), a combination of anti-PD-1 and anti-CTLA-4 antibodies, or PBS (negative control). [Figure 15] Figures 15A and 15B show tumor volumes in B16 melanoma syngeneic tumor-bearing mice treated with IL-12(F60A) / PD-L2-Fc immunocytokine (#30), PD-L2-Fc / IL-12(F60A) immunocytokine (#34), or PBS (negative control). Black arrows indicate the day of injection. Figure 15A shows the mean tumor volume for all mouse groups, with the mean tumor size (±STD) at the time of administration of the first treatment indicated in parentheses. Figure 15B shows tumor volumes for individual mice receiving the indicated IL-12 immunocytokine. [Figure 16] Figures 16A and 16B show tumor volumes in LL2 lung cancer syngeneic mice treated with IL-12(F60A) / PD-L2-Fc immunocytokine (#30), PD-L2-Fc / IL-12(F60A) immunocytokine (#34), or PBS (negative control). Black arrows indicate the day of injection. Figure 16A shows the mean tumor volume for all mouse groups, with the mean tumor size (±STD) at the time of administration of the first treatment indicated in parentheses. Figure 16B shows the tumor volume for individual mice that received the indicated IL-12 immunocytokine. DETAILED DESCRIPTION OF THE INVENTION

[0031] Cytokines are key mediators of innate and adaptive immunity. However, cytokine therapy (e.g., for the treatment of cancer) has shown limited success because severe toxicity limits dose setting far below the therapeutically effective dose. Immunocytokines are constructs in which a cytokine is fused to an antibody, antigen-binding fragment, ligand-Fc fusion protein, or receptor-Fc fusion protein (collectively referred to as "ligand / receptor-Fc fusion protein" or "ligand / receptor-hinge-Fc fusion protein" hereinafter). Recognition of the target antigen by the antibody or antigen-binding fragment (e.g., antibody fragment, ligand, or receptor) within the immunocytokine enables the cytokine to be delivered to target cells (e.g., tumor cells or immune effector cells) or tissues, thereby reducing nonspecific (off-target) cytokine activity and / or associated toxicity (e.g., toxicity to healthy cells or tissues) and concentrating the cytokine therapeutic effect at the target site (e.g., disease site). Immunocytokines can be activated by transactivation, which requires specific binding of an antibody or antigen-binding fragment to a target antigen on tumor cells, or by cisactivation, which requires specific binding of an antibody or antigen-binding fragment to a target antigen on immune cells. Many immunocytokines developed today have cytokine moieties fused to the N- or C-terminus of the heavy or light chain of a full-length antibody (e.g., Hu14.8-IL2, NHS-IL2LT, NHS-IL12, BC1-IL12; see, e.g., Figures 1C-1E) or to the N- or C-terminus of an antigen-binding fragment (e.g., L19-IL2 or F16-IL2, e.g., diabodies, scFvs). Therefore, cytokine-receptor binding / activation can still occur in the absence of antibody-antigen recognition, which can lead to off-target toxicity.

[0032] The present invention provides immunocytokines with unique configurations that address the problems faced by current cytokine / immunocytokine therapies. In particular, the immunocytokines of the present invention comprise a cytokine portion (e.g., a cytokine or a variant thereof) bound to an antigen-binding fragment (e.g., a ligand, a receptor, a VHH, an scFv, or an Fab) and an Fc domain subunit or a portion thereof (e.g., a CH2-CH3 fragment, or only CH2, or only CH3), e.g., an antigen-binding polypeptide comprising a VH-VL-cytokine-Fc subunit, or a VL-VH-cytokine-Fc subunit), a Fab, or a portion thereof (e.g., a hinge region between an scFv and an Fc domain subunit). By placing the Fc domain subunit in the hinge region between a full-length antibody (e.g., an antigen-binding polypeptide comprising a VH-CH1-cytokine-Fc subunit) or in the hinge region between a ligand (or receptor) and an Fc domain subunit (e.g., an antigen-binding polypeptide comprising a ligand-cytokine-Fc subunit or a receptor-cytokine-Fc subunit), the nonspecific activity of the cytokine (i.e., antibody- or antigen-binding fragment-independent binding) is reduced and the specific activity (i.e., antibody- or antigen-binding fragment-dependent binding) is increased. Without being bound by theory, it is believed that in the absence of target antigen binding by the antigen-binding fragment, steric hindrance between the antigen-binding fragment (e.g., ligand, receptor, VHH, scFv, Fab) and the Fc domain or a portion thereof reduces the ability of the cytokine or its variant to reach its receptor or "masks" the cytokine or its variant from binding to its receptor. On the other hand, upon target antigen binding, the cytokine becomes activated. Surprisingly, unlike other immunocytokine designs that "expose" the cytokine moiety at its N- or C-terminus, the unique immunocytokine configuration of the present invention requires that the antigen-binding fragment (e.g., ligand, receptor, VHH, scFv, or Fab) first binds to its target antigen before binding of the cytokine moiety to its receptor can occur, thus ensuring that cytokine receptor activation is entirely target antigen binding dependent (on-target).This enhanced targeting specificity design allows cytokines to be safely delivered to target sites (e.g., tumor cells or immune cells) to achieve therapeutic effects.

[0033] Furthermore, the inventors of the present application have discovered that certain immunocytokines, such as those constructed with a parent antigen-binding protein (e.g., an antibody or fragment thereof, or a receptor- or ligand-Fc fusion protein) and a cytokine moiety with opposing effects in modulating the immune response, have demonstrated significantly better toxicity profiles and therapeutic efficacy. For example, when the IL-12 cytokine (pro-inflammatory) was located in the hinge region of a PD-L2 extracellular domain-hinge-Fc fusion protein, the resulting IL-12 / PD-L2-Fc immunocytokine not only specifically targeted IL-12 activity (e.g., IL-12 receptor-binding activity and / or IL-12 pro-inflammatory activity) to PD-1+ target cells, but also stimulated PD-1 inhibitory immune checkpoint signaling through PD-L2-PD-1 binding, thus creating an immunosuppressive signal that counteracted and "balanced" the immunostimulatory activity of IL-12 (hereinafter also referred to as a "balancing immunocytokine"). Any agonist antibody or ligand (e.g., PD-L2, PD-L1, CD80, or CD86) that can activate or stimulate an immunoinhibitory signaling pathway (e.g., by binding to an inhibitory immune checkpoint molecule such as PD-1 or CTLA-4), or any antagonist antibody, ligand, or receptor that can reduce or block an immunostimulatory signaling pathway (e.g., by binding to a stimulatory immune checkpoint molecule such as CD27 or CD28 or an immunostimulatory receptor such as IL-2R), can be used in combination with an immunostimulatory cytokine or variant thereof (e.g., IL-2, IL-12, or IL-23) to construct a balanced immunocytokine with any of the immunocytokine configurations described herein.Any antagonist antibody, ligand, or receptor capable of reducing or blocking immunosuppressive signaling pathways (e.g., by binding to inhibitory immune checkpoint molecules such as PD-1 or CTLA-4), or any agonist antibody or ligand (e.g., CD70, CD80, CD86, or IL-2) capable of activating or stimulating immunostimulatory signaling pathways (e.g., by binding to stimulatory immune checkpoint molecules such as CD27 or CD28 or immunostimulatory receptors such as IL-2R), can be used in combination with an immunosuppressive cytokine or variant thereof (e.g., IL-10, IL-27, IL-35, TGF-β) to construct a balanced immunocytokine with any of the immunocytokine configurations described herein. Such designs to "balance" the immune response add an additional layer of regulation to fine-tune the biological activity and toxicity of the immunocytokines described herein, in addition to their unique targeting specificity designs.

[0034] The immunocytokines described herein can also treat a variety of advanced and / or hard-to-treat cancer types (e.g., TNBC, melanoma, lung cancer) by inhibiting cancer metastasis, treating or delaying tumor progression of cancer types resistant to current immunotherapies (e.g., anti-PD-1 therapy, anti-CTLA-4 therapy, or combinations thereof), and / or extending the lifespan of such patients. The immunocytokines described herein have an excellent safety profile with significantly reduced side effects compared to other immunocytokine formats, such as C'Fc fusions.

[0035] In combination with this unique immunocytokine structural design, various cytokine mutants were also generated and screened for those located in the hinge region that have little or no activity (e.g., cytokine receptor binding activity and / or cytokine biological activity) in the absence of target antigen binding, but are "rescued" or "manifest" increased activity in the presence of target antigen binding. Various immunocytokines were obtained that can specifically target cytokine (e.g., IFN-α2b, IL-12, IL-23, IL-10, or IFN-γ) activity toward target cells (e.g., PD-1+ cells, CD4+ T cells, or CD8+ T cells), while exhibiting minimal or no cytokine activity toward non-target cells (e.g., PD-1 cells, CD8+ T cells, or CD4+ T cells).

[0036] Accordingly, one aspect of the present application provides an immunocytokine comprising: a) an antigen-binding protein (e.g., an antibody such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) that specifically recognizes a target antigen (e.g., a cell surface antigen, receptor, or ligand); and b) a cytokine or variant thereof, wherein the antigen-binding protein comprises, from N' to C': an antigen-binding polypeptide (e.g., an antibody heavy chain, or an antigen-binding fragment-hinge-Fc fusion polypeptide such as a ligand / receptor-hinge-Fc fusion polypeptide) comprising an antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, or VH), a hinge region, and an Fc domain subunit or a portion thereof (e.g., CH2+CH3, or CH2 only), and wherein the cytokine or variant thereof is located in (e.g., at or within the N', C', or N') of the hinge region.

[0037] In some embodiments, the parent antigen-binding protein (i.e., the antigen-binding protein scaffold used in the construction of the immunocytokines described herein) is an antibody (e.g., a full-length antibody) or fragment thereof that specifically recognizes a target antigen. In some embodiments, the antigen-binding polypeptide comprising the hinge region is a heavy chain of an antibody, and wherein the cytokine or variant thereof is located in the hinge region of the heavy chain. Thus, in some embodiments, the present invention provides immunocytokines comprising: a) an antibody (e.g., a full-length antibody) that specifically recognizes a target antigen; and b) a cytokine or variant thereof, wherein the antibody comprises a heavy chain comprising a hinge region, and wherein the cytokine or variant thereof is located in the hinge region (e.g., at the N' thereof, at the C' thereof, or within The immunocytokines described herein, in some embodiments, comprise: (a) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VH domain, an optional first CH1, a cytokine portion in the hinge region, and a first subunit of an Fc domain or a portion thereof (e.g., CH2+CH3, or CH2 only); (b) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VH domain, an optional second CH1, a hinge region, and F; and (c) a first VL domain and any and (d) a fourth antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VL domain and an optional second CL; wherein the first VH and first VL and optionally the first CH1 and first CL form a first antigen-binding domain (e.g., Fab) that specifically recognizes a first target antigen; and wherein the second VH and second VL and optionally the second CH1 and second CL form a second antigen-binding domain (e.g., Fab) that specifically recognizes a second target antigen. In some embodiments, the antigen-binding polypeptide comprising the hinge region is an scFv-hinge-Fc fusion polypeptide.The immunocytokines described herein, in some embodiments, comprise: (a) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: (a first VH domain and a first VL domain) or (a first VL domain and a first VH domain), a cytokine portion located in the hinge region, and a first subunit of an Fc domain or a portion thereof (e.g., CH2+CH3, or CH2 only); and (b) a second VH domain and a second VL domain, or (a second VL domain and a first VH domain), a cytokine portion located in the hinge region, and a first subunit of an Fc domain or a portion thereof (e.g., CH2+CH3, or CH2 only). and a second antigen-binding polypeptide comprising a first VH and a second VH domain, a hinge region, and a second subunit of an Fc domain or a portion thereof (e.g., CH2+CH3, or CH2 only); wherein the first VH and first VL form a first antigen-binding domain (e.g., scFv) that specifically recognizes a first target antigen; and wherein the second VH and second VL form a second antigen-binding domain (e.g., scFv) that specifically recognizes a second target antigen. In some embodiments, the antigen-binding polypeptide comprising the hinge region is a VHH-hinge-Fc fusion polypeptide. In some embodiments, the immunocytokine described herein comprises: (a) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VHH, a cytokine portion in the hinge region, and a first subunit of the Fc domain or a portion thereof (e.g., CH2+CH3, or CH2 only); and (b) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VHH, a hinge region, and a second subunit of the Fc domain or a portion thereof (e.g., CH2+CH3, or CH2 only); wherein the first VHH specifically recognizes a first target antigen; and wherein the second VHH specifically recognizes a second target antigen.

[0038] In some embodiments, the parent antigen-binding protein is an antigen-binding fragment-hinge-Fc fusion protein. In some embodiments, the antigen-binding fragment is an scFv. In some embodiments, the antigen-binding fragment is a VHH. In some embodiments, the antigen-binding fragment is a ligand and the target antigen is a receptor specifically recognized by the ligand. In some embodiments, the antigen-binding fragment is a receptor and the target antigen is a ligand specifically recognized by the receptor. Thus, in some embodiments, the present invention provides an immunocytokine comprising: a) an antigen-binding fragment-hinge-Fc fusion protein that specifically recognizes a target antigen (e.g., a cell surface antigen, receptor, or ligand); and b) a cytokine or variant thereof, wherein the antigen-binding fragment-hinge-Fc fusion protein comprises, from N' to C': an antigen-binding fragment-hinge-Fc fusion polypeptide comprising an antigen-binding fragment (e.g., a ligand, receptor, VHH, or scFv), a hinge region, and an Fc domain subunit or portion thereof, and wherein the cytokine or variant thereof is located in (e.g., at or within the N', C', or N' of) the hinge region. In some embodiments, the immunocytokines described herein comprise: (a) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first antigen-binding fragment (e.g., a ligand, receptor, VHH, or scFv), a portion of the cytokine in the hinge region, and a first subunit of the Fc domain or a portion thereof (e.g., CH2+CH3, or CH2 only); and (b) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second antigen-binding fragment (e.g., a ligand, receptor, VHH, or scFv), a hinge region, and a second subunit of the Fc domain or a portion thereof (e.g., CH2+CH3, or CH2 only); wherein the first antigen-binding fragment specifically recognizes a first target antigen (e.g., a cell surface antigen, receptor, or ligand); and wherein the second antigen-binding fragment specifically recognizes a second target antigen (e.g., a cell surface antigen, receptor, or ligand).

[0039] Also provided are isolated nucleic acids encoding such immunocytokines, vectors containing such nucleic acids, host cells containing such nucleic acids or vectors, methods for making such immunocytokines, pharmaceutical compositions and products containing such immunocytokines, methods for selectively activating the activity of cytokines or variants thereof against cells expressing target antigens with such immunocytokines or pharmaceutical compositions thereof, and methods for treating diseases (e.g., cancer, viral infections, autoimmune diseases) with such immunocytokines or pharmaceutical compositions thereof.

[0040] I. Definition The practice of the present invention will employ, unless specifically indicated to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below by way of example, and such techniques are fully explained in the literature. For example, Current Protocols in Molecular Biology or Current Protocols in Immunology,John Wiley & Sons,New York,NY(2009);Ausubel et al.,Short Protocols in Molecular Biology,3rd ed.,John Wiley & Sons,1995;Sambrook and Russell,Molecular Cloning:A Laboratory Manual(3rd Edition,2001);Maniatis et al.,Molecular Cloning:A Laboratory Manual(1982);DNA Cloning: A Practical Approach,vol.I&II(D.Glover,ed.);Oligonucleotide Synthesis(N.Gait,ed.,1984);Nucleic Acid Hybridization(B.Hames & S.Higgins,eds.,1985);Transcription and Translation(B.Hames & S.Higgins,eds.,1984);Animal Cell Culture(R.Freshney,ed.,1986);Perbal,A See Practical Guide to Molecular Cloning (1984) and other similar references.

[0041] The term "immunocytokine," as used herein, refers to an antigen-binding protein (e.g., an antibody, or antigen-binding fragment (e.g., a ligand, receptor, or antibody fragment)) format that is fused to a cytokine molecule. The antigen-binding protein (e.g., an antibody, or antigen-binding fragment (e.g., a ligand, receptor, or antibody fragment)) format can be any of those described herein, and the cytokine can be fused to the antigen-binding protein format directly or by a linker or by chemical conjugation.

[0042] The term "cytokine storm," also known as "cytokine cascade" or "hypercytokinemia," is a potentially fatal immune response that typically consists of a positive feedback loop between cytokines and immune cells, and is accompanied by highly elevated levels of various cytokines (e.g., IFN-γ, IL-10, IL-6, CCL2, etc.).

[0043] As used herein, when an antigen-binding protein (e.g., an antibody, antigen-binding fragment, or ligand) is referred to as an "antagonist" of a target antigen (e.g., a receptor or immune checkpoint molecule), it means that, upon target antigen binding, the antigen-binding protein (e.g., an antibody, antigen-binding fragment, or ligand) blocks, inhibits, or reduces the biological activity of the target antigen (e.g., blocks receptor signaling). For example, an anti-PD-1 antagonist antibody is an antibody that reduces or blocks PD-1 signaling. When an antigen-binding protein (e.g., an antibody, antigen-binding fragment, or ligand) is referred to as an "agonist" of a target antigen (e.g., a receptor or immune checkpoint molecule), it means that, upon target antigen binding, the antigen-binding protein (e.g., an antibody, antigen-binding fragment, or ligand) stimulates, activates, or enhances the biological activity of the target antigen (e.g., activates receptor signaling). For example, wild-type PD-L2 ligand (e.g., the extracellular domain) is an agonist that activates PD-1 signaling.

[0044] As used herein, "treatment" or "treating" refers to an approach for obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, ameliorating the disease state, providing remission (partial or complete) of the disease, reducing the dose of one or more other medications required to treat the disease, slowing the progression of the disease, improving quality of life, and / or prolonging survival. "Treatment" also encompasses the reduction of the pathological consequences of a disease. The methods of the present invention contemplate any one or more of these aspects of treatment. For example, an individual is successfully "treated" if one or more symptoms associated with a viral infection are alleviated or eliminated, including, but not limited to, reducing the proliferation of (or destroying) the infectious virus, reducing symptoms caused by the disease (e.g., cytokine storm), improving the quality of life of an individual affected by the disease, reducing the dose of other medications required to treat the disease, and / or prolonging the survival of the individual.

[0045] The term "prevent," and similar phrases such as "prevented," "preventing," and the like, refer to an approach for preventing, inhibiting, or reducing the likelihood of a disease or condition, such as cancer, recurring. It also refers to delaying the recurrence of a disease or condition, or delaying the recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar phrases also refer to reducing the intensity, effect, symptoms, and / or burden of a disease or condition before the disease or condition recurs.

[0046] As used herein, "delaying" the onset of a disease means to postpone, prevent, slow, retard, stabilize, and / or postpone the onset of the disease. This delay can be of varying lengths of time depending on the disease under treatment and / or the individual's medical history. A method that "delays" the onset of a disease is one that reduces the probability of disease onset in a given time frame and / or reduces the extent of disease in a given time frame when compared to the absence of the method. Such comparisons are typically based on clinical trials using a statistically significant number of individuals. The onset of cancer may be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Onset may also refer to disease (e.g., cancer) progression, which may be initially undetectable and includes emergence, recurrence, and development.

[0047] As used herein, the term "effective amount" refers to an amount of an agent or combination of agents sufficient to treat, such as ameliorating, alleviating, mitigating, and / or delaying one or more of the symptoms of, the specified disorder, condition, or disease. With respect to cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and / or a reduction in the rate of tumor growth (such as tumor growth inhibition), or to prevent or delay other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay onset. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition may (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow to some extent, and preferably stop cancer cell invasion into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the appearance and / or recurrence of tumors; (vii) alleviate to some extent one or more symptoms associated with cancer; (viii) stimulate or activate immune cells (e.g., immune effector cells) for an immune response, such as by producing one or more cytokines, or for immune cell proliferation and / or differentiation; and / or (ix) prevent, reduce, or eliminate an inflammatory or autoimmune response, such as by inhibiting pro-inflammatory cytokine secretion.In the case of a viral infection, an effective amount of an agent may inhibit (i.e., reduce to some extent, preferably neutralize) viral activity; control and / or attenuate and / or inhibit inflammation or cytokine storm induced by said viral pathogen; prevent, arrest and / or ameliorate at least one symptom of said viral infection or damage to said subject or said subject's organs or tissues resulting from or associated with said viral infection from worsening; control, reduce and / or inhibit cell necrosis in infected and / or non-infected tissues and / or organs; control, ameliorate and / or prevent infiltration of inflammatory cells (e.g., NK cells, cytotoxic T cells, neutrophils) in infected or non-infected tissues and / or organs; and / or stimulate or activate immune cells (e.g., immune effector cells) for an immune response, such as by producing one or more cytokines, or for immune cell proliferation and / or differentiation.

[0048] As used herein, "individual" or "subject" refers to a mammal, including, but not limited to, a human, cow, horse, cat, dog, rodent, or primate. In some embodiments, the individual is a human.

[0049] The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof so long as they exhibit the desired antigen-binding activity. The term "antibody" includes traditional four-chain antibodies, single-domain antibodies, and antigen-binding fragments thereof.

[0050] 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 of these basic heterotetrameric units, together with an additional polypeptide called the J chain, and contain ten antigen-binding sites, while IgA antibodies contain two to five of these basic four-chain units, which can combine with the J chain to form multivalent aggregates. In the case of IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain contains an N-terminal variable domain (V H ), followed by three constant domains (C for each of the α and γ chains) H ), and four C for μ and ε isotypes H Each L chain has an N-terminal variable domain (V L ), followed by a constant domain at the other end. L is V H Align with C L is the first constant domain of the heavy chain (C H 1). Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain. H and V L pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. L 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. Immunoglobulins are characterized by the constant domains of their heavy chains (C H) can be assigned to different classes or isotypes depending on the amino acid sequence. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain designated α, δ, ε, γ, and μ. The γ and α classes are H They are further divided into subclasses based on relatively minor differences in sequence and function; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0051] An "isolated" antibody (or construct) is one that has been identified, separated, and / or recovered from components of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free from association with any other components from its production environment. Contaminant components of its production environment, such as those arising from recombinantly transfected cells, are materials that would typically interfere with research, diagnostic, or therapeutic uses for the antibody, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to greater than 95% by weight of the antibody, and in some embodiments, greater than 99% by weight, as determined, for example, by the Lowry assay; (2) 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 using Coomassie blue, or preferably silver staining. Isolated antibody (or construct) includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated polypeptide, antibody, or construct will be prepared by at least one purification step.

[0052] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy and light chains are respectively referred to as "V H " and "V LThese domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen-binding sites. Heavy chain-only antibodies from camelid species have a single heavy chain variable region, which is called a "V" domain. H H". Therefore, V H H is a special kind of V H is.

[0053] The term "variable" refers to the fact that certain sections of the variable domains differ extensively in sequence among antibodies. V domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the entire width of the variable domains. Instead, variability is concentrated in three sections, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs), in both the heavy and light chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy and light chain variable domains each contain four FR regions that largely adopt a β-sheet configuration, linked by three CDRs that form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions, and the CDRs from the other chain contribute to forming the antigen-binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding an antibody to an antigen but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0054] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in the present invention can be produced by, for example, the hybridoma method (Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2002) nded. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004), and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); US Patent No. 5,545,807; US Patent No. 5,545,806; US Patent No. 5,569,825; US Patent No. 5,625,126; US Patent No. 5,633,425; al.,Bio / Technology 10:779-783(1992);Lonberg et al.,Nature 368:856-859(1994);Morrison,Nature 368:812-813(1994);Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0055] 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, full-length four-chain antibodies include those having heavy and light chains, including an Fc region. Full-length heavy-chain-only antibodies include those having a heavy chain variable domain (V H H) and 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, the intact antibody may have one or more effector functions. For the purposes of the present invention, the term "full length antibody" should be understood to also include a full length antibody scaffold or parent full length antibody (e.g., a full length four chain antibody, or a full length heavy chain only antibody) having a cytokine portion with the hinge region located therein (see, e.g., Figures 2A-2D).

[0056] An "antibody fragment," "antigen-binding domain," or "antigen-binding fragment" comprises a portion of an intact antibody, preferably the antigen-binding and / or variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, 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 (scFv) molecules; single-domain antibodies (V H These include antibodies with specificity (e.g., H), and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies yields two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a designation reflecting their ability to be readily crystallized. The Fab fragment contains the entire L chain along with the variable domain of the H chain (VH ), and the first constant domain of one heavy chain (C H 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities, still capable of cross-linking antigen. The Fab' fragment is C H They differ from Fab fragments by having several additional residues at the carboxy terminus of one domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which one or more 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 couplings of antibody fragments are also known. It should be understood that, for the purposes of the present invention, the expression "antigen-binding domain" or "antigen-binding fragment" also includes a ligand capable of specifically recognizing a target receptor, or a receptor capable of specifically recognizing a target ligand.

[0057] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain is the C H 1. C H 2 and C H 3 domains (collectively, C H ) and light chain CHL (or C L ) domain.

[0058] The "heavy chain" of an antibody (immunoglobulin) can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (a crystallizable fragment). The Fd region is V Hand CH1 domains, which combine with the light chain to form the Fab-antigen-binding fragment. The Fc fragment is responsible for immunoglobulin effector functions, including, for example, complement fixation and binding to cognate Fc receptors on effector cells. The hinge region, found in IgG, IgA, and IgD immunoglobulin classes, acts as a flexible spacer that allows the Fab portion to move freely in space relative to the Fc region. In contrast to the constant region, the hinge domain is structurally diverse, varying in both sequence and length between immunoglobulin classes and subclasses. For heavy-chain-only antibodies, the "heavy chain" refers to the heavy chain variable domain (V H H), hinge region, and Fc region. For the purposes of the present invention, the term "heavy chain" should also be understood to include heavy chains comprising a VH domain, hinge region, and Fc domain or a portion thereof (e.g., a VL-VH-hinge-Fc domain subunit, or a VH-VL-hinge-Fc domain subunit), and heavy chains comprising a cytokine moiety located in the hinge region (e.g., the heavy chain of a full-length four-chain antibody, a VH-hinge-Fc-containing antibody, or a heavy chain-only antibody) (see, e.g., Figures 2A-3C).

[0059] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0060] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy-chain and one light-chain variable domain in tight, noncovalent association. The folding of these two domains generates six hypervariable loops (three loops from each heavy and light chain) that contribute to antigen binding by amino acid residues and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

[0061] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a V linked to a single polypeptide chain. H and V L Preferably, the scFv polypeptide is an antibody fragment comprising a V H Domains and V L Because scFvs further contain a polypeptide linker between the domains, they are able to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0062] The term "diabody" refers to a V domain that is fragmented such that interchain, but not intrachain, pairing of V domains occurs, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. H Domains and V L This refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains. Bispecific diabodies are small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains. H and V L Diabodies are heterodimers of two "crossover" sFv fragments whose domains are present on different polypeptide chains. Diabodies are described in further detail in, for example, EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0063] The monoclonal antibodies of this specification particularly include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more chains is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The term "humanized antibody" is used as a subtype of "chimeric antibody."

[0064] "Humanized" forms of non-human (e.g., llama or camelid) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. In some embodiments, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR (defined below) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit, camel, llama, alpaca, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual substitutions of FR residues which improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR typically does not exceed six in the H chain and three in the L chain. The humanized antibody also optionally will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0065] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or an antibody generated using any of the techniques for generating human antibodies as disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Human monoclonal antibodies can also be prepared using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomouse, that have been modified to produce such antibodies in response to antigen challenge, but in which the endogenous gene locus has been disabled (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), regarding human antibodies generated by human B cell hybridoma technology.

[0066] The terms "hypervariable region," "HVR," or "HV," as used herein, refer to regions in an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, single-domain antibodies comprise three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 (or CDR3) exhibits the highest degree of diversity among the three HVRs and is believed to play a unique role in conferring precise specificity to antibodies. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0067] The term "complementarity determining region" or "CDR" is used to refer to a hypervariable region as defined by the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0068] Several HVR designations are in use and are encompassed herein. Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia, instead, refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are listed in Table A below.

[0069] [Table 1]

[0070] HVRs may include "extended HVRs" as follows: L 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) and V H 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the variable domain. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0071] The phrases "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the antibody sequences in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FRs or HVRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues may be determined for a given antibody by aligning regions of homology in the antibody sequence with sequences according to the "standard" Kabat numbering system.

[0072] Unless otherwise indicated herein, the numbering of residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra. "EU index as in Kabat" refers to the numbering of residues in a human IgG1 EU antibody.

[0073] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0074] "Human consensus framework" or "acceptor human framework" refers to the human immunoglobulin V L or V H The framework corresponds to the most frequently occurring amino acid residue in a series of alternative framework sequences. L or V H The set of sequence choices is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5 thSubgroups as in Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). For example, V L For VH, the subgroup can be subgroup kappa I, kappa II, kappa III, or kappa IV as in Kabat et al., supra. Additionally, for VH, the subgroup can be subgroup I, subgroup II, or subgroup III as in Kabat et al. Alternatively, a human consensus framework can be derived from the above at specific residues, such as when human framework residues are selected based on their homology with the donor framework by aligning the donor framework sequence with a panel of different human framework sequences. An acceptor human framework "derived" from a human immunoglobulin framework or human consensus framework can comprise the same amino acid sequence, or it can have pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer.

[0075] An "affinity matured" antibody is one that has one or more modifications in one or more of its CDRs that result in an improved affinity of the antibody for an antigen compared to a parent antibody that does not possess such one or more modifications. In some embodiments, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity matured antibodies. H and V LAffinity maturation by domain shuffling has been described. Random mutagenesis of CDR and / or framework residues has been described, for example, by Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0076] The term "epitope" refers to a protein determinant capable of specific binding to an antibody or antigen-binding fragment (e.g., ligand, receptor, VHH, scFv, Fab, etc.). Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0077] As used herein, the terms "specifically bind," "specifically recognize," or "specific for" refer to a measurable and reproducible interaction, such as the binding between a target and an antigen-binding protein (or between a cytokine and a cytokine receptor), that is determinative of the presence of a target (or cytokine) in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antigen-binding protein (such as a Fab) that specifically binds to a target (which may be an epitope) is an antigen-binding protein that binds this target with higher affinity, avidity, more readily, and / or with a longer duration than it binds other targets. A cytokine that specifically binds to a cytokine receptor is a cytokine that binds this cytokine receptor with higher affinity, avidity, more readily, and / or with a longer duration than it binds other cytokine receptors. In some embodiments, the extent to which an antigen binding protein (or cytokine) binds to an unrelated target (or unrelated cytokine receptor) is less than about 10% of the binding of the antigen binding protein (or cytokine) to the target (or cytokine receptor as measured), as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antigen binding protein that specifically binds a target (or a cytokine that specifically binds a cytokine receptor) has a binding affinity of ≦10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≦10 -12 Dissociation constant of M (K D). In some embodiments, the antigen binding protein (or cytokine receptor) specifically binds an epitope on a protein (or cytokine) that is conserved among proteins from different species. In some embodiments, specific binding can, but need not, include exclusive binding. The binding specificity of an antigen binding protein (or cytokine and cytokine receptor) can be determined experimentally by any protein binding method known in the art. Such methods include, but are not limited to, Western blot, ELISA test, RIA test, ECL test, IRMA test, EIA test, BIACORE™ test, and peptide scan.

[0078] The term "specificity" refers to the selective recognition of an antigen-binding protein for a specific epitope of an antigen. Natural antibodies, for example, are monospecific. The term "multispecificity," as used herein, means that an antigen-binding protein has multiepitope specificity (i.e., specific binding to two, three, or more different epitopes on a single biomolecule, or specific binding to epitopes on two, three, or more different biomolecules). "Bispecificity," as used herein, means that an antigen-binding protein has two different antigen-binding specificities. Unless otherwise specified, the antigens bound by a bispecific antibody may be listed in any order. For example, the terms "anti-CD3 / HER2," "anti-HER2 / CD3," "CD3xHER2," and "HER2xCD3" may be used interchangeably to refer to a bispecific antibody that specifically binds to both CD3 and HER2. The term "monospecificity," as used herein, refers to an antigen-binding protein that has one or more binding sites, each of which binds to the same epitope of the same antigen.

[0079] The term "valent," as used herein, refers to the presence of a specified number of binding sites in an antigen-binding protein. For example, a natural antibody, or a full-length antibody, has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" refer to the presence of two, three, four, five, and six binding sites, respectively, in an antigen-binding protein.

[0080] "Antibody effector function" refers to a biological activity attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region) and varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. A "reduced or minimized" antibody effector function means that it is reduced by at least 50% (alternatively, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) from the wild-type or unmodified antibody. Determination of antibody effector function is readily determinable and measurable by one of skill in the art. In preferred embodiments, the antibody effector functions of complement fixation, complement-dependent cytotoxicity, and antibody-dependent cellular cytotoxicity are affected. In some embodiments, effector function is abolished by a mutation in the constant region that abolishes glycosylation, e.g., an "effectorless mutation." In some embodiments, the effectorless mutation is HThe N297A or DANA mutation (D265A + N297A) in the two regions is shown in Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001). Alternatively, additional mutations that result in reduced or eliminated effector function include K322A and L234A / L235A (LALA). Alternatively, effector function can be reduced or eliminated through production techniques, such as expression in non-glycosylated host cells (e.g., E. coli) or host cells that result in an altered glycosylation pattern that is ineffective or less effective in promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5):3466-3473 (2003)).

[0081] "Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling specific binding of these cytotoxic effector cells to antigen-bearing target cells and subsequent killing of the target cells by cytotoxins. Antibodies "arm" the cytotoxic cells and are required for target cell killing by this mechanism. The primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS USA 95:652-656 (1998).

[0082] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed. Antibody variants with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Pat. No. 6,194,551 B1 and WO 99 / 51642, the contents of which are specifically incorporated herein by reference. See also Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0083] The terms "Fc region," "fragment crystallizable region," "Fc fragment," or "Fc domain" are used herein to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from Pro230, to its carboxyl-terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the antibody or Fc fusion protein, or by recombinantly engineering a nucleic acid encoding the heavy chain of the antibody or Fc fusion protein. Accordingly, a composition of intact antibodies can include antibody populations in which all K447 residues have been removed, antibody populations in which none of the K447 residues have been removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native sequence Fc regions for use in the immunocytokines described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0084] The term IgG "isotype" or "subclass," as used herein, refers to any of the subclasses of immunoglobulins defined by the chemical and antigenic properties of their constant regions. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different immunoglobulin classes are well known and are outlined, for example, in Abbas et al. Cellular and Mol. Immunology, 4th ed. (WB Saunders, Co., 2000).

[0085] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody or Fc fusion protein. A preferred FcR is a native-sequence human FcR. Furthermore, a preferred FcR is one that binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternatively spliced ​​forms of these receptors). FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

[0086] The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is involved in the transfer of maternal IgG to the fetus. Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994). Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18:(12):592-8 (1997); Ghetie et al., Nature Biotechnology 15(7):637-40 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6 (2004); WO 2004 / 92219 (Hinton et al.)). In vivo binding to FcRn and serum half-life of human FcRn high affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides having variant Fc regions are administered. WO 2004 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcR. See, e.g., Shields See also, et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0087] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody, antigen-binding fragment (ligand, receptor, VHH, scFv, etc.), or cytokine) and its binding partner (e.g., an antigen (cell surface molecule, receptor, ligand, etc.), or cytokine receptor). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair. Binding affinity is measured by the K d , K. off , K. on , or K a The term "K" can be used to refer to off " as used herein refers to the unit s -1The term "K" is intended to refer to the off-rate constant for dissociation of an antibody (or antigen-binding fragment) from an antibody (or antigen-binding fragment) / antigen complex (e.g., a ligand-receptor complex), or the off-rate constant for dissociation of a cytokine from a cytokine / cytokine receptor complex, as determined from a kinetic selection setup, expressed as on ", as used herein, refers to the unit M -1 s -1 The equilibrium dissociation constant "K" is intended to refer to the on-rate constant for the association of an antibody (or antigen-binding fragment) with an antigen to form an antibody (or antigen-binding fragment) / antigen complex, or the on-rate constant for the association of a cytokine with a cytokine receptor to form a cytokine / cytokine receptor complex, which is expressed as D " or "K d " as used herein refers to the dissociation constant of a particular antibody (or antigen-binding fragment)-antigen interaction (or cytokine-cytokine receptor interaction), and represents the concentration of antigen (or cytokine) required to occupy half of all antibody binding domains (or antigen-binding fragments) present at equilibrium in a solution of antibody (or antigen-binding fragment) molecules (or cytokine receptors), and is expressed in units of M. off / K on is equal to K d Measurement of K assumes that all of the binding agent is in solution. When the antibody (or antigen-binding fragment) is anchored to the cell wall, for example in a yeast expression system, the corresponding equilibrium rate constant is expressed as EC50, which is K d gives a good approximation of the affinity constant K a is the dissociation constant K d is the reciprocal of the unit M -1 It is expressed as the dissociation constant (K D or K d) is used as an indicator of the affinity of an antibody (or antigen-binding fragment) for an antigen (or a cytokine for a cytokine receptor). For example, simple analysis is possible by the Scatchard method using antibodies (or antigen-binding fragments) labeled with various markers, or by using a commercially available measurement kit, BIACORE™ X (manufactured by Amersham Biosciences), or a similar kit, following the user manual and experimental procedures provided with the kit. The K that can be derived using these methods D The value is expressed in units of M (Mol). An antibody or antigen-binding fragment thereof (or cytokine) that specifically binds to a target (or cytokine receptor) has a binding activity of, for example, ≦10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≦10 -12 Dissociation constant of M (K d ).

[0088] 50% inhibitory concentration (IC 50 IC ) is a measure of the effectiveness of a substance (such as an antibody or antigen-binding fragment) in inhibiting a specific biological or biochemical function. It indicates how much of a particular drug or other substance (such as an antibody or antigen-binding fragment, an inhibitor) is needed to inhibit a given biological process by half. This value is typically expressed as a molar concentration. 50 The EC2000 standard for agonist drugs or other substances (such as antibodies, antigen-binding fragments, or cytokines) is 50 " is equivalent to EC 50 Also refers to the plasma concentration required to achieve 50% of the maximal effect in vivo. As used herein, "IC 50 " is used to refer to the effective concentration of an antibody or antigen-binding fragment required to neutralize 50% of the antigen's biological activity in vitro. 50 or EC 50can be measured by bioassays such as inhibition of ligand binding by FACS analysis (competitive binding assays), cell-based cytokine release assays, or amplified luminescence proximity homogeneous assays (AlphaLISA).

[0089] As used herein, a "covalent bond" refers to a stable bond between two atoms that share one or more electrons. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As used herein, a "peptide bond" refers to a covalent bond formed between a carboxyl group of an amino acid and an amine group of an adjacent amino acid. A "disulfide bond" refers to a covalent bond formed between two sulfur atoms, such as the combination of two Fc fragments (or cytokine subunits) through one or more disulfide bonds. One or more disulfide bonds may be formed between two fragments by linking thiol groups present in the two fragments. In some embodiments, one or more disulfide bonds can be formed between one or more cysteines of two Fc fragments. A disulfide bond can be formed by oxidation of two thiol groups. In some embodiments, the covalent linkage is a direct covalent linkage. In some embodiments, the covalent linkage is a direct peptide bond or disulfide bond.

[0090] "Percent (%) amino acid sequence identity" and "homology," with respect to peptide, polypeptide, or antibody sequences, are defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in a particular peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including which algorithms are needed to achieve maximal alignment over the full length of the sequences under comparison.

[0091] As used herein, the "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide that donates its amine group to form a peptide bond with the carboxyl group of an adjacent amino acid residue. The "N-terminus" of a polypeptide, as used herein, refers to the first amino acid of the polypeptide that donates its carboxyl group to form a peptide bond with the amine group of an adjacent amino acid residue.

[0092] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that has been 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. Preferably, an isolated nucleic acid is free from association with all components associated with the production environment. An isolated nucleic acid molecule encoding a construct, polypeptide, or antibody described herein is in a form other than the form or setting in which it is found in nature. Thus, an isolated nucleic acid molecule is distinguished from nucleic acids encoding the constructs, polypeptides, and antibodies described herein that are naturally present in a cell. Isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that ordinarily contains the nucleic acid molecule, but that is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0093] The term "control sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0094] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0095] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as autonomously replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0096] The terms "transfected" or "transformed" or "transduced," as used herein, refer to the process for transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0097] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and its progeny without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are encompassed herein.

[0098] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation is to be administered. Such formulations are sterile. A "sterile" formulation is aseptic or free of all living microorganisms and their spores.

[0099] It is understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0100] As used herein, reference to "about" a value or parameter includes (and represents) a variation on the value or parameter itself. For example, the reference to "about X" includes the reference to "X."

[0101] As used herein, the phrase "not" a value or parameter generally means and describes "other than" the value or parameter. For example, "this method is not used to treat cancer type X" means that the method is used to treat cancer types other than X.

[0102] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0103] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0104] II. Immunocytokines In one aspect, the present invention provides an immunocytokine comprising a cytokine or variant thereof located in the hinge region of an antibody heavy chain or in the hinge region between an antigen-binding fragment (e.g., a ligand, receptor, or antibody fragment) and an Fc domain subunit or a portion thereof. The parent antibody that serves as a scaffold for constructing the immunocytokines described herein can be any antibody or antigen-binding fragment format that includes a heavy chain containing a hinge region, such as a full-length four-chain antibody fused to an Fc domain subunit or a portion thereof (e.g., CH2+CH3, or CH2 only) via the hinge region, a heavy chain-only antibody, or an antigen-binding fragment (e.g., scFv, Fab). The parent antigen-binding protein that serves as a scaffold in constructing the immunocytokines described herein may also be an antigen-binding fragment-hinge-Fc fusion protein, such as an antigen-binding fragment-hinge-Fc fusion protein comprising two antigen-binding fragment-hinge-Fc fusion polypeptides each comprising, from N' to C': an antigen-binding fragment (e.g., a ligand or receptor), a hinge region, and an Fc domain subunit or portion thereof.

[0105] In some embodiments, the immunocytokine comprises: a) an antigen binding protein (e.g., an antibody, such as a full-length antibody, or an antigen binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof. are provided, wherein the antigen-binding protein comprises, from N' to C': an antigen-binding polypeptide (e.g., an antibody heavy chain, or an antigen-binding fragment-hinge-Fc fusion polypeptide such as a ligand / receptor-hinge-Fc fusion polypeptide) comprising an antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, or VH), a hinge region, and an Fc domain subunit or portion thereof (e.g., CH2+CH3, or CH2 only), and wherein the cytokine or variant thereof is located in the hinge region (e.g., at the N', at the C', or within the N'). In some embodiments, the antigen-binding protein is an antibody (e.g., a full-length antibody) that specifically recognizes a target antigen, the antigen-binding polypeptide comprising the hinge region is the heavy chain of the antibody, and the cytokine or variant thereof is located in the hinge region of the heavy chain. Thus, in some embodiments, an immunocytokine is provided, comprising: a) an antibody (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment (e.g., scFv, Fab) fused to an Fc domain subunit or portion thereof via a hinge region) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the antibody comprises a heavy chain including a hinge region, and wherein the cytokine or variant thereof is located at the hinge region (e.g., within the hinge region, between the C-terminus of the CH1 of the heavy chain of the full-length antibody and the N-terminus of the hinge region, or between the C-terminus of the antigen-binding fragment and the N-terminus of the hinge region).In some embodiments, an immunocytokine is provided comprising: a) an antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the antibody comprises a heavy chain, and wherein the heavy chain comprises, from N-terminus to C-terminus: a VH domain, optionally a CH1 domain, a cytokine or a variant thereof in the hinge region, a CH2 domain, and optionally a CH3 domain.In some embodiments, the polypeptide comprises: (a) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VH domain, an optional first CH1, a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof located in a first hinge region (e.g., the cytokine portion is between the C-terminus of the CH1 and the N-terminus of the hinge region, or between the C-terminus of the hinge region and the N-terminus of the Fc domain subunit, or within the hinge region), and a first subunit of an Fc domain or a portion thereof (e.g., CH2+CH3, or CH2 only); (b) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VH domain, an optional second CH1, a second hinge region, and a second subunit of an Fc domain or a portion thereof (e.g., CH2+CH3, or CH2 only); and (c) an N-terminus to and (d) a fourth antigen-binding polypeptide comprising, from the N-terminus to the C-terminus: a second VL domain and an optional second CL; wherein the first VH and first VL and optionally the first CH1 and first CL bind to a first target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-L1). , CD3, CD4, CD123, or CD8); and wherein the second VH and second VL and optionally the second CH1 and second CL form a second antigen-binding domain (e.g., Fab) that specifically recognizes a second target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8). In some embodiments, the immunocytokine described herein comprises a full-length parent antibody (hereinafter also referred to as a "full-length antibody immunocytokine").Thus, in some embodiments, an immunocytokine is provided that includes: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the cytokine or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, the antigen-binding protein is an antigen-binding fragment-hinge-Fc fusion protein, such as an scFv-hinge-Fc fusion protein, a VHH-hinge-Fc fusion protein, a ligand-hinge-Fc fusion protein, or a receptor-hinge-Fc fusion protein (hereinafter, the corresponding immunocytokine is also referred to as an "Fc fusion protein immunocytokine"). In some embodiments, the antigen-binding fragment is a ligand, and the target antigen is a receptor that is specifically recognized by the ligand. In some embodiments, the antigen-binding fragment is a receptor, and the target antigen is a ligand that is specifically recognized by the receptor. In some embodiments, the parent antigen-binding fragment-hinge-Fc fusion protein used as a scaffold in constructing the immunocytokines described herein comprises two antigen-binding fragment-hinge-Fc fusion polypeptides, each comprising, from N' to C': an antigen-binding fragment (e.g., a ligand, a receptor, a VHH, or an scFv), a hinge region, and an Fc domain subunit or portion thereof.Thus, in some embodiments, an immunocytokine is provided that includes: a) an antigen-binding fragment-hinge-Fc fusion protein that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof. wherein the antigen-binding fragment-hinge-Fc fusion protein comprises an antigen-binding fragment-hinge-Fc fusion polypeptide comprising, from N' to C': an antigen-binding fragment (e.g., a ligand, receptor, VHH, or scFv), a hinge region, and an Fc domain subunit or portion thereof (e.g., CH2+CH3, or CH2 only), and wherein the cytokine or variant thereof is located in (e.g., at or within the N', C', or N') the hinge region.In some embodiments, the first antigen-binding polypeptide comprises: (a) from N-terminus to C-terminus: a first antigen-binding fragment (e.g., a ligand, receptor, VHH, or scFv), a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof located in a first hinge region (e.g., the cytokine portion is between the C-terminus of the antigen-binding fragment and the N-terminus of the hinge region, between the C-terminus of the hinge region and the N-terminus of the Fc domain subunit, or within the hinge region), and a first subunit of the Fc domain or a portion thereof (e.g., CH2+CH3, or CH2 only); (b) from N-terminus to C-terminus: a Provided are immunocytokines comprising a second antigen-binding polypeptide comprising two antigen-binding fragments (e.g., a ligand, receptor, VHH, or scFv), a second hinge region, and a second subunit of an Fc domain or a portion thereof (e.g., CH2+CH3, or CH2 only); wherein the first antigen-binding fragment specifically recognizes a first target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and wherein the second antigen-binding fragment specifically recognizes a second target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8). In some embodiments, in the presence of binding of an antigen binding protein (e.g., an antibody (e.g., a full-length antibody), or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to a target antigen, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the cytokine or variant thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antigen binding protein (e.g., an antibody (e.g., a full-length antibody), or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to the target antigen.In some embodiments, in the absence of binding of an antigen-binding protein (e.g., an antibody (e.g., a full-length antibody), or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to a target antigen, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the cytokine or variant thereof located in the hinge region is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0% or less) of the activity of the corresponding cytokine or variant thereof in the free state. In some embodiments, the cytokine or variant thereof is a cytokine variant, wherein the activity of the cytokine variant in the free state (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) is about 80% or less (e.g., about any of 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% or less) of the activity of the corresponding wild-type cytokine in the free state. In some embodiments, the antibody (e.g., a full-length antibody or an antigen-binding fragment fused to an Fc domain or portion thereof) comprises two heavy chains each comprising a hinge region, and only one heavy chain comprises the cytokine or variant thereof located in the hinge region. In some embodiments, the antigen binding protein (e.g., an antibody such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) comprises two antigen-binding polypeptides (e.g., antibody heavy chains, or antigen-binding fragment-hinge-Fc fusion polypeptides such as a ligand / receptor-hinge-Fc fusion polypeptide), each comprising a hinge region, and wherein only one of the antigen-binding polypeptides comprises a cytokine or variant thereof located in the hinge region (e.g., at or within the N', C', or N'). In some embodiments, the antigen. A binding protein (e.g., an antibody such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) comprises two antigen-binding polypeptides (e.g., antibody heavy chains, or antigen-binding fragment-hinge-Fc fusion polypeptides such as a ligand / receptor-hinge-Fc fusion polypeptide), each comprising a hinge region, and wherein each antigen-binding polypeptide comprises a cytokine or variant thereof located in the hinge region (e.g., at its N', its C', or within it). In some embodiments, an antibody (e.g., a full-length antibody, or an antigen-binding fragment fused to an Fc domain or portion thereof) comprises two heavy chains, each comprising a hinge region, and each heavy chain comprises a cytokine or variant thereof located in the hinge region. In some embodiments, the immunocytokine comprises two or more (e.g., 2, 3, 4, 5, or more) cytokines or variants thereof, wherein the two or more cytokines or variants thereof are located in tandem (e.g., linked via optional peptide linkers) in the hinge region of an antigen-binding polypeptide (e.g., an antibody heavy chain or an antigen-binding fragment-hinge-Fc fusion polypeptide such as a ligand / receptor-hinge-Fc fusion polypeptide). In some embodiments, the cytokine or variants thereof is a monomeric cytokine or variants thereof, such as IL-2 or IFN-α (e.g., IFN-α2a, IFN-α2b, IFN-α2c). In some embodiments, the cytokine or variants thereof is a dimeric cytokine or variants thereof. In some embodiments, the cytokine or variants thereof is a homodimeric cytokine or variants thereof, such as IL-10 or IFN-γ. In some embodiments, the cytokine or variants thereof is a heterodimeric cytokine or variants thereof, such as IL-12 or IL-23. In some embodiments, both subunits of the dimeric cytokine or variant thereof are located in tandem (e.g., linked via an optional peptide linker) in the hinge region of an antigen-binding polypeptide (e.g., the heavy chain of an antibody or an antigen-binding fragment-hinge-Fc fusion polypeptide such as a ligand / receptor-hinge-Fc fusion polypeptide).In some embodiments, the antigen-binding protein (e.g., an antibody such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) comprises two antigen-binding polypeptides (e.g., heavy chains or antigen-binding fragment-hinge-Fc fusion polypeptides such as a ligand / receptor-hinge-Fc fusion polypeptide), each comprising a hinge region, wherein one subunit of the dimeric cytokine or variant thereof is located in the hinge region of one antigen-binding polypeptide (e.g., heavy chain or antigen-binding fragment-hinge-Fc fusion polypeptide such as a ligand / receptor-hinge-Fc fusion polypeptide), and the other subunit of the dimeric cytokine or variant thereof is located in the hinge region of the other antigen-binding polypeptide (e.g., heavy chain or antigen-binding fragment-hinge-Fc fusion polypeptide such as a ligand / receptor-hinge-Fc fusion polypeptide). In some embodiments, the parent antigen binding protein (e.g., an antibody such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) is a homodimer. In some embodiments, the parent antigen binding protein (e.g., an antibody such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) is a heterodimer. In some embodiments, two or more cytokines or variants thereof are the same. In some embodiments, two or more cytokines or variants thereof are different. In some embodiments, the antigen binding protein (e.g., an antibody or fragment thereof, or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) is monospecific. In some embodiments, the antigen binding protein (e.g., an antibody or fragment thereof, or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) is multispecific.In some embodiments, the cytokine or variant thereof is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IL-35, IFN-α, IFN-β, IFN-γ, TNF-α, TGF-β, erythropoietin, VEGF, thrombopoietin, G-CSF, M-CSF, SCF, and GM-CSF. In some embodiments, the cytokine or variant thereof is located between the C-terminus of the antigen-binding fragment of the antigen-binding polypeptide (e.g., scFv, VHH, ligand, receptor) and the N-terminus of the hinge region. In some embodiments, the heavy chain of the antibody (e.g., a full-length antibody) comprises constant region 1 (CH1), and the cytokine or variant thereof is located between the C-terminus of CH1 of the antibody heavy chain and the N-terminus of the hinge region. In some embodiments, the cytokine or variant thereof is located between the C-terminus of the hinge region and the N-terminus of the Fc domain subunit (or a portion thereof) of an antigen-binding polypeptide (e.g., an antibody heavy chain, or an antigen-binding fragment-hinge-Fc fusion protein). In some embodiments, the cytokine or variant thereof is located within the hinge region of an antigen-binding polypeptide (e.g., an antibody heavy chain, or fragment thereof, or an antigen-binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) (e.g., replacing an internal portion of the hinge region, inserted within the hinge region and introducing one or more additional hinge or linker amino acids, or inserted within the hinge region without deleting any hinge region amino acids). In some embodiments, an antigen-binding polypeptide, such as an Fc domain subunit or portion thereof (e.g., an antibody heavy chain or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein), comprises a knob-into-hole (KIH) mutation (e.g., each antigen-binding polypeptide (e.g., a heavy chain of a full-length antibody) comprises a KIH mutation).In some embodiments, the antigen binding protein is an agonist antibody (or antigen-binding fragment thereof) or ligand (e.g., PD-L2, PD-L1, CD80, or CD86) that can activate or stimulate an immunoinhibitory signaling pathway (e.g., by binding to an inhibitory immune checkpoint molecule such as PD-1 or CTLA-4 on a target cell), and the cytokine or variant thereof is an immunostimulatory cytokine or variant thereof (e.g., IL-2, IL-12, or IL-23). ​​In some embodiments, the antigen binding protein is an antagonist antibody (or antigen-binding fragment thereof), ligand, or receptor that can reduce or block an immunostimulatory signaling pathway (e.g., by binding to a stimulatory immune checkpoint molecule such as CD27 or CD28, or an immunostimulatory receptor such as IL-2R), and the cytokine or variant thereof is an immunostimulatory cytokine or variant thereof (e.g., IL-2, IL-12, or IL-23). In some embodiments, the antigen binding protein is an antagonist antibody (or antigen-binding fragment thereof), ligand, or receptor capable of reducing or blocking an immunoinhibitory signaling pathway (e.g., by binding to an inhibitory immune checkpoint molecule such as PD-1 or CTLA-4), and the cytokine or variant thereof is an immunoinhibitory cytokine or variant thereof (e.g., IL-10, IL-27, IL-35, TGF-β). In some embodiments, the antigen binding protein is an agonist antibody (or antigen-binding fragment thereof) or ligand (e.g., CD70, CD80, CD86, or IL-2) capable of activating or stimulating an immunostimulatory signaling pathway (e.g., by binding to a stimulatory immune checkpoint molecule such as CD27 or CD28, or an immunostimulatory receptor such as IL-2R), and the cytokine or variant thereof is an immunoinhibitory cytokine or variant thereof (e.g., IL-10, IL-27, IL-35, TGF-β).

[0106] In some embodiments, an immunocytokinin is provided, comprising: a) an antigen binding protein (e.g., an antibody, such as a full-length antibody, or an antigen binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof. provided herein are antigen-binding polypeptides, wherein the antigen-binding protein comprises, from N' to C': an antigen-binding polypeptide (e.g., an antibody heavy chain, or an antigen-binding fragment-hinge-Fc fusion polypeptide such as a ligand / receptor-hinge-Fc fusion polypeptide) comprising an antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, or VH), a hinge region, and an Fc domain subunit or portion thereof (e.g., CH2+CH3, or CH2 only), wherein the cytokine or variant thereof is located in (e.g., at or within the N', C', or N' of) the hinge region;and wherein in the presence of binding of an antigen binding protein (e.g., an antibody (e.g., a full-length antibody), or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to a target antigen, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the cytokine or variant thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antigen binding protein (e.g., an antibody (e.g., a full-length antibody), or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to the target antigen. In some embodiments, an immunocytokinin is provided, comprising: a) an antigen binding protein (e.g., an antibody, such as a full-length antibody, or an antigen binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof. provided herein are antigen-binding polypeptides, wherein the antigen-binding protein comprises, from N' to C': an antigen-binding polypeptide (e.g., an antibody heavy chain, or an antigen-binding fragment-hinge-Fc fusion polypeptide such as a ligand / receptor-hinge-Fc fusion polypeptide) comprising an antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, or VH), a hinge region, and an Fc domain subunit or portion thereof (e.g., CH2+CH3, or CH2 only), wherein the cytokine or variant thereof is located in (e.g., at or within the N', C', or N' of) the hinge region;and wherein, in the absence of binding of an antigen-binding protein (e.g., an antibody (e.g., a full-length antibody), or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to a target antigen, the activity (binding affinity to the corresponding cytokine receptor or a subunit thereof, and / or biological activity) of the cytokine or variant thereof located in the hinge region is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0% or less) of the activity of the corresponding cytokine or variant thereof in the free state. In some embodiments, an immunocytokinin is provided, comprising: a) an antigen binding protein (e.g., an antibody, such as a full-length antibody, or an antigen binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof. provided herein are antigen-binding polypeptides, wherein the antigen-binding protein comprises, from N' to C': an antigen-binding polypeptide (e.g., an antibody heavy chain, or an antigen-binding fragment-hinge-Fc fusion polypeptide such as a ligand / receptor-hinge-Fc fusion polypeptide) comprising an antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, or VH), a hinge region, and an Fc domain subunit or portion thereof (e.g., CH2+CH3, or CH2 only), wherein the cytokine or variant thereof is located in (e.g., at or within the N', C', or N' of) the hinge region;wherein in the presence of binding of an antigen-binding protein (e.g., an antibody (e.g., a full-length antibody), or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to a target antigen, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the cytokine or variant thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antigen-binding protein (e.g., an antibody (e.g., a full-length antibody), or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to the target antigen;and wherein, in the absence of binding of an antigen-binding protein (e.g., an antibody (e.g., a full-length antibody), or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to a target antigen, the activity (binding affinity to the corresponding cytokine receptor or a subunit thereof, and / or biological activity) of the cytokine or variant thereof located in the hinge region is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0% or less) of the activity of the corresponding cytokine or variant thereof in the free state. In some embodiments, the antigen-binding protein is an antibody that specifically recognizes a target antigen, the antigen-binding polypeptide comprising a hinge region is a heavy chain of the antibody, and the cytokine or variant thereof is located in the hinge region of the heavy chain. In some embodiments, the antigen-binding fragment is a ligand, and the target antigen is a receptor specifically recognized by the ligand. In some embodiments, the antigen-binding fragment is a receptor, and the target antigen is a ligand specifically recognized by the receptor. In some embodiments, an immunocytokine is provided, comprising: a) an antibody (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof via a hinge region) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the antibody comprises a heavy chain including a hinge region, and wherein the cytokine or variant thereof is located in the hinge region (e.g., within the hinge region, between the C-terminus of the CH1 of the heavy chain of the full-length antibody and the N-terminus of the hinge region, or between the C-terminus of the antigen-binding fragment and the N-terminus of the hinge region);and wherein in the presence of binding of the antibody (or antigen-binding fragment) to the target antigen, the activity of the cytokine or variant thereof (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antibody (or antigen-binding fragment) to the target antigen. In some embodiments, an immunocytokine is provided, comprising: a) an antibody (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof via a hinge region) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the antibody comprises a heavy chain including a hinge region, and wherein the cytokine or variant thereof is located within the hinge region (e.g., within the hinge region) of the full-length antibody. and in the absence of binding of the antibody (or antigen-binding fragment) to a target antigen, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the cytokine or variant thereof located in the hinge region is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding cytokine or variant thereof in the free state. In some embodiments, a) an antibody (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused via a hinge region to an Fc domain subunit or portion thereof) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, CD123, HER2, PD-1, CD3, CD4, or CD8);b) immunocytokines comprising a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the antibody comprises a heavy chain including a hinge region, and wherein the cytokine or variant thereof is located in the hinge region (e.g., within the hinge region, between the C-terminus of the CH1 of the heavy chain of a full-length antibody and the N-terminus of the hinge region, or between the C-terminus of an antigen-binding fragment and the N-terminus of the hinge region); and wherein in the presence of binding of the antibody (or antigen-binding fragment) to a target antigen, the activity of the cytokine or variant thereof (binding affinity to a corresponding cytokine receptor or subunit thereof, and / or biological activity) is increased compared to the activity in the absence of binding of the antibody (or antigen-binding fragment) to the target antigen. and in the absence of binding of the antibody (or antigen-binding fragment) to a target antigen, the activity (binding affinity to a corresponding cytokine receptor or subunit thereof, and / or biological activity) of the cytokine or variant thereof located in the hinge region is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding cytokine or variant thereof in the free state. Partial implementation; In some embodiments, an immunocytokine is provided that includes: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, CD123, HER2, PD-1, CD3, CD4, or CD8); and b) a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the cytokine or variant thereof is located in (e.g., within the hinge region or CH) of the hinge region of the heavy chain of the full-length antibody. 1 and the N-terminus of the hinge region); and wherein in the presence of binding of the full-length antibody to the target antigen, the activity of the cytokine or variant thereof (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the full-length antibody to the target antigen. In some embodiments, an immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, CD123, HER2, PD-1, CD3, CD4, or CD8); and b) a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the cytokine or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). and wherein, in the absence of binding of the full-length antibody to a target antigen, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the cytokine or variant thereof located in the hinge region of the heavy chain of the full-length antibody is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding cytokine or variant thereof in the free state.In some embodiments, an immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, CD123, HER2, PD-1, CD3, CD4, or CD8); and b) a cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the cytokine or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region); and wherein, in the presence of binding of the full-length antibody to the target antigen, the activity of the cytokine or variant thereof (binding affinity to a corresponding cytokine receptor or subunit thereof, and / or biological activity) is greater than or equal to the activity of the full-length antibody binding to the target antigen. and wherein, in the absence of binding of the full-length antibody to a target antigen, the activity of a cytokine or variant thereof located in the hinge region of the heavy chain of the full-length antibody (as determined by a corresponding cytokine receptor or In some embodiments, the cytokine or variant thereof is a cytokine variant, wherein the activity (binding affinity to a corresponding cytokine receptor or a subunit thereof, and / or biological activity) of the corresponding wild-type cytokine in the free state is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding cytokine or variant thereof in the free state. In some embodiments, the cytokine or variant thereof is a cytokine variant, wherein the activity (binding affinity to a corresponding cytokine receptor or a subunit thereof, and / or biological activity) of the corresponding wild-type cytokine in the free state is about 80% or less (e.g., about any of 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%) of the activity of the corresponding wild-type cytokine in the free state. In some embodiments, the antigen binding protein (eg, an antibody or fragment thereof, or an antigen binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) is monospecific.In some embodiments, the antigen binding protein is multispecific (e.g., bispecific). In some embodiments, the parent antigen binding protein (e.g., an antibody such as a full-length antibody or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) is a homodimer. In some embodiments, the parent antigen binding protein is a heterodimer. In some embodiments, the antigen binding protein is an agonist. In some embodiments, the antigen binding protein is an antagonist.

[0107] In some embodiments, an immunocytokine is provided that includes: a) an antigen binding protein (e.g., an antibody, such as a full-length antibody, or an antigen binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); b) a first cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof; and c) a second cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein wherein the antigen-binding protein comprises, from N' to C': two antigen-binding polypeptides (e.g., antibody heavy chains, or antigen-binding fragment-hinge-Fc fusion polypeptides such as a ligand / receptor-hinge-Fc fusion polypeptide), each comprising an antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, or VH), a hinge region, and an Fc domain subunit or portion thereof (e.g., CH2+CH3, or CH2 only), wherein a first cytokine or variant thereof is located in the hinge region of the first antigen-binding polypeptide (e.g., at or within the N', C', or N' of the first cytokine or variant thereof), and wherein a second cytokine or variant thereof is located in the hinge region of the second antigen-binding polypeptide (e.g., at or within the N', C', or N' of the second cytokine or variant thereof). In some embodiments, the antigen-binding protein is an antibody that specifically recognizes a target antigen, the antigen-binding polypeptide comprising a hinge region is a heavy chain of the antibody, and the cytokine or variant thereof is located in the hinge region of the heavy chain. In some embodiments, the antigen-binding fragment is a ligand, and the target antigen is a receptor specifically recognized by the ligand. In some embodiments, the antigen-binding fragment is a receptor, and the target antigen is a ligand specifically recognized by the receptor.In some embodiments, the antibody comprises a combination of: a) an antibody (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof via a hinge region) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); b) a first cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof; and c) a second cytokine (e.g., IL-2, IFN-α (e.g., and an immunocytokine comprising an IFN-α2b, IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the antibody comprises two heavy chains each comprising a hinge region, and wherein a first cytokine or variant thereof is located in the hinge region of the first heavy chain of the antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region), and a second cytokine or variant thereof is located in the hinge region of the second heavy chain of the antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, the antibody comprises: a) an antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); b) a first cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof; and c) a second cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23). or a variant thereof, wherein the antibody comprises two heavy chains, wherein the first heavy chain comprises, from N-terminus to C-terminus: a VH domain, optionally a CH1 domain, a first cytokine or a variant thereof in a first hinge region, a CH2 domain, and optionally a CH3 domain; and wherein the second heavy chain comprises, from N-terminus to C-terminus: a VH domain, optionally a CH1 domain, a second cytokine or a variant thereof in a second hinge region, a CH2 domain, and optionally a CH3 domain.In some embodiments, the antibody comprises a combination of a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); b) a first cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof; and c) a second cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, and a second cytokine or variant thereof, wherein the first cytokine or variant thereof is located in the hinge region of a first heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region), and the second cytokine or variant thereof is located in the hinge region of a second heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, the antigen binding protein (e.g., an antibody or fragment thereof, or an antigen-binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) is monospecific. In some embodiments, the antigen binding protein is multispecific (e.g., bispecific). In some embodiments, the parent antigen binding protein (e.g., an antibody such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) is a homodimer. In some embodiments, the parent antigen binding protein is a heterodimer. In some embodiments, a first heavy chain and a first light chain of the antibody form a first antigen-binding fragment (e.g., a first Fab) that specifically recognizes a first target antigen, and a second heavy chain and a second light chain of the antibody form a second antigen-binding fragment (e.g., a second Fab) that specifically recognizes a second target antigen.In some embodiments, in the presence of binding of an antigen binding protein (e.g., an antibody such as a full-length antibody, or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to a target antigen (e.g., binding of a first antigen-binding fragment such as a Fab or ligand to a first target antigen, and / or binding of a second antigen-binding fragment such as a Fab or ligand to a second target antigen), activity of the first and / or second cytokine or variant thereof (e.g., binding to a corresponding cytokine receptor or subunit thereof) is inhibited. The affinity, and / or biological activity) of the antibody is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antigen-binding protein (e.g., an antibody such as a full-length antibody, or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to the target antigen (wherein the antigen-binding fragment, such as a Fab or ligand, does not bind to the target antigen). In some embodiments, in the absence of binding of an antigen-binding protein (e.g., an antibody such as a full-length antibody, or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to a target antigen (e.g., an antigen-binding fragment such as a Fab or ligand does not bind to the target antigen), the first and / or second cytokines located in the hinge region of each antigen-binding polypeptide (e.g., an antibody heavy chain, or an antigen-binding fragment-hinge-Fc polypeptide) bind to the target antigen. The activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the corresponding first and / or second cytokine or variant thereof is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0% or less) of the activity of the corresponding first and / or second cytokine or variant thereof in the free state.In some embodiments, the first and / or second cytokine or variant thereof is a cytokine variant, wherein the activity of the cytokine variant in the free state (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) is about 80% or less (e.g., about any of 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% or less) of the activity of the corresponding wild-type cytokine in the free state. In some embodiments, the two cytokines or variants thereof are the same. In some embodiments, the two cytokines or variants thereof are different. In some embodiments, the first cytokine or variant thereof is IL-2 or a variant thereof (e.g., SEQ ID NO: 2). In some embodiments, the second cytokine or variant thereof is IL-12 or a variant thereof (e.g., SEQ ID NO: 36). In some embodiments, the antigen binding protein is an agonist. In some embodiments, the antigen binding protein is an antagonist.

[0108] In some embodiments, an immunocytokine is provided that includes: a) an antigen binding protein (e.g., an antibody, such as a full-length antibody, or an antigen binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); b) a first cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof; and c) a second cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof. and wherein the antigen-binding protein comprises, from N' to C': an antigen-binding polypeptide (e.g., an antibody heavy chain, or an antigen-binding fragment-hinge-Fc fusion polypeptide such as a ligand / receptor-hinge-Fc fusion polypeptide) comprising an antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, or VH), a hinge region, and an Fc domain subunit or portion thereof (e.g., CH2+CH3, or CH2 only), and wherein a first cytokine or variant thereof and a second cytokine or variant thereof are located in tandem (e.g., connected via an optional peptide linker) in the hinge region of the antigen-binding polypeptide (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, the antigen-binding protein is an antibody that specifically recognizes a target antigen, the antigen-binding polypeptide comprising the hinge region is the heavy chain of the antibody, and the cytokine or variant thereof is located in the hinge region of the heavy chain. In some embodiments, the antigen-binding fragment is a ligand and the target antigen is a receptor that is specifically recognized by that ligand. In some embodiments, the antigen-binding fragment is a receptor and the target antigen is a ligand that is specifically recognized by that receptor.In some embodiments, the antibody comprises: a) an antibody (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused via a hinge region to an Fc domain subunit or portion thereof) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); b) a first cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof; and c) a second cytokine. and a first cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or variant thereof, wherein the antibody comprises a heavy chain including a hinge region, and wherein the first cytokine or variant thereof and the second cytokine or variant thereof are located in tandem (e.g., connected via an optional peptide linker) in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, the antibody comprises: a) an antibody (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused via a hinge region to an Fc domain subunit or portion thereof) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); b) a first cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof; and c) a first cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof; and ) a second cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or variant thereof, and wherein the antibody comprises a heavy chain comprising, from N-terminus to C-terminus: a VH domain, optionally a CH1 domain, the first cytokine or variant thereof in the hinge region, optionally a linker (e.g., a peptide linker), the second cytokine or variant thereof, a CH2 domain, and optionally a CH3 domain.In some embodiments, the antibody comprises a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); b) a first cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof; and c) a second cytokine (e.g., IL-2, IFN-α (e.g., IFN-α2b), IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof. and a second cytokine or variant thereof, wherein the first cytokine or variant thereof and the second cytokine or variant thereof are located in tandem (e.g., linked via an optional peptide linker) in the hinge region of a heavy chain of a full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, in the presence of binding of the antigen binding protein (e.g., an antibody such as a full-length antibody, or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to the target antigen, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the first and second cytokines or variants thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more) compared to the activity in the absence of binding of the antigen binding protein (e.g., an antibody such as a full-length antibody, or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to the target antigen.In some embodiments, in the absence of binding of the antigen binding protein (e.g., an antibody such as a full-length antibody, or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to a target antigen, the activity (corresponding sequence) of the first and second cytokines or variants thereof located in the hinge region of the antigen-binding polypeptide (e.g., an antibody heavy chain, or an antigen-binding fragment-hinge-Fc fusion protein) is not observed. The binding affinity to a cytokine receptor or a subunit thereof, and / or biological activity) of the corresponding first and second cytokines or variants thereof in the free state is about 70% or less (e.g., about any of about 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0% or less). In some embodiments, the first and / or second cytokine or variant thereof is a cytokine variant, wherein the activity of the cytokine variant in the free state (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) is about 80% or less (such as about any of 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% or less) of the activity of the corresponding wild-type cytokine in the free state. In some embodiments, the antigen binding protein (e.g., an antibody or fragment thereof, or an antigen-binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) is monospecific. In some embodiments, the antigen binding protein is multispecific (e.g., bispecific). In some embodiments, the parent antigen binding protein (e.g., an antibody, such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) is a homodimer. In some embodiments, the parent antigen binding protein is a heterodimer. In some embodiments, the two cytokines or variants thereof are the same. In some embodiments, the two cytokines or variants thereof are different. In some embodiments, the first cytokine or variant thereof is IL-2 or a variant thereof (eg, SEQ ID NO: 2).In some embodiments, the second cytokine or variant thereof is IL-12 or a variant thereof (e.g., SEQ ID NO: 36). In some embodiments, the antigen binding protein is an agonist. In some embodiments, the antigen binding protein is an antagonist.

[0109] In some embodiments, the cytokine or variant thereof is a dimeric cytokine or variant thereof. In some embodiments, the cytokine or variant thereof is a homodimeric cytokine or variant thereof, such as IL-10 or IFN-γ. In some embodiments, the cytokine or variant thereof is a heterodimeric cytokine or variant thereof, such as IL-12 or IL-23. In some embodiments, an immunocytokine is provided comprising: a) an antigen binding protein (e.g., an antibody such as a full-length antibody, or an antigen binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a dimeric cytokine (e.g., IFN-γ, IL-10, IL-12, or IL-23) or variant thereof, wherein the antigen binding protein is, from N' to C': an antigen binding fragment (e.g., a ligand, receptor, VHH , scFv, or VH), a hinge region, and an antigen-binding polypeptide (e.g., an antibody heavy chain, or an antigen-binding fragment-hinge-Fc fusion polypeptide such as a ligand / receptor-hinge-Fc fusion polypeptide) comprising an Fc domain subunit or portion thereof (e.g., CH2+CH3, or CH2 only), and wherein the subunits of the dimeric cytokine or variant thereof are both located in tandem (e.g., at the N', C', or within) the hinge region of the antigen-binding polypeptide (e.g., linked via an optional peptide linker such as any of SEQ ID NOs: 227-229). In some embodiments, the antigen-binding protein is an antibody that specifically recognizes a target antigen, the antigen-binding polypeptide comprising the hinge region is the heavy chain of the antibody, and the cytokine or variant thereof is located in the hinge region of the heavy chain. In some embodiments, the antigen-binding fragment is a ligand, and the target antigen is a receptor that is specifically recognized by the ligand. In some embodiments, the antigen-binding fragment is a receptor and the target antigen is a ligand that is specifically recognized by that receptor.In some embodiments, an immunocytokine is provided, comprising: a) an antibody (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof via a hinge region) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a dimeric cytokine (e.g., IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the antibody comprises a heavy chain including a hinge region, and wherein the subunits of the dimeric cytokine or variant thereof are both located in tandem at the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region) (e.g., connected via an optional peptide linker, such as any of SEQ ID NOs: 227-229). In some embodiments, an immunocytokine is provided comprising: a) an antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a dimeric cytokine (e.g., IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the antibody comprises a heavy chain comprising, from N-terminus to C-terminus: a VH domain, optionally a CH1 domain, a hinge region [a first subunit of the dimeric cytokine or variant thereof, optionally a linker (e.g., a peptide linker), a second subunit of the dimeric cytokine or variant thereof], a CH2 domain, and optionally a CH3 domain.In some embodiments, an immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a dimeric cytokine (e.g., IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the subunits of the dimeric cytokine or variant thereof are both located in tandem (e.g., connected via an optional peptide linker, such as any of SEQ ID NOs: 227-229) in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, in the presence of an antigen binding protein (e.g., an antibody such as a full-length antibody, or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) binding to a target antigen, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the dimeric cytokine or variant thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more) compared to the activity in the absence of the antigen binding protein (e.g., an antibody such as a full-length antibody, or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) binding to the target antigen.In some embodiments, in the absence of binding of an antigen-binding protein (e.g., an antibody such as a full-length antibody, or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to a target antigen, the activity (binding affinity to a corresponding cytokine receptor or subunit thereof, and / or biological activity) of a dimeric cytokine or variant thereof located in the hinge region of an antigen-binding polypeptide (e.g., an antibody heavy chain or antigen-binding fragment-hinge-Fc fusion polypeptide) is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0% or less) of the activity of the corresponding dimeric cytokine or variant thereof in the free state. In some embodiments, the dimeric cytokine or variant thereof is a dimeric cytokine variant, and the activity of the dimeric cytokine variant in the free state (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) is about 80% or less (e.g., about any of 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% or less) of the activity of the corresponding wild-type dimeric cytokine in the free state. In some embodiments, the antigen binding protein (e.g., an antibody or fragment thereof, or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) is monospecific. In some embodiments, the antigen binding protein is multispecific (e.g., bispecific). In some embodiments, the parent antigen binding protein (e.g., an antibody such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) is a homodimer. In some embodiments, the parent antigen binding protein is a heterodimer. In some embodiments, the cytokine or variant thereof is a homodimeric cytokine or variant thereof, such as IL-10 or IFN-γ, hi some embodiments, the cytokine or variant thereof is a heterodimeric cytokine or variant thereof, such as IL-12 or IL-23.In some embodiments, the antigen binding protein is an agonist. In some embodiments, the antigen binding protein is an antagonist.

[0110] In some embodiments, an immunocytokine is provided that includes: a) an antigen binding protein (e.g., an antibody, such as a full-length antibody, or an antigen binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a dimeric cytokine (e.g., IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the antigen binding protein is comprised of, from N' to C': the antigen binding fragment (e.g., ligand, receptor, VHH, scFv, or VH), the hinge, and the dimeric cytokine (e.g., IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof. and two antigen-binding polypeptides (e.g., antibody heavy chains, or antigen-binding fragment-hinge-Fc fusion polypeptides such as ligand / receptor-hinge-Fc fusion polypeptides), each comprising an Fc domain subunit or portion thereof (e.g., CH2+CH3, or CH2 only); wherein one subunit of the dimeric cytokine or variant thereof is located in the hinge region of the first antigen-binding polypeptide (e.g., at or within the N', C', or N') and the other subunit of the dimeric cytokine or variant thereof is located in the hinge region of the second antigen-binding polypeptide (e.g., at or within the N', C', or N'). In some embodiments, the antigen-binding protein is an antibody that specifically recognizes a target antigen, the antigen-binding polypeptide comprising the hinge region is the heavy chain of the antibody, and the cytokine or variant thereof is located in the hinge region of the heavy chain. In some embodiments, the antigen-binding fragment is a ligand, and the target antigen is a receptor that is specifically recognized by the ligand. In some embodiments, the antigen-binding fragment is a receptor and the target antigen is a ligand that is specifically recognized by that receptor.In some embodiments, an immunocytokinin comprises: a) an antibody (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof via a hinge region) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a dimeric cytokine (e.g., IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof. The invention also provides a dimeric cytokine or a variant thereof comprising two heavy chains each comprising a hinge region, wherein one subunit of the dimeric cytokine or a variant thereof is located in the hinge region of the first heavy chain (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region), and the other subunit of the dimeric cytokine or a variant thereof is located in the hinge region of the second heavy chain (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, an immunocytokine is provided comprising: a) an antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a dimeric cytokine (e.g., IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein the antibody comprises two heavy chains, wherein a first heavy chain comprises, from N-terminus to C-terminus: a VH domain, optionally a CH1 domain, a first subunit of the dimeric cytokine or a variant thereof located at a first hinge region, a CH2 domain, and optionally a CH3 domain, and wherein a second heavy chain comprises, from N-terminus to C-terminus: a VH domain, optionally a CH1 domain, a second subunit of the dimeric cytokine or a variant thereof located at a second hinge region, a CH2 domain, and optionally a CH3 domain.In some embodiments, an immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) a dimeric cytokine (e.g., IFN-γ, IL-10, IL-12, or IL-23) or a variant thereof, wherein one subunit of the dimeric cytokine or variant thereof is located in the hinge region of a first heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region), and the other subunit of the dimeric cytokine or variant thereof is located in the hinge region of a second heavy chain of the full-length antibody (e.g., between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, the antigen binding protein (e.g., an antibody or fragment thereof, or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) is monospecific. In some embodiments, the antigen binding protein is multispecific (e.g., bispecific). In some embodiments, the parent antigen binding protein (e.g., an antibody such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) is a homodimer. In some embodiments, the parent antigen binding protein is a heterodimer. In some embodiments, a first heavy chain and a first light chain of the antibody form a first antigen-binding fragment (e.g., a first Fab) that specifically recognizes a first target antigen, and a second heavy chain and a second light chain of the antibody form a second antigen-binding fragment (e.g., a second Fab) that specifically recognizes a second target antigen.In some embodiments, in the presence of binding of an antigen binding protein (e.g., an antibody such as a full-length antibody, or a ligand / receptor-hinge-Fc fusion protein) or antigen binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to a target antigen (e.g., binding of a first antigen binding fragment such as a Fab or ligand to a first target antigen, and / or binding of a second antigen binding fragment such as a Fab or ligand to a second target antigen), the activity of the dimeric cytokine or variant thereof (e.g., binding affinity to the corresponding cytokine receptor or subunit thereof, and and / or biological activity) is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antigen-binding protein (e.g., an antibody such as a full-length antibody, or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to the target antigen (e.g., an antigen-binding fragment such as a Fab or ligand does not bind to the target antigen). In some embodiments, in the absence of binding of an antigen-binding protein (e.g., an antibody such as a full-length antibody, or a ligand / receptor-hinge-Fc fusion protein) or antigen-binding fragment (e.g., a ligand, receptor, VHH, scFv, Fab) to a target antigen (e.g., an antigen-binding fragment such as a Fab or ligand does not bind to the target antigen), a dimer in which each subunit is located at the hinge region of each antigen-binding polypeptide (e.g., an antibody heavy chain, or an antigen-binding fragment-hinge-Fc polypeptide) forms. The activity of the cytokine or variant thereof (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding dimeric cytokine or variant thereof in the free state.In some embodiments, the dimeric cytokine or variant thereof is a cytokine variant, wherein the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the dimeric cytokine variant in the free state is about 80% or less (e.g., about any of 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% or less) of the activity of the corresponding wild-type dimeric cytokine in the free state. In some embodiments, the cytokine or variant thereof is a homodimeric cytokine or variant thereof, such as IL-10 or IFN-γ. In some embodiments, the cytokine or variant thereof is a heterodimeric cytokine or variant thereof, such as IL-12 or IL-23. In some embodiments, the antigen binding protein (e.g., an antibody or fragment thereof, or an antigen-binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) is monospecific. In some embodiments, the antigen binding protein is multispecific (e.g., bispecific). In some embodiments, the parent antigen binding protein (e.g., an antibody such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) is a homodimer. In some embodiments, the parent antigen binding protein is a heterodimer. In some embodiments, the antigen binding protein is an agonist. In some embodiments, the antigen binding protein is an antagonist.

[0111] In some embodiments, the cytokine or variant thereof is IL-2 or a variant thereof. In some embodiments, an IL-2 immunocytokine is provided, comprising: a) an antigen binding protein (e.g., an antibody, such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the antigen binding protein is a nucleotide sequence consisting of a sequence identical to that of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: and an antigen-binding polypeptide (e.g., an antibody heavy chain, or an antigen-binding fragment-hinge-Fc fusion polypeptide such as a ligand / receptor-hinge-Fc fusion polypeptide) comprising an antigen-binding fragment (e.g., a ligand such as the PD-L2 extracellular domain, a receptor, a VHH, an scFv, or a VH), a hinge region, and an Fc domain subunit or portion thereof (e.g., CH2+CH3, or CH2 only), and wherein IL-2 or a variant thereof is located in the hinge region (e.g., at the N', C', or within the N'). In some embodiments, the antigen-binding protein is an antibody (e.g., a full-length antibody) that specifically recognizes a target antigen, the antigen-binding polypeptide comprising the hinge region is the heavy chain of the antibody, and IL-2 or a variant thereof is located in the hinge region of the heavy chain. In some embodiments, the antigen-binding fragment is a ligand, and the target antigen is a receptor that is specifically recognized by the ligand. In some embodiments, the antigen-binding fragment is a receptor and the target antigen is a ligand that is specifically recognized by that receptor.In some embodiments, an IL-2 immunocytokine is provided, comprising: a) an antibody (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof via a hinge region) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the antibody comprises a heavy chain including a hinge region, and wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, an IL-2 immunocytokine is provided, comprising: a) an antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) anti-IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the antibody comprises a heavy chain, and wherein the heavy chain comprises, from N-terminus to C-terminus: a VH domain, optionally a CH1 domain, IL-2 or a variant thereof in the hinge region, a CH2 domain, and optionally a CH3 domain. In some embodiments, an IL-2 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region).In some embodiments, an IL-2 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) an IL-2 variant comprising one or more mutations at a position selected from the group consisting of L18, Q22, F24, K35, R38, F42, K43, E61, and P65 relative to wild-type IL-2 comprising the sequence of SEQ ID NO: 1, wherein the IL-2 variant is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IL-2 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) an IL-2 variant that comprises one or more mutations selected from the group consisting of F24A, R38D, K43E, E61R, and P65L relative to wild-type IL-2 comprising the sequence of SEQ ID NO: 1, wherein the IL-2 variant is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, an IL-2 immunocytokine is provided comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) an IL-2 variant that comprises R38D / K43E / E61R mutations relative to wild-type IL-2 comprising the sequence of SEQ ID NO: 1, wherein the IL-2 variant is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IL-2 immunocytokine is provided comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) an IL-2 variant comprising the sequence of SEQ ID NO: 2, wherein the IL-2 variant is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region).In some embodiments, the activity of the IL-2 variant in its free state (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) is about 80% or less (e.g., about any of 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% or less) of the activity of the corresponding wild-type IL-2 in its free state. In some embodiments, the antigen binding protein (e.g., an antibody or fragment thereof, or an antigen-binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) is monospecific. In some embodiments, the antigen binding protein is multispecific (e.g., bispecific). In some embodiments, the parent antigen binding protein (e.g., an antibody, such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) is a homodimer. In some embodiments, the parent antigen binding protein is a heterodimer. In some embodiments, the antigen binding protein is an agonist. In some embodiments, the antigen binding protein is an antagonist. In some embodiments, the hinge region comprises any of the sequences of SEQ ID NOs: 40-47, 50-52, and 55-59.

[0112] In some embodiments, the antigen-binding protein is an anti-HER2 antibody. Accordingly, in some embodiments, an IL-2 immunocytokine ("IL-2 / anti-HER2 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes HER2 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the antibody comprises a heavy chain comprising a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IL-2 / anti-HER2 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes HER2; and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59) (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region); and wherein the anti-HER2 antibody comprises: i) a VH-CDR1 comprising the sequence of SEQ ID NO: 188; ii) a VH-CDR2 comprising the sequence of SEQ ID NO: 189; iii) a VH-CDR3 comprising the sequence of SEQ ID NO: 190; iv) a VL-CDR1 comprising the sequence of SEQ ID NO: 191; v) a VL-CDR2 comprising the sequence of SEQ ID NO: 192; and vi) a VL-CDR3 comprising the sequence of SEQ ID NO: 193. In some embodiments, the anti-HER2 antibody, or antigen-binding fragment thereof, comprises a VH comprising the sequence of SEQ ID NO: 150 and a VL comprising the sequence of SEQ ID NO: 151. In some embodiments, the parent anti-HER2 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 152 or 153 and two light chains each comprising the sequence of SEQ ID NO: 154. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO: 2. In some embodiments, the heavy chain comprising the IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 156.In some embodiments, an IL-2 / anti-HER2 immunocytokine is provided that comprises: two light chains comprising the sequence of SEQ ID NO: 154, one heavy chain comprising an IL-2 variant located in its hinge region comprising the sequence of SEQ ID NO: 156, and one heavy chain comprising the sequence of SEQ ID NO: 155. In some embodiments, in the presence of binding of the antibody (or antigen-binding fragment) to HER2, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IL-2 cytokine or variant thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antibody (or antigen-binding fragment) to HER2. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to HER2, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IL-2 cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the corresponding IL-2 cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is homodimeric. In some embodiments, the antibody is heterodimeric. In some embodiments, the antibody is an agonistic antibody. In some embodiments, the antibody is an antagonist antibody.

[0113] In some embodiments, the antigen-binding protein is an anti-CD3 antibody. Accordingly, in some embodiments, an IL-2 immunocytokine ("IL-2 / anti-CD3 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes CD3 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the antibody comprises a heavy chain comprising a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IL-2 / anti-CD3 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes CD3; and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59) (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region); and wherein the anti-CD3 antibody comprises: i) a VH-CDR1 comprising the sequence of SEQ ID NO: 85; ii) a VH-CDR2 comprising the sequence of SEQ ID NO: 86; iii) a VH-CDR3 comprising the sequence of SEQ ID NO: 87; iv) a VL-CDR1 comprising the sequence of SEQ ID NO: 88; v) a VL-CDR2 comprising the sequence of SEQ ID NO: 89; and vi) a VL-CDR3 comprising the sequence of SEQ ID NO: 90. In some embodiments, the anti-CD3 antibody comprises a VH comprising the sequence of SEQ ID NO: 91 and a VL comprising the sequence of SEQ ID NO: 92. In some embodiments, the parent anti-CD3 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 93 and two light chains each comprising the sequence of SEQ ID NO: 94. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO: 2. In some embodiments, the heavy chain comprising the IL-2 variant located in its hinge region comprises the sequence of SEQ ID NO: 96. In some embodiments, an IL-2 / anti-CD3 immunocytokine is provided comprising: two light chains comprising the sequence of SEQ ID NO: 94, one heavy chain comprising an IL-2 variant located in its hinge region comprising the sequence of SEQ ID NO: 96, and one heavy chain comprising the sequence of SEQ ID NO: 95.In some embodiments, in the presence of the antibody (or antigen-binding fragment) binding to CD3, the activity of the IL-2 cytokine or variant thereof (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of the antibody (or antigen-binding fragment) binding to CD3. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to CD3, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IL-2 cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the corresponding IL-2 cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is homodimeric. In some embodiments, the antibody is heterodimeric. In some embodiments, the antibody is an agonistic antibody. In some embodiments, the antibody is an antagonist antibody.

[0114] In some embodiments, the antigen-binding protein is an anti-PD-1 antibody. Accordingly, in some embodiments, an IL-2 immunocytokine ("IL-2 / anti-PD-1 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes PD-1 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the antibody comprises a heavy chain that includes a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IL-2 or variant thereof is located in (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region) of the heavy chain (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59). In some embodiments, an IL-2 / anti-PD-1 immunocytokine is provided comprising: a) a full-length antibody that specifically recognizes PD-1; and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region); and wherein the anti-PD-1 antibody comprises VH-CDR1, VH-CDR2, and VH-CDR3 of a VH comprising the sequence of SEQ ID NO: 102, and VL-CDR1, VL-CDR2, and VL-CDR3 of a VL comprising the sequence of SEQ ID NO: 103. In some embodiments, the anti-PD-1 antibody comprises a VH comprising the sequence of SEQ ID NO: 102 and a VL comprising the sequence of SEQ ID NO: 103. In some embodiments, the parent anti-PD-1 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 104 or 105 and two light chains each comprising the sequence of SEQ ID NO: 106. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO: 2. In some embodiments, the heavy chain comprising the IL-2 variant located in its hinge region comprises the sequence of SEQ ID NO: 108. In some embodiments, an IL-2 / anti-PD-1 immunocytokine is provided comprising: two light chains comprising the sequence of SEQ ID NO: 106, one heavy chain comprising an IL-2 variant located in its hinge region comprising the sequence of SEQ ID NO: 108, and one heavy chain comprising the sequence of SEQ ID NO: 107.In some embodiments, in the presence of the antibody (or antigen-binding fragment) binding to PD-1, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IL-2 cytokine or variant thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of the antibody (or antigen-binding fragment) binding to PD-1. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to PD-1, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IL-2 cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the corresponding IL-2 cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is homodimeric. In some embodiments, the antibody is heterodimeric. In some embodiments, the antibody is an agonistic antibody. In some embodiments, the antibody is an antagonist antibody.

[0115] In some embodiments, the antigen-binding protein is an anti-CD4 antibody. Accordingly, in some embodiments, an IL-2 immunocytokine ("IL-2 / anti-CD4 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes CD4 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the antibody comprises a heavy chain comprising a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IL-2 / anti-CD4 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes CD4; and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59) (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region); and wherein the anti-CD4 antibody comprises: i) a VH-CDR1 comprising the sequence of SEQ ID NO: 67; ii) a VH-CDR2 comprising the sequence of SEQ ID NO: 68; iii) a VH-CDR3 comprising the sequence of SEQ ID NO: 69; iv) a VL-CDR1 comprising the sequence of SEQ ID NO: 70; v) a VL-CDR2 comprising the sequence of SEQ ID NO: 71; and vi) a VL-CDR3 comprising the sequence of SEQ ID NO: 72. In some embodiments, the anti-CD4 antibody comprises a VH comprising the sequence of SEQ ID NO: 73 and a VL comprising the sequence of SEQ ID NO: 74. In some embodiments, the parent anti-CD4 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 75 or 76 and two light chains each comprising the sequence of SEQ ID NO: 77. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO: 2. In some embodiments, the heavy chain comprising the IL-2 variant located in its hinge region comprises the sequence of SEQ ID NO: 79. In some embodiments, an IL-2 / anti-CD4 immunocytokine is provided comprising: two light chains comprising the sequence of SEQ ID NO: 77, one heavy chain comprising an IL-2 variant located in its hinge region comprising the sequence of SEQ ID NO: 79, and one heavy chain comprising the sequence of SEQ ID NO: 78.In some embodiments, in the presence of the antibody (or antigen-binding fragment) binding to CD4, the activity of the IL-2 cytokine or variant thereof (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of the antibody (or antigen-binding fragment) binding to CD4. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to CD4, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IL-2 cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the corresponding IL-2 cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is homodimeric. In some embodiments, the antibody is heterodimeric. In some embodiments, the antibody is an agonistic antibody. In some embodiments, the antibody is an antagonist antibody.

[0116] In some embodiments, the antigen-binding protein is an anti-CD8 antibody. Accordingly, in some embodiments, an IL-2 immunocytokine ("IL-2 / anti-CD8 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes CD8 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the antibody comprises a heavy chain comprising a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IL-2 / anti-CD8 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes CD8; and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59) (e.g., within the hinge region, between the C-terminus of CH1 and the N-terminus of the hinge region); and wherein the anti-CD8 antibody comprises VH-CDR1, VH-CDR2, and VH-CDR3 of a VH comprising the sequence of SEQ ID NO: 114, and VL-CDR1, VL-CDR2, and VL-CDR3 of a VL comprising the sequence of SEQ ID NO: 115. In some embodiments, the anti-CD8 antibody comprises a VH comprising the sequence of SEQ ID NO: 114 and a VL comprising the sequence of SEQ ID NO: 115. In some embodiments, the parent anti-CD8 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 116 and two light chains each comprising the sequence of SEQ ID NO: 117. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO: 2. In some embodiments, the heavy chain comprising the IL-2 variant located in its hinge region comprises the sequence of SEQ ID NO: 119. In some embodiments, an IL-2 / anti-CD8 immunocytokine is provided comprising: two light chains comprising the sequence of SEQ ID NO: 117, one heavy chain comprising an IL-2 variant located in its hinge region comprising the sequence of SEQ ID NO: 119, and one heavy chain comprising the sequence of SEQ ID NO: 118.In some embodiments, in the presence of the antibody (or antigen-binding fragment) binding to CD8, the activity of the IL-2 cytokine or variant thereof (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of the antibody (or antigen-binding fragment) binding to CD8. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to CD8, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IL-2 cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the corresponding IL-2 cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is homodimeric. In some embodiments, the antibody is heterodimeric. In some embodiments, the antibody is an agonistic antibody. In some embodiments, the antibody is an antagonist antibody.

[0117] In some embodiments, the antigen-binding protein is an anti-CTLA-4 antibody. Accordingly, in some embodiments, an IL-2 immunocytokine ("IL-2 / anti-CTLA-4 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes CTLA-4 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the antibody comprises a heavy chain that includes a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IL-2 / anti-CTLA-4 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes CTLA-4; and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59) (e.g., within the hinge region, between the C-terminus of CH1 and the N-terminus of the hinge region); and wherein the anti-CTLA-4 antibody comprises VH-CDR1, VH-CDR2, and VH-CDR3 of a VH comprising the sequence of SEQ ID NO: 125, and VL-CDR1, VL-CDR2, and VL-CDR3 of a VL comprising the sequence of SEQ ID NO: 126. In some embodiments, the anti-CTLA-4 antibody comprises a VH comprising the sequence of SEQ ID NO: 125 and a VL comprising the sequence of SEQ ID NO: 126. In some embodiments, the parent anti-CTLA-4 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 127 or 128 and two light chains each comprising the sequence of SEQ ID NO: 129. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO: 2. In some embodiments, the heavy chain comprising the IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 131. In some embodiments, an IL-2 / anti-CTLA-4 immunocytokine is provided comprising: two light chains comprising the sequence of SEQ ID NO: 129, one heavy chain comprising an IL-2 variant located in the hinge region comprising the sequence of SEQ ID NO: 131, and one heavy chain comprising the sequence of SEQ ID NO: 130.In some embodiments, in the presence of the antibody (or antigen-binding fragment) binding to CTLA-4, the activity of the IL-2 cytokine or variant thereof (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of the antibody (or antigen-binding fragment) binding to CTLA-4. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to CTLA-4, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IL-2 cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the corresponding IL-2 cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is a homodimer. In some embodiments, the antibody is a heterodimer. In some embodiments, the antibody is an agonist antibody. In some embodiments, the antibody is an antagonist antibody.

[0118] In some embodiments, the antigen binding protein is an anti-PD-L1 antibody. Accordingly, in some embodiments, an IL-2 immunocytokine ("IL-2 / anti-PD-L1 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes PD-L1 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), where the antibody comprises a heavy chain that includes a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and where the IL-2 or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region, between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IL-2 / anti-PD-L1 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes PD-L1; and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the IL-2 or variant thereof is located in (e.g., within the hinge region, between the C-terminus of CH1 and the N-terminus of the hinge region) the hinge region of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59); and wherein the anti-PD-L1 antibody comprises i) a VH-CDR1 comprising the sequence of SEQ ID NO: 243; ii) a VH-CDR2 comprising the sequence of SEQ ID NO: 244; iii) a VH-CDR3 comprising the sequence of SEQ ID NO: 245; iv) a VL-CDR1 comprising the sequence of SEQ ID NO: 246; v) a VL-CDR2 comprising the sequence of SEQ ID NO: 247; and vi) a VL-CDR3 comprising the sequence of SEQ ID NO: 248. In some embodiments, the anti-PD-L1 antibody comprises a VH comprising the sequence of SEQ ID NO: 137 and a VL comprising the sequence of SEQ ID NO: 138. In some embodiments, the parent anti-PD-L1 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 139 and two light chains each comprising the sequence of SEQ ID NO: 140. In some embodiments, the parent anti-PD-L1 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 141 and two light chains each comprising the sequence of SEQ ID NO: 142. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO: 2. In some embodiments, the heavy chain comprising the IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 144.In some embodiments, an IL-2 / anti-PD-L1 immunocytokine is provided that comprises: two light chains comprising the sequence of SEQ ID NO: 140, one heavy chain comprising an IL-2 variant located in its hinge region comprising the sequence of SEQ ID NO: 144, and one heavy chain comprising the sequence of SEQ ID NO: 143. In some embodiments, in the presence of binding of the antibody (or antigen-binding fragment) to PD-L1, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IL-2 cytokine or variant thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more) compared to the activity in the absence of binding of the antibody (or antigen-binding fragment) to PD-L1. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to PD-L1, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IL-2 cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the corresponding IL-2 cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is a homodimer. In some embodiments, the antibody is a heterodimer.

[0119] In some embodiments, the antigen-binding protein is an anti-CD25 antibody. Accordingly, in some embodiments, an IL-2 immunocytokine ("IL-2 / anti-CD25 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes CD25 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the antibody comprises a heavy chain comprising a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IL-2 / anti-CD25 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes CD25; and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), wherein the IL-2 or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59) (e.g., within the hinge region, between the C-terminus of CH1 and the N-terminus of the hinge region); and wherein the anti-CD25 antibody comprises VH-CDR1, VH-CDR2, and VH-CDR3 of a VH comprising the sequence of SEQ ID NO: 162, and VL-CDR1, VL-CDR2, and VL-CDR3 of a VL comprising the sequence of SEQ ID NO: 163. In some embodiments, the anti-CD25 antibody comprises a VH comprising the sequence of SEQ ID NO: 162 and a VL comprising the sequence of SEQ ID NO: 163. In some embodiments, the parent anti-CD25 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 164 and two light chains each comprising the sequence of SEQ ID NO: 165. In some embodiments, the parent anti-CD25 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 166 and two light chains each comprising the sequence of SEQ ID NO: 167. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO: 2. In some embodiments, the heavy chain comprising the IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 169.In some embodiments, an IL-2 / anti-CD25 immunocytokine is provided that comprises: two light chains comprising the sequence of SEQ ID NO: 165, one heavy chain comprising an IL-2 variant located in its hinge region comprising the sequence of SEQ ID NO: 169, and one heavy chain comprising the sequence of SEQ ID NO: 168. In some embodiments, in the presence of binding of the antibody (or antigen-binding fragment) to CD25, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IL-2 cytokine or variant thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antibody (or antigen-binding fragment) to CD25. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to CD25, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IL-2 cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the corresponding IL-2 cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is homodimeric. In some embodiments, the antibody is heterodimeric. In some embodiments, the antibody is an agonistic antibody. In some embodiments, the antibody is an antagonist antibody.

[0120] In some embodiments, the antigen binding protein is a PD-L2 (e.g., a PD-L2 extracellular domain)-hinge-Fc fusion protein. Accordingly, in some embodiments, an IL-2 immunocytokine (an "IL-2 / PD-L2-Fc immunocytokine") is provided that comprises: a) a PD-L2-hinge-Fc fusion polypeptide comprising, from N' to C': a PD-L2 (e.g., SEQ ID NO: 176) that specifically recognizes PD-1, a hinge region, and an Fc domain subunit or portion thereof; and b) IL-2 or a variant thereof (e.g., SEQ ID NO: 1 or 2), and wherein the IL-2 or variant thereof is located in (e.g., within the hinge region or between the C-terminus of PD-L2 and the N-terminus of the hinge region) of the PD-L2-hinge-Fc fusion polypeptide (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59). In some embodiments, the parent PD-L2-hinge-Fc fusion protein comprises two PD-L2-hinge-Fc fusion polypeptides each comprising the sequence of SEQ ID NO: 177. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO: 2. In some embodiments, the PD-L2-hinge-Fc fusion polypeptide comprising an IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 180. In some embodiments, an IL-2 / PD-L2-Fc immunocytokine is provided comprising: the PD-L2-hinge-Fc fusion polypeptide comprising an IL-2 variant located in the hinge region comprises the sequence of SEQ ID NO: 180, and the PD-L2-hinge-Fc fusion polypeptide comprises the sequence of SEQ ID NO: 179. In some embodiments, in the presence of binding of a PD-L2 ligand to PD-1, the activity of the IL-2 cytokine or variant thereof (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of a PD-L2 ligand to PD-1.In some embodiments, in the absence of PD-L2 ligand binding to PD-1, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IL-2 cytokine or variant thereof located in the hinge region is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the corresponding IL-2 cytokine or variant thereof in the free state. In some embodiments, the PD-L2-hinge-Fc fusion protein is monospecific. In some embodiments, the PD-L2-hinge-Fc fusion protein is multispecific (e.g., bispecific). In some embodiments, the PD-L2-hinge-Fc fusion protein is a homodimer. In some embodiments, the PD-L2-hinge-Fc fusion protein is a heterodimer. In some embodiments, the PD-L2 moiety is an agonist capable of stimulating or enhancing PD-1 signaling. In some embodiments, the PD-L2 moiety is an antagonist capable of reducing or blocking PD-1 signaling.

[0121] In some embodiments, the cytokine or variant thereof is IFN-α (e.g., IFN-α2b) or a variant thereof. In some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine is provided, comprising: a) an antigen binding protein (e.g., an antibody, such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the antigen The binding protein comprises, from N' to C': an antigen-binding polypeptide (e.g., an antibody heavy chain or an antigen-binding fragment-hinge-Fc fusion polypeptide such as a ligand / receptor-hinge-Fc fusion polypeptide) comprising an antigen-binding fragment (e.g., a ligand such as the PD-L2 extracellular domain, a receptor, a VHH, an scFv, or a VH), a hinge region, and an Fc domain subunit or portion thereof (e.g., CH2+CH3, or CH2 only), and wherein the IFN-α or variant thereof is located in the hinge region (e.g., at the N', the C', or within the N'). In some embodiments, the antigen-binding protein is an antibody (e.g., a full-length antibody) that specifically recognizes a target antigen, the antigen-binding polypeptide comprising the hinge region is the heavy chain of the antibody, and the IFN-α or variant thereof is located in the hinge region of the heavy chain. In some embodiments, the antigen-binding fragment is a ligand, and the target antigen is a receptor specifically recognized by the ligand. In some embodiments, the antigen-binding fragment is a receptor and the target antigen is a ligand that is specifically recognized by that receptor.In some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine is provided, comprising: a) an antibody (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof via a hinge region) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) IFN-α (e.g., IFN-α2b) or a variant thereof, wherein the antibody comprises a heavy chain including a hinge region, and wherein the IFN-α (e.g., IFN-α2b) or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine is provided comprising: a) an antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) IFN-α (e.g., IFN-α2b) or a variant thereof, wherein the antibody comprises a heavy chain, and wherein the heavy chain comprises, from N-terminus to C-terminus: a VH domain, optionally a CH1 domain, the IFN-α (e.g., IFN-α2b) or a variant thereof in the hinge region, a CH2 domain, and optionally a CH3 domain. In some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine is provided comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) IFN-α (e.g., IFN-α2b) or a variant thereof, wherein the IFN-α (e.g., IFN-α2b) or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region).In some embodiments, the antibody comprises: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) an IFN-α comprising the sequence of SEQ ID NO: 3, which is selected from the group consisting of R22, L26, F27, L30, K31, D32, R33, H34, D35, F36, S68, T79, K83, Y85, Y89, R120, K121, Y122, Q124, Y and an IFN-α (e.g., IFN-α2b) variant comprising one or more mutations at positions selected from the group consisting of K129, K131, E132, R144, and E146, wherein the IFN-α (e.g., IFN-α2b) variant is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine is provided comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) an IFN-α (e.g., IFN-α2b) variant comprising one or more mutations selected from the group consisting of L30A, K31A, D32A, R33A, H34A, and D35A relative to an IFN-α comprising the sequence of SEQ ID NO: 3, wherein the IFN-α (e.g., IFN-α2b) variant is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) an IFN-α (e.g., IFN-α2b) variant comprising a sequence set forth in any of SEQ ID NOs: 4-9, wherein the IFN-α (e.g., IFN-α2b) variant is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region).In some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine is provided comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) an IFN-α (e.g., IFN-α2b) variant comprising a L30A mutation relative to an IFN-α comprising the sequence of SEQ ID NO: 3, wherein the IFN-α (e.g., IFN-α2b) variant is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine is provided comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) an IFN-α (e.g., IFN-α2b) variant comprising the sequence of SEQ ID NO: 4, wherein the IFN-α (e.g., IFN-α2b) variant is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the free IFN-α (e.g., IFN-α2b) variant is about 80% or less (e.g., about any of 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% or less) of the activity of the corresponding wild-type IFN-α (e.g., IFN-α2b) in the free state. In some embodiments, the antigen-binding protein (e.g., an antibody or fragment thereof, or an antigen-binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) is monospecific. In some embodiments, the antigen-binding protein is multispecific (e.g., bispecific). In some embodiments, the parent antigen-binding protein (e.g., an antibody, such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) is a homodimer. In some embodiments, the parent antigen-binding protein is a heterodimer. In some embodiments, the antigen binding protein is an agonist.In some embodiments, the antigen binding protein is an antagonist. In some embodiments, the hinge region comprises any of the sequences of SEQ ID NOs: 40-47, 50-52, and 55-59.

[0122] In some embodiments, the antigen-binding protein is an anti-HER2 antibody. Thus, in some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine ("IL-2 / anti-HER2 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes HER2 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the antibody comprises a heavy chain comprising a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IFN-α or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-HER2 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes HER2; and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOS: 3-9), wherein the IFN-α or variant thereof is located in (e.g., the hinge region of) the heavy chain of the full-length antibody (e.g., any of SEQ ID NOS: 40-47, 50-52, and 55-59). and wherein the anti-HER2 antibody comprises i) a VH-CDR1 comprising the sequence of SEQ ID NO: 188; ii) a VH-CDR2 comprising the sequence of SEQ ID NO: 189; iii) a VH-CDR3 comprising the sequence of SEQ ID NO: 190; iv) a VL-CDR1 comprising the sequence of SEQ ID NO: 191; v) a VL-CDR2 comprising the sequence of SEQ ID NO: 192; and vi) a VL-CDR3 comprising the sequence of SEQ ID NO: 193. In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a VH comprising the sequence of SEQ ID NO: 150 and a VL comprising the sequence of SEQ ID NO: 151. In some embodiments, the parent anti-HER2 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 152 or 153 and two light chains each comprising the sequence of SEQ ID NO: 154. In some embodiments, the IFN-α (e.g., IFN-α2b) variant comprises the sequence of SEQ ID NO: 4. In some embodiments, the heavy chain comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO:157.In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-HER2 immunocytokine is provided that comprises: two light chains comprising the sequence of SEQ ID NO: 154, one heavy chain comprising an IFN-α variant located in the hinge region comprising the sequence of SEQ ID NO: 157, and one heavy chain comprising the sequence of SEQ ID NO: 155. In some embodiments, in the presence of binding of the antibody (or antigen-binding fragment) to HER2, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α cytokine or variant thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antibody (or antigen-binding fragment) to HER2. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to HER2, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding IFN-α cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is a homodimer. In some embodiments, the antibody is a heterodimer. In some embodiments, the antibody is an agonist antibody. In some embodiments, the antibody is an antagonist antibody.

[0123] In some embodiments, the antigen-binding protein is an anti-CD3 antibody. Thus, in some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine ("IL-2 / anti-CD3 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes CD3 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the antibody comprises a heavy chain comprising a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IFN-α or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-CD3 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes CD3; and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the IFN-α or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59) (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region); and wherein the anti-CD3 antibody comprises: i) a VH-CDR1 comprising the sequence of SEQ ID NO: 85; ii) a VH-CDR2 comprising the sequence of SEQ ID NO: 86; iii) a VH-CDR3 comprising the sequence of SEQ ID NO: 87; iv) a VL-CDR1 comprising the sequence of SEQ ID NO: 88; v) a VL-CDR2 comprising the sequence of SEQ ID NO: 89; and vi) a VL-CDR3 comprising the sequence of SEQ ID NO: 90. In some embodiments, the anti-CD3 antibody comprises a VH comprising the sequence of SEQ ID NO: 91 and a VL comprising the sequence of SEQ ID NO: 92. In some embodiments, the parent anti-CD3 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 93 and two light chains each comprising the sequence of SEQ ID NO: 94. In some embodiments, the IFN-α (e.g., IFN-α2b) variant comprises the sequence of SEQ ID NO: 4. In some embodiments, the heavy chain comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 97.In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-CD3 immunocytokine is provided that comprises: two light chains comprising the sequence of SEQ ID NO: 94, one heavy chain comprising an IFN-α variant located in its hinge region comprising the sequence of SEQ ID NO: 97, and one heavy chain comprising the sequence of SEQ ID NO: 95. In some embodiments, in the presence of binding of the antibody (or antigen-binding fragment) to CD3, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) cytokine or variants thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antibody (or antigen-binding fragment) to CD3. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to CD3, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding IFN-α cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is a homodimer. In some embodiments, the antibody is a heterodimer. In some embodiments, the antibody is an agonist antibody. In some embodiments, the antibody is an antagonist antibody.

[0124] In some embodiments, the antigen-binding protein is an anti-PD-1 antibody. Accordingly, in some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine ("IFN-α / anti-PD-1 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes PD-1 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the antibody comprises a heavy chain that includes a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IFN-α (e.g., IFN-α2b) or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an anti-PD-1 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes PD-1; and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the IFN-α (e.g., IFN-α2b) or variant thereof is located in (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region) the hinge region of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59); and wherein the anti-PD-1 antibody comprises VH-CDR1, VH-CDR2, and VH-CDR3 of a VH comprising the sequence of SEQ ID NO: 102, and VL-CDR1, VL-CDR2, and VL-CDR3 of a VL comprising the sequence of SEQ ID NO: 103. In some embodiments, the anti-PD-1 antibody comprises a VH comprising the sequence of SEQ ID NO: 102 and a VL comprising the sequence of SEQ ID NO: 103. In some embodiments, the parent anti-PD-1 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 104 or 105 and two light chains each comprising the sequence of SEQ ID NO: 106. In some embodiments, the IFN-α (e.g., IFN-α2b) variant comprises the sequence of SEQ ID NO: 4. In some embodiments, the heavy chain comprising the IFN-α (e.g., IFN-α2b) variant located in the hinge region comprises the sequence of SEQ ID NO: 109.In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-PD-1 immunocytokine is provided that comprises: two light chains comprising the sequence of SEQ ID NO: 106, one heavy chain comprising an IFN-α variant located in its hinge region comprising the sequence of SEQ ID NO: 109, and one heavy chain comprising the sequence of SEQ ID NO: 107. In some embodiments, in the presence of binding of the antibody (or antigen-binding fragment) to PD-1, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) or variants thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antibody (or antigen-binding fragment) to PD-1. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to PD-1, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding IFN-α (e.g., IFN-α2b) or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is a homodimer. In some embodiments, the antibody is a heterodimer. In some embodiments, the antibody is an agonist antibody. In some embodiments, the antibody is an antagonist antibody.

[0125] In some embodiments, the antigen-binding protein is an anti-CD4 antibody. Thus, in some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine ("IFN-α / anti-CD4 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes CD4 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the antibody comprises a heavy chain comprising a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IFN-α or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-CD4 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes CD4; and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOS: 3-9), wherein the IFN-α or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOS: 40-47, 50-52, and 55-59) (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region); and wherein the anti-CD4 antibody comprises: i) a VH-CDR1 comprising the sequence of SEQ ID NO: 67; ii) a VH-CDR2 comprising the sequence of SEQ ID NO: 68; iii) a VH-CDR3 comprising the sequence of SEQ ID NO: 69; iv) a VL-CDR1 comprising the sequence of SEQ ID NO: 70; v) a VL-CDR2 comprising the sequence of SEQ ID NO: 71; and vi) a VL-CDR3 comprising the sequence of SEQ ID NO: 72. In some embodiments, the anti-CD4 antibody comprises a VH comprising the sequence of SEQ ID NO: 73 and a VL comprising the sequence of SEQ ID NO: 74. In some embodiments, the parent anti-CD4 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 75 or 76 and two light chains each comprising the sequence of SEQ ID NO: 77. In some embodiments, the IFN-α (e.g., IFN-α2b) variant comprises the sequence of SEQ ID NO: 4. In some embodiments, the heavy chain comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 80.In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-CD4 immunocytokine is provided that comprises: two light chains comprising the sequence of SEQ ID NO: 77, one heavy chain comprising an IFN-α variant located in its hinge region comprising the sequence of SEQ ID NO: 80, and one heavy chain comprising the sequence of SEQ ID NO: 78. In some embodiments, in the presence of binding of the antibody (or antigen-binding fragment) to CD4, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) cytokine or variants thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antibody (or antigen-binding fragment) to CD4. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to CD4, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding IFN-α (e.g., IFN-α2b) cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is a homodimer. In some embodiments, the antibody is a heterodimer. In some embodiments, the antibody is an agonist antibody. In some embodiments, the antibody is an antagonist antibody.

[0126] In some embodiments, the antigen-binding protein is an anti-CD8 antibody. Thus, in some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine ("IFN-α / anti-CD8 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes CD8 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the antibody comprises a heavy chain comprising a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IFN-α or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-CD8 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes CD8; and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the IFN-α or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59) (e.g., within the hinge region, between the C-terminus of CH1 and the N-terminus of the hinge region); and wherein the anti-CD8 antibody comprises VH-CDR1, VH-CDR2, and VH-CDR3 of the VH comprising the sequence of SEQ ID NO: 114, and VL-CDR1, VL-CDR2, and VL-CDR3 of the VL comprising the sequence of SEQ ID NO: 115. In some embodiments, the anti-CD8 antibody comprises a VH comprising the sequence of SEQ ID NO: 114 and a VL comprising the sequence of SEQ ID NO: 115. In some embodiments, the parent anti-CD8 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 116 and two light chains each comprising the sequence of SEQ ID NO: 117. In some embodiments, the IFN-α (e.g., IFN-α2b) variant comprises the sequence of SEQ ID NO: 4. In some embodiments, the heavy chain comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 120.In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-CD8 immunocytokine is provided that comprises: two light chains comprising the sequence of SEQ ID NO: 117, one heavy chain comprising an IFN-α variant located in its hinge region comprising the sequence of SEQ ID NO: 120, and one heavy chain comprising the sequence of SEQ ID NO: 118. In some embodiments, in the presence of binding of the antibody (or antigen-binding fragment) to CD8, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) cytokine or variants thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antibody (or antigen-binding fragment) to CD8. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to CD8, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding IFN-α cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is homodimeric. In some embodiments, the antibody is heterodimeric. In some embodiments, the antibody is an agonist antibody. In some embodiments, the antibody is an antagonist antibody.

[0127] In some embodiments, the antigen-binding protein is an anti-CTLA-4 antibody. Thus, in some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine ("IFN-α / anti-CTLA-4 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes CTLA-4 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the antibody comprises a heavy chain that includes a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IFN-α or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-CTLA-4 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes CTLA-4; and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the IFN-α or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59) (e.g., within the hinge region, between the C-terminus of CH1 and the N-terminus of the hinge region); and wherein the anti-CTLA-4 antibody comprises VH-CDR1, VH-CDR2, and VH-CDR3 of the VH comprising the sequence of SEQ ID NO: 125, and VL-CDR1, VL-CDR2, and VL-CDR3 of the VL comprising the sequence of SEQ ID NO: 126. In some embodiments, the anti-CTLA-4 antibody comprises a VH comprising the sequence of SEQ ID NO: 125 and a VL comprising the sequence of SEQ ID NO: 126. In some embodiments, the parent anti-CTLA-4 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 127 or 128 and two light chains each comprising the sequence of SEQ ID NO: 129. In some embodiments, the IFN-α (e.g., IFN-α2b) variant comprises the sequence of SEQ ID NO: 4. In some embodiments, the heavy chain comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 132.In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-CTLA-4 immunocytokine is provided that comprises: two light chains comprising the sequence of SEQ ID NO: 129, one heavy chain comprising an IFN-α variant located in the hinge region comprising the sequence of SEQ ID NO: 132, and one heavy chain comprising the sequence of SEQ ID NO: 130. In some embodiments, in the presence of binding of the antibody (or antigen-binding fragment) to CTLA-4, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) cytokine or variants thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antibody (or antigen-binding fragment) to CTLA-4. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to CTLA-4, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding IFN-α cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is a homodimer. In some embodiments, the antibody is a heterodimer. In some embodiments, the antibody is an agonist antibody. In some embodiments, the antibody is an antagonist antibody.

[0128] In some embodiments, the antigen binding protein is an anti-PD-L1 antibody. Accordingly, in some embodiments, there is provided an IFN-α (e.g., IFN-α2b) immunocytokine (an "IFN-α / anti-PD-L1 immunocytokine") comprising: a) an antibody that specifically recognizes PD-L1 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the antibody comprises a heavy chain that comprises a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IFN-α or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region, between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-PD-L1 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes PD-L1; and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the IFN-α or variant thereof is located in the hinge region (e.g., the hinge region) of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59). and wherein the anti-PD-L1 antibody comprises i) a VH-CDR1 comprising the sequence of SEQ ID NO: 243; ii) a VH-CDR2 comprising the sequence of SEQ ID NO: 244; iii) a VH-CDR3 comprising the sequence of SEQ ID NO: 245; iv) a VL-CDR1 comprising the sequence of SEQ ID NO: 246; v) a VL-CDR2 comprising the sequence of SEQ ID NO: 247; and vi) a VL-CDR3 comprising the sequence of SEQ ID NO: 248. In some embodiments, the anti-PD-L1 antibody comprises a VH comprising the sequence of SEQ ID NO: 137 and a VL comprising the sequence of SEQ ID NO: 138. In some embodiments, the parent anti-PD-L1 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 139 and two light chains each comprising the sequence of SEQ ID NO: 140. In some embodiments, the parent anti-PD-L1 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 141 and two light chains each comprising the sequence of SEQ ID NO: 142. In some embodiments, the IFN-α (e.g., IFN-α2b) variant comprises the sequence of SEQ ID NO: 4.In some embodiments, the heavy chain comprising the IFN-α (e.g., IFN-α2b) variant located in its hinge region comprises the sequence of SEQ ID NO: 145. In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-PD-L1 immunocytokine is provided comprising: two light chains comprising the sequence of SEQ ID NO: 140, one heavy chain comprising the IFN-α variant located in its hinge region comprising the sequence of SEQ ID NO: 145, and one heavy chain comprising the sequence of SEQ ID NO: 143. In some embodiments, in the presence of the antibody (or antigen-binding fragment) binding to PD-L1, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) cytokine or variant thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more) compared to the activity in the absence of the antibody (or antigen-binding fragment) binding to PD-L1. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to PD-L1, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding IFN-α cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is a homodimer. In some embodiments, the antibody is a heterodimer.

[0129] In some embodiments, the antigen-binding protein is an anti-CD25 antibody. Thus, in some embodiments, an IFN-α (e.g., IFN-α2b) immunocytokine ("IFN-α / anti-CD25 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes CD25 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the antibody comprises a heavy chain comprising a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein the IFN-α or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-CD25 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes CD25; and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), wherein the IFN-α or variant thereof is located in the hinge region of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59) (e.g., within the hinge region, between the C-terminus of CH1 and the N-terminus of the hinge region); and wherein the anti-CD25 antibody comprises VH-CDR1, VH-CDR2, and VH-CDR3 of the VH comprising the sequence of SEQ ID NO: 162, and VL-CDR1, VL-CDR2, and VL-CDR3 of the VL comprising the sequence of SEQ ID NO: 163. In some embodiments, the anti-CD25 antibody comprises a VH comprising the sequence of SEQ ID NO: 162 and a VL comprising the sequence of SEQ ID NO: 163. In some embodiments, the parent anti-CD25 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 164 and two light chains each comprising the sequence of SEQ ID NO: 165. In some embodiments, the parent anti-CD25 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 166 and two light chains each comprising the sequence of SEQ ID NO: 167. In some embodiments, the IFN-α (e.g., IFN-α2b) variant comprises the sequence of SEQ ID NO: 4. In some embodiments, the heavy chain comprising the IFN-α (e.g., IFN-α2b) variant located in the hinge region comprises the sequence of SEQ ID NO: 170.In some embodiments, an IFN-α (e.g., IFN-α2b) / anti-CD25 immunocytokine is provided that comprises: two light chains comprising the sequence of SEQ ID NO: 165, one heavy chain comprising an IFN-α variant located in its hinge region comprising the sequence of SEQ ID NO: 170, and one heavy chain comprising the sequence of SEQ ID NO: 168. In some embodiments, in the presence of binding of the antibody (or antigen-binding fragment) to CD25, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) cytokine or variants thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the antibody (or antigen-binding fragment) to CD25. In some embodiments, in the absence of binding of the antibody (or antigen-binding fragment) to CD25, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) cytokine or variant thereof located in the hinge region of the heavy chain is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding IFN-α cytokine or variant thereof in the free state. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is a homodimer. In some embodiments, the antibody is a heterodimer. In some embodiments, the antibody is an agonist antibody. In some embodiments, the antibody is an antagonist antibody.

[0130] In some embodiments, the antigen binding protein is a PD-L2 (e.g., PD-L2 extracellular domain)-hinge-Fc fusion protein. Accordingly, in some embodiments, there is provided an IFN-α (e.g., IFN-α2b) immunocytokine (an "IFN-α / PD-L2-Fc immunocytokine") comprising: a) a PD-L2-hinge-Fc fusion polypeptide comprising, from N' to C': a PD-L2 that specifically recognizes PD-1 (e.g., SEQ ID NO: 176), a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and an Fc domain subunit or portion thereof; and b) IFN-α (e.g., IFN-α2b) or a variant thereof (e.g., any of SEQ ID NOs: 3-9), and wherein the IFN-α or variant thereof is located in the hinge region of the PD-L2-hinge-Fc fusion polypeptide (e.g., within the hinge region, or between the C-terminus of PD-L2 and the N-terminus of the hinge region). In some embodiments, the parent PD-L2-hinge-Fc fusion protein comprises two PD-L2-hinge-Fc fusion polypeptides each comprising the sequence of SEQ ID NO: 177. In some embodiments, the IFN-α (e.g., IFN-α2b) variant comprises the sequence of SEQ ID NO: 4. In some embodiments, the PD-L2-hinge-Fc fusion polypeptide comprising the IFN-α (e.g., IFN-α2b) variant located in the hinge region comprises the sequence of SEQ ID NO: 182. In some embodiments, an IFN-α / PD-L2-Fc immunocytokine is provided, comprising: the PD-L2-hinge-Fc fusion polypeptide comprising the IFN-α variant located in the hinge region comprises the sequence of SEQ ID NO: 182, and the PD-L2-hinge-Fc fusion polypeptide comprises the sequence of SEQ ID NO: 181. In some embodiments, in the presence of binding of a PD-L2 ligand to PD-1, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) cytokine or variant thereof is increased by at least about 20% (such as at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to activity in the absence of binding of a PD-L2 ligand to PD-1.In some embodiments, in the absence of PD-L2 ligand binding to PD-1, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) of the IFN-α (e.g., IFN-α2b) cytokine or variant thereof located in the hinge region is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding IFN-α cytokine or variant thereof in the free state. In some embodiments, the PD-L2-hinge-Fc fusion protein is monospecific. In some embodiments, the PD-L2-hinge-Fc fusion protein is multispecific (e.g., bispecific). In some embodiments, the PD-L2-hinge-Fc fusion protein is a homodimer. In some embodiments, the PD-L2-hinge-Fc fusion protein is a heterodimer. In some embodiments, the PD-L2 moiety is an agonist capable of stimulating or enhancing PD-1 signaling. In some embodiments, the PD-L2 moiety is an antagonist capable of reducing or blocking PD-1 signaling.

[0131] In some embodiments, the cytokine or variant thereof is IFN-γ or a variant thereof. In some embodiments, an IFN-γ immunocytokine is provided, comprising: a) an antigen binding protein (e.g., an antibody such as a full-length antibody, or an antigen binding fragment-hinge-Fc fusion protein such as a ligand / receptor-hinge-Fc fusion protein) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) IFN-γ or a variant thereof (e.g., SEQ ID NO: 18 or 19), wherein the antigen binding protein is, from N' to C': an antigen binding fragment (e.g., a ligand such as the PD-L2 extracellular domain, a receptor, a VHH, a s and an antigen-binding polypeptide (e.g., an antibody heavy chain, or an antigen-binding fragment-hinge-Fc fusion polypeptide such as a ligand / receptor-hinge-Fc fusion polypeptide) comprising an Fc domain subunit or portion thereof (e.g., CH2+CH3, or CH2 only), and wherein the subunits of IFN-γ or a variant thereof (e.g., any of SEQ ID NOs: 10-17) are both located in tandem in the hinge region (e.g., at or within the N', C', or N' chains) (e.g., linked via an optional peptide linker, such as any of SEQ ID NOs: 227-229). In some embodiments, the antigen-binding protein is an antibody (e.g., a full-length antibody) that specifically recognizes a target antigen, the antigen-binding polypeptide comprising the hinge region is a heavy chain of the antibody, and both subunits of IFN-γ or a variant thereof (e.g., any of SEQ ID NOS: 10-17) are located in tandem in the hinge region of the heavy chain (e.g., linked via an optional peptide linker, such as any of SEQ ID NOS: 227-229). In some embodiments, the antigen-binding fragment is a ligand, and the target antigen is a receptor specifically recognized by that ligand. In some embodiments, the antigen-binding fragment is a receptor, and the target antigen is a ligand specifically recognized by that receptor.In some embodiments, an IFN-γ immunocytokine is provided, comprising: a) an antibody (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof via a hinge region) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) IFN-γ or a variant thereof (e.g., SEQ ID NO: 18 or 19), wherein the antibody comprises a heavy chain including a hinge region, and wherein both subunits of the IFN-γ or variant thereof (e.g., any of SEQ ID NOs: 10-17) are located in tandem (e.g., connected via an optional peptide linker, such as any of SEQ ID NOs: 227-229) in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-γ immunocytokine is provided, comprising: a) an antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) IFN-γ or a variant thereof (e.g., SEQ ID NO: 18 or 19), wherein the antibody comprises a heavy chain comprising, from N-terminus to C-terminus: a VH domain, optionally a CH1 domain, a first subunit of IFN-γ or a variant thereof (e.g., any of SEQ ID NOs: 10-17), optionally a linker (e.g., a peptide linker), a second subunit of IFN-γ or a variant thereof (e.g., any of SEQ ID NOs: 10-17) in the hinge region, a CH2 domain, and optionally a CH3 domain.In some embodiments, an IFN-γ immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) IFN-γ or a variant thereof (e.g., SEQ ID NO: 18 or 19), wherein both subunits of the IFN-γ or variant thereof (e.g., any of SEQ ID NOs: 10-17) are located in tandem (e.g., connected via an optional peptide linker, such as any of SEQ ID NOs: 227-229) in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-γ immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) an IFN-γ variant that includes one or more mutations within one or both IFN-γ subunits at positions selected from the group consisting of V5, S20, D21, V22, A23, D24, N25, G26, H111, and Q115 relative to a wild-type IFN-γ subunit comprising the sequence of SEQ ID NO: 10, wherein both subunits of the IFN-γ variant are located in tandem (e.g., connected via an optional peptide linker, such as any of SEQ ID NOs: 227-229) in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region).In some embodiments, the antibody comprises: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) one or both of the IFN-γ subunits have, within the IFN-γ subunits, one or more of the following mutations: S20A, D21A, D21K, V22A, A23S, A23E, A23Q, A23V, D24A, D24E, N25A, N26A, N30A, N31A, N32A, N33A, N34A, N35A, N36A, N37A, N38A, N39A, N40A, N41A, N42A, N43A, N44A, N45A, N46A, N47A, N48A, N49A, N50A, N51A, N52A, N53A, N54A, N55A, N56A, N57A, N58A, N59A, N60A, N61A, N62A, N63A, N64A, N65A, N66A, N67A, N68A, N69A, N69B, N69C, N69D, N69E, N69F, N69H, N69I, N69I, N69J, N69K, N69K, N69I, N70A, N71A, N72A, N73A, N74A, N75A, N76A, N77A, N78A, N79A, N79A, N80A, N81A, N82A, N83A, N84A and an IFN-γ mutant comprising one or more mutations selected from the group consisting of H111D, H112D, H113D, H114D, H115D, H116D, H117D, H118D, H119D, H120D, H121D, H122D, H123D, H124D, H125K, H126D, H127D, H128D, H129D, H130D, H131D, H132D, H133D, H134D, H135D, H136D, H137D, H138D, H139D, H140D, H141D, H142D, H143D, H144D, H145D, H146D, H147D, H148D, H149D, H150D, H151D, H152D, H153D, H154D, H155D, H156D, H157D, H158D, H159D, H160D, H161D, H162D, H163D, H164D, H165D, H166D, H167D, H168D, H169D, H170D, H171D, H172D, H173D, H174D, H175D, H176D, H177D, H178D, H179D, H180D, H181D, H182D, H183D, H184D, H185D, H186D, H187D, H188D, H189D, H189D, H189D, H189D In some embodiments, the antibody comprises: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) one or both of the IFN-γ subunits have, within the IFN-γ subunits, one or more of the following mutations relative to a wild-type IFN-γ subunit comprising the sequence of S20A / D21A, D21K, V22A / A23S, D24A / N25A, A23E / D24E / N25K ... and an IFN-γ variant comprising one or more mutations selected from the group consisting of A23V, Q, and A23V, wherein both subunits of the IFN-γ variant are located in tandem in the hinge region of a heavy chain of a full-length antibody (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region) (e.g., connected via an optional peptide linker, such as any of SEQ ID NOs: 227-229).In some embodiments, an IFN-γ immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) an IFN-γ variant, wherein one or both subunits of the IFN-γ variant comprise the sequence of any of SEQ ID NOs: 11-17, and wherein both subunits of the IFN-γ variant are located in tandem (e.g., connected via an optional peptide linker, such as any of SEQ ID NOs: 227-229) in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-γ immunocytokine is provided that includes: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) an IFN-γ variant that includes an A23V mutation within one or both IFN-γ subunits relative to a wild-type IFN-γ subunit comprising the sequence of SEQ ID NO: 10, wherein both subunits of the IFN-γ variant are located in tandem (e.g., connected via an optional peptide linker, such as any of SEQ ID NOs: 227-229) in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region). In some embodiments, an IFN-γ immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) an IFN-γ variant, wherein one or both subunits of the IFN-γ variant comprise the sequence of SEQ ID NO: 13, and wherein both subunits of the IFN-γ variant are located in tandem (e.g., connected via an optional peptide linker, such as any of SEQ ID NOs: 227-229) in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region).In some embodiments, the two subunits of IFN-γ or a variant thereof are connected by a linker (e.g., any of SEQ ID NOs: 227-229). In some embodiments, an IFN-γ immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, HER2, PD-1, CD3, CD4, CD123, or CD8); and b) an IFN-γ variant comprising the sequence of SEQ ID NO: 19, wherein the IFN-γ variant is located in the hinge region of the heavy chain of the full-length antibody (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). In some embodiments, the activity of the IFN-γ variant in its free state (binding affinity to the corresponding cytokine receptor or subunit thereof, and / or biological activity) is about 80% or less (e.g., about any of 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% or less) of the activity of the corresponding wild-type IFN-γ in its free state. In some embodiments, the antigen-binding protein (e.g., an antibody or fragment thereof, or an antigen-binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) is monospecific. In some embodiments, the antigen-binding protein is multispecific (e.g., bispecific). In some embodiments, the parent antigen-binding protein (e.g., an antibody, such as a full-length antibody, or an antigen-binding fragment-hinge-Fc fusion protein, such as a ligand / receptor-hinge-Fc fusion protein) is a homodimer. In some embodiments, the parent antigen-binding protein is a heterodimer. In some embodiments, the antigen-binding protein is an agonist. In some embodiments, the antigen binding protein is an antagonist. In some embodiments, the hinge region comprises any of the sequences of SEQ ID NOs: 40-47, 50-52, and 55-59.

[0132] In some embodiments, the antigen-binding protein is an anti-HER2 antibody. Thus, in some embodiments, an IFN-γ immunocytokine ("IFN-γ / anti-HER2 immunocytokine") is provided, comprising: a) an antibody that specifically recognizes HER2 (e.g., a full-length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof); and b) IFN-γ or a variant thereof (e.g., SEQ ID NO: 18 or 19), wherein the antibody comprises a heavy chain comprising a hinge region (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59), and wherein both subunits of the IFN-γ or variant thereof (e.g., any of SEQ ID NOs: 10-17) are located in tandem in the hinge region of the heavy chain (e.g., within the hinge region or between the C-terminus of the CH1 and the N-terminus of the hinge region) (e.g., connected via an optional peptide linker, such as any of SEQ ID NOs: 227-229). In some embodiments, an IFN-γ / anti-HER2 immunocytokine is provided, comprising: a) a full-length antibody that specifically recognizes HER2; and b) IFN-γ or a variant thereof (e.g., SEQ ID NO: 18 or 19), wherein both subunits of the IFN-γ or variant thereof (e.g., any of SEQ ID NOs: 10-17) are located in the hinge region of the heavy chain of the full-length antibody (e.g., any of SEQ ID NOs: 40-47, 50-52, and 55-59) (e.g., within the hinge region or between the C-terminus of CH1 and the N-terminus of the hinge region). and wherein the anti-HER2 antibody comprises i) a VH-CDR1 comprising the sequence of SEQ ID NO: 188; ii) a VH-CDR2 comprising the sequence of SEQ ID NO: 189; iii) a VH-CDR3 comprising the sequence of SEQ ID NO: 190; iv) a VL-CDR1 comprising the sequence of SEQ ID NO: 191; v) a VL-CDR2 comprising the sequence of SEQ ID NO: 192; and vi) a VL-CDR3 comprising the sequence of SEQ ID NO: 193. In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a VH comprising the sequence of SEQ ID NO: 150 and a VL comprising the sequence of SEQ ID NO: 151.In some embodiments, the parent anti-HER2 antibody comprises two heavy chains each comprising the sequence of SEQ ID NO: 152 or 153 and two light chains each comprising the sequence of SEQ ID NO: 154. In some embodiments, one or both subunits of the IFN-γ variant comprise the sequence of SEQ ID NO: 13. In some embodiments, the IFN-γ variant comprises the sequence of SEQ ID NO: 19. In some embodiments, the heavy chain comprising the IFN-γ variant located in the hinge region com...

Claims

1. an immunocytokine comprising: a) an antigen-binding protein that specifically recognizes a target antigen; and b) an IL-12 variant, wherein the antigen-binding protein comprises an antigen-binding polypeptide comprising, from N' to C': an antigen-binding fragment, a hinge region, and an Fc domain subunit, and the IL-12 variant is located in the hinge region; The IL-12 variant comprises, from N-terminus to C-terminus, a p35 subunit-linker-p40 subunit or a p40 subunit-linker-p35 subunit; and the p35 subunit is a wild-type p35 subunit comprising the sequence of SEQ ID NO:29 or a p35 subunit having at least 95% amino acid sequence identity to SEQ ID NO:29; the p40 subunit is a mutant p40 subunit having either an E59A / F60A mutation, or an E59A mutation, or an F60A mutation relative to a wild-type p40 subunit having the sequence of SEQ ID NO: 30, and the p40 subunit has at least 95% amino acid sequence identity with SEQ ID NO: 30; the activity of the IL-12 variant in the free state is 80% or less of the activity of the corresponding wild-type IL-12 in the free state, and in the presence of the antigen binding protein bound to the target antigen, the activity of the IL-12 variant is increased by at least 20% compared to the absence of the antigen binding protein bound to the target antigen; The IL-12 variant located in the hinge region refers to an antigen-binding polypeptide comprising, from N-terminus to C-terminus: (i) antigen-binding fragment-optional linker-IL-12 variant-optional linker-hinge-CH2-CH3; (ii) antigen-binding fragment-optional linker-hinge-optional linker-IL12 variant-optional linker-CH2-CH3; or (iii) an immunocytokine comprising an antigen-binding fragment-optional linker-hinge N' portion-IL12 variant-optional linker-hinge C' portion-CH2-CH3; and wherein the antigen-binding fragment in (i), (ii) or (iii) is selected from a VHH, scFv, Fab, ligand or receptor that binds to the target antigen.

2. In the presence of binding of said antigen binding protein to said target antigen, the activity of said IL-12 variant is increased by at least 20% compared to the activity in the absence of binding of said antigen binding protein to said target antigen; or In the absence of binding of the antigen binding protein to the target antigen, the activity of the IL-12 variant located in the hinge region is 70% or less of the activity of the corresponding IL-12 variant in the free state; or The immunocytokine according to claim 1, wherein the activity of the IL-12 mutant in the free state is 80% or less of the activity of the corresponding wild-type IL-12 in the free state.

3. The immunocytokine of claim 1 or 2, wherein the antigen-binding protein comprises two antigen-binding polypeptides each comprising a hinge region, and only one of the antigen-binding polypeptides comprises the IL-12 variant located in the hinge region.

4. The immunocytokine according to any one of claims 1 to 3, wherein the p40 subunit of the IL-12 mutant comprises the sequence of any one of SEQ ID NOs: 31 to 33.

5. The immunocytokine of claim 4, wherein the p40 subunit of the IL-12 mutant comprises the sequence of SEQ ID NO: 31 or 33.

6. The immunocytokine of claim 1, wherein the IL-12 mutant comprises the sequence of SEQ ID NO: 36, 275 or 331.

7. The immunocytokine according to any one of claims 1 to 6, wherein the antigen-binding protein is an antibody that specifically recognizes PD1, the antigen-binding polypeptide constituting the hinge region is a heavy chain of the antibody, and the IL-12 mutant is located in the hinge region of the heavy chain.

8. an anti-PD-1 antibody, comprising: (i) VH-CDR1, VH-CDR2, and VH-CDR3 of VH comprising the sequence of SEQ ID NO: 102, and VL-CDR1, VL-CDR2, and VL-CDR3 of VL comprising the sequence of SEQ ID NO: 103; or (ii) a VH comprising the sequence of SEQ ID NO: 102, and a VL comprising the sequence of SEQ ID NO: 103; or (iii) a light chain comprising the sequence of SEQ ID NO: 106, wherein the IL-12 variant is an IL-12 variant comprising the sequence of SEQ ID NO: 36, and the heavy chain comprising the IL-12 variant located in the hinge region comprises the sequence of any one of SEQ ID NOs: 268 to 273; Furthermore, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 in (i) are defined by Kabat numbering. The immunocytokine according to claim 7.

9. The immunocytokine according to any one of claims 1 to 6, wherein the antigen-binding fragment is PD-L2 or PD-L1.

10. The immunocytokine of any one of claims 9, wherein the PD-L2 or PD-L1 comprises the sequence of SEQ ID NO: 176, SEQ ID NO: 250, any of SEQ ID NOs: 255-261, or any of SEQ ID NOs: 262-265. (i) the IL-12 variant comprises the sequence of SEQ ID NO: 36, and the antigen-binding polypeptide comprising the IL-12 variant located in the hinge region comprises the sequence of SEQ ID NO: 186; or (ii) the IL-12 variant comprises the sequence of SEQ ID NO: 275, and the antigen-binding polypeptide comprising the IL-12 variant located in the hinge region comprises the sequence of SEQ ID NO: 274; The immunocytokine according to claim 9.

12. 12. The immunocytokine of claim 10 or 11, wherein the antigen-binding protein comprises a second antigen-binding polypeptide located in the hinge region that does not comprise the cytokine or a variant thereof, and the second antigen-binding polypeptide comprises the sequence of any of SEQ ID NOs: 178, 179, 181, and 185.

13. An isolated nucleic acid encoding the immunocytokine of any one of claims 1 to 12.

14. A pharmaceutical composition comprising an immunocytokine according to any one of claims 1 to 12, and optionally a pharmaceutically acceptable carrier.

15. An immunocytokine according to any one of claims 1 to 12 for treating a disease or disorder in an individual.

16. The immunocytokine of claim 15, wherein the disease or disorder is cancer or an infectious disease.

17. the cancer is selected from the group consisting of lung cancer, liver cancer, kidney cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bile duct cancer, squamous cell carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, and Merkel cell carcinoma; The immunocytokine of claim 16.

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