Combination of cytokine fusion proteins and CD8 antigen-binding molecules

Abstract

Provided herein are fusion polypeptides with IL-2 or IL-21 that contain anti-CD8 antigen binding molecules and CD8 antigen binding molecules to selectively regulate the function of CD8+ T cells over other immune cells. The disclosure provides methods of using IL-2 fusion proteins in combination with IL-21 fusion proteins to treat diseases.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 335,697, filed April 27, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] CD8+ T cells expressing the αβ T cell receptor are a large subset of major histocompatibility (MHC) class I-restricted T cells that mediate adaptive immunity against various pathogens and cancers. Furthermore, they can also be pathogenic and cause disease in certain autoimmune and inflammatory conditions. Modulating CD8+ T cell function can be therapeutically effective by activating CD8+ T cell function in the setting of infection and cancer or by inhibiting CD8+ T cell function in the setting of certain autoimmune diseases.

[0003] The activation and differentiation of CD8+ T cells are largely controlled by soluble immunoregulatory proteins such as cytokines. The biological activity of cytokines is typically mediated by binding with very high affinity to their respective cytokine receptors on the cell surface, resulting in their ability to potently stimulate downstream signaling and induce various cellular processes that regulate immune cell phenotype and function. Cytokines typically have pleiotropic effects, triggering multiple downstream cellular events, such as activation, proliferation, survival, apoptosis, and the secretion of other immunoregulatory proteins. Furthermore, because cytokine receptors are expressed on multiple immune cell subsets, cytokines act not only on CD8+ T cells but also on other immune and non-immune cells that express their receptors.

[0004] A challenge with cytokine immunotherapy is that in some cases, while activating immune cells to enhance immune responses, the same cytokines can also activate counterregulatory pathways, as exemplified by IL-2 and IFNγ. These counterregulatory pathways can activate regulatory T cell responses and inhibitory pathways. Therefore, there remains a need for improved therapeutic agents for targeting CD8+ T cells without activating regulatory T cell responses and inhibitory pathways. Summary of the Invention

[0005] In some aspects, the present disclosure provides a method of treating a disease in a subject, the method comprising administering to the subject an effective amount of (a) an IL-21 fusion protein comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human CD8, and (ii) an IL-21 polypeptide; and (b) administering an IL-2 fusion protein comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to human CD8, and (ii) an IL-2 polypeptide; Treating a subject thereby.

[0006] In some aspects, the present disclosure provides methods for treating a disease in a subject who has been treated or is being treated with an IL-21 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds human CD8 and an IL-21 polypeptide, the method comprising administering to the subject an effective amount of an IL-2 fusion protein comprising (i) an antibody or antigen-binding fragment thereof that specifically binds human CD8, and (ii) an IL-2 polypeptide, thereby treating the subject.

[0007] In some aspects, the present disclosure provides methods for treating a disease in a subject who has been treated or is being treated with an IL-2 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds human CD8 and an IL-2 polypeptide, the method comprising administering to the subject an effective amount of an IL-21 fusion protein comprising (i) an antibody or antigen-binding fragment thereof that specifically binds human CD8, and (ii) an IL-21 polypeptide, thereby treating the subject.

[0008] In some aspects, the present disclosure provides kits comprising: (a) an IL-21 fusion protein comprising (i) an antibody or antigen-binding fragment thereof that specifically binds human CD8, and (ii) an IL-21 polypeptide; and (b) one or more containers comprising (i) an antibody or antigen-binding fragment thereof that specifically binds human CD8, and (ii) an IL-2 fusion protein comprising an IL-2 polypeptide, and optionally a pharmaceutically acceptable carrier, and instructions for administering the IL-21 and IL-2 fusion proteins concurrently, sequentially, or simultaneously to a subject in need thereof.

[0009] In some aspects, the present disclosure provides kits comprising a container containing an IL-21 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds human CD8 and an IL-21 polypeptide, optionally a pharmaceutically acceptable carrier, and instructions for administering the IL-21 fusion protein to a subject in need thereof who has been treated or is being treated with an IL-2 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds human CD8 and an IL-2 polypeptide, thereby treating the subject.

[0010] In some aspects, the present disclosure provides kits that include a container containing an IL-2 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds human CD8 and an IL-2 polypeptide, optionally a pharmaceutically acceptable carrier, and instructions for administering the IL-2 fusion protein to a subject in need thereof who has been treated or is being treated with an IL-21 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds human CD8 and an IL-21 polypeptide, thereby treating the subject.

[0011] In some aspects, the present disclosure provides a use for administering an IL-21 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds human CD8 and an IL-21 polypeptide to a subject in need thereof, wherein the subject has received or is undergoing treatment with an IL-2 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds human CD8 and an IL-2 polypeptide.

[0012] In some aspects, the present disclosure provides a use for administering an IL-2 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds human CD8 and an IL-21 polypeptide to a subject in need thereof, wherein the subject has been treated or is undergoing treatment with an IL-21 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds human CD8 and an IL-21 polypeptide.

[0013] In any of the foregoing or related embodiments, the subject in need thereof has a disease. In some embodiments, the disease is cancer. In some embodiments, the disease is an infectious disease.

[0014] In any of the foregoing or related embodiments, the treatment induces an immune response in the subject. In some embodiments, the immune response is a T cell response.

[0015] In any of the foregoing or related embodiments, the treatment delays cancer progression in the subject. In some embodiments, the treatment reduces tumor volume in the subject. In some embodiments, the treatment reduces or inhibits tumor growth in the subject. In some embodiments, the treatment delays cancer progression, reduces tumor volume, and / or reduces or inhibits tumor growth in the subject more than treatment with either an IL-2 fusion protein or an IL-21 fusion protein alone.

[0016] In any of the foregoing or related embodiments, the antibodies or antigen-binding fragments thereof for the IL-21 fusion protein and the IL-2 fusion protein are identical. In some embodiments, the antibodies or antigen-binding fragments thereof for the IL-21 fusion protein and the IL-2 fusion protein are different. In some embodiments, the antibodies or antigen-binding fragments thereof for the IL-21 fusion protein and the IL-2 fusion protein bind to cells expressing a human CD8ab heterodimer on their surface with an EC50 of less than 1000 nM. In some embodiments, the antibodies or antigen-binding fragments thereof for the IL-21 fusion protein and the IL-2 fusion protein bind to human CD8+ T cells.

[0017] In some embodiments, the antibodies or antigen-binding fragments of the IL-21 and IL-2 fusion proteins bind to different epitopes on CD8ab or CD8b. In some embodiments, the antibodies or antigen-binding fragments of the IL-21 and IL-2 fusion proteins do not compete for binding to CD8ab or CD8b. In some embodiments, the IL-21 fusion protein comprises a first antibody that specifically binds to human CD8 or an antigen-binding fragment thereof, and the IL-2 fusion protein comprises a second antibody that specifically binds to human CD8 or an antigen-binding fragment thereof, and the first and second antibodies bind to different epitopes on CD8ab or CD8b. In some embodiments, the first and second antibodies do not cross-compete for binding to CD8ab or CD8b.

[0018] In some embodiments, the antibodies or antigen-binding fragments of the IL-21 fusion proteins and IL-2 fusion proteins comprise a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein: the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 35, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 36; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 177, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 178, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 179, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 180, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 181, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 182; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 225, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 226, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 227, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 228; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 229, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 230, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 231, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 225, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 232, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 234, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 235, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 236; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 225, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 232, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 228; or The VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 229, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 237, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 231, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42.

[0019] In some embodiments, the antibodies or antigen-binding fragments of the IL-21 fusion proteins and IL-2 fusion proteins comprise a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein: the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 53, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 54, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 35, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 36; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 55, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 56, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 55, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 50, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 183, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 184, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 179, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 180, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 181, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 182; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 238, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 239, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 228; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 240, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 241, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 242, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 238, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 234, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 235, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 236; or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 238, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 228; or The VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 240, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 244, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 242, and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42.

[0020] In some embodiments, the VH domain and the VL domain comprise an amino acid sequence selected from the group consisting of: (a) SEQ ID NOs: 58 and 59, respectively; (b) SEQ ID NOs: 247 and 248, respectively; (c) SEQ ID NOs: 62 and 63, respectively; (d) SEQ ID NOs: 64 and 65, respectively; (e) SEQ ID NOs: 66 and 67, respectively; (f) SEQ ID NOs: 68 and 69, respectively; (g) SEQ ID NOs: 70 and 71, respectively; (h) SEQ ID NOs: 72 and 73, respectively; (i) SEQ ID NOs: 185 and 186, respectively; (j) SEQ ID NOs: 245 and 246, respectively; (k) SEQ ID NOs: 249 and 250, respectively; (l) SEQ ID NOs: 251 and 252, respectively; (m) SEQ ID NOs: 253 and 254, respectively; (n) SEQ ID NOs: 255 and 256, respectively; and (o) SEQ ID NOs: 257 and 258, respectively.

[0021] In any of the foregoing or related embodiments, (i) the IL-2 polypeptide is linked to the antibody or antigen-binding fragment directly or via a linker, and / or (ii) the IL-2 polypeptide is linked to the antibody or antigen-binding fragment directly or via a linker. In some embodiments, (i) the IL-2 polypeptide is linked to the antibody or antigen-binding fragment by a Gly-Ser linker, and / or (ii) the IL-2 polypeptide is linked to the antibody or antigen-binding fragment by a Gly-Ser linker.

[0022] In any of the preceding or related embodiments, the IL-2 fusion protein, the IL-21 fusion protein, or both the IL-2 fusion protein and the IL-21 fusion protein, respectively, comprises: Two antibody heavy chain polypeptides comprising, from N-terminus to C-terminus, a structure according to formula [I]: VH-CH1-hinge-CH2-CH3 [I] and two antibody light chain polypeptides comprising, from N-terminus to C-terminus, a structure according to formula [II]: VL-CL [II] where VH is a VH domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2-CH3 are antibody Fc domains, VL is a VL domain, and CL is an antibody constant light chain domain; The N-terminus of the IL-2 or IL-21 polypeptide is fused to the C-terminus of one of the two CH3 domains.

[0023] In some embodiments, one or both of the antibody heavy chain polypeptides comprise the following amino acid substitutions: L234A, L235A, and G237A (as numbered according to the EU index). In some embodiments, a first antibody heavy chain polypeptide comprises amino acid substitutions Y349C and T366W, and a second antibody heavy chain polypeptide comprises amino acid substitutions S354C, T366S, L368A, and Y407V (as numbered according to the EU index).

[0024] In any of the foregoing or related embodiments, the IL-2 polypeptide has a binding affinity for IL-2Rα that is 50% or more reduced compared to the binding affinity of a human IL-2 polypeptide comprising SEQ ID NO: 81. In some embodiments, the IL-2 polypeptide comprises one or more amino acid mutations that reduce the binding affinity of the IL-2 polypeptide compared to a human IL-2 polypeptide comprising SEQ ID NO: 81. In some aspects, the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 81 having one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 81, wherein the one, two, three, four, or five substitution(s) comprise substitution(s) at a position selected from the group consisting of Q11, H16, L18, L19, D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, 192, T123, Q126, S127, 1129, and S130 of SEQ ID NO: 81. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 having one of the following sets of amino acid substitutions (relative to the sequence of SEQ ID NO: 81): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A, and N88S; R38E, F42A, and N88A; R38E, F42A, and N88G; R38E, F42A, and N88R; R38E, F42A, and N88T; R38E, F42A, and N88D; R38E, F42A, and V91E; R38E, F42A, and D84H; R38E, F42A, and D84K; R38E, F42A, and D84R;H16D, R38E and F42A;H16E, R38E and F42A;R38E, F42A and Q126S;R38D, F42A and N88S;R38D, F42A and N88A;R38D, F42A and N88G;R38D, F42A and N88R;R38D, F42A and N88T;R38D, F4 2A and N88D; R38D, F42A and V91E; R38D, F42A, and D84H; R38D, F42A, and D84K; R38D, F42A, and D84R; H16D, R38D, and F42A; H16E, R38D, and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S;R38A, F42K, and N88A; R38A, F42K, and N88G; R38A, F42K, and N88R; R38A, F42K, and N88T; R38A, F42K, and N88D; R38A, F42K, and V91E; R38A, F42K, and D84H; R38A, F42K, and D84K; R38A, F42K, and D84R; H16D, R38A, and F42K; H16E, R38A, and F42K; R3 In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81, with an additional amino acid substitution at position C125 compared to SEQ ID NO: 81. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 having one of the following sets of amino acid substitutions (relative to the sequence of SEQ ID NO: 81): R38E, F42A, and C125A; R38D, F42A, and C125A; F42A, E62Q, and C125A; R38A, F42K, and C125A; R38E, F42A, N88S, and C125A; R38E, F42A, N88A, and C125A; R38E, F42A, N88G, and C125A; R38E, F42A, N88R, and C125A; R38E, F42A, N88D, and C125A; R38E, F42A, N88R, and C125A. A, N88T, and C125A; R38E, F42A, V91E, and C125A; R38E, F42A, D84H, and C125A; R38E, F42A, D84K, and C125A; R38E, F42A, D84R, and C125A; H16D, R38E, F42A, and C125A; H16E , R38E, F42A, and C125A;R38E, F42A, C125A and Q126S;R38D, F42A, N88S, and C125A;R38D, F42A, N88A, and C125A;R38D, F42A, N88G, and C125A;R38D, F42A, N88R, and C125A;R38D, F42A, N88T, and C125A; R38D, F42A, N88D, and C125A; R38D, F42A, V91E, and C125A; R38D, F42A, D84H, and C125A; R38D, F42A, D84K, and C125A; R38D, F42A, D84R, and C125A; H16D, R38D, F42A, and C125A; H16E, R38D, F42A, and C125A; R38D, F42A, C125A, and Q126S; R38A, F42K, N88S, and C125A; R38A, F42K, N88G, and C125A; R38A, F42K, N88R, and C125A; R38A, F42K, N88T, and C125A; R38A, F42K, N88D, and C125A; R38A, F42K, N88A, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H, and C125A; R38A, F42K, D84K, and C125A; R38A, F42K, D84R, and C125A; H16D, R38A, F42K, and C125A; H16E, R38A, F42K, and C125A; R38A, F42K, C12 5A and Q126S; F42A, E62Q, N88S, and C125A; F42A, E62Q, N88A, and C125A; F42A, E62Q, N88G, and C125A; F42A, E62Q, N88R, and C125A; F42A, E62Q, N88T, and C125A; F42A, E62Q, N88D, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A; F42A, E62Q, and D84K, and C125A; F42A, E62Q, and D84R, and C125A; H1 6D, F42A, and E62Q, and C125A; H16E, F42A, E62Q, and C125A; F42A, E62Q, C125A, and Q126S; F42A, N88S, and C125A; F42A, N88A, and C125A; F42A, N88G, and C125A; F42A, N88R, and C125A; F42A, N88T, and C125A; F42A, N88D, and C125A; F42A, V91E, and C125A; F42A, D84H, and C125A; F42A, D84K, and C125A; F42A, D84R, and C125A;H16D, F42A, and C125A; H16E, F42A, and C125A; and F42A, C125A and Q126S. In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 80 or SEQ ID NO: 297. In some embodiments, the IL-2 polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 80, 85-155, 190-216, 297, and 354-383.

[0025] In any of the foregoing or related embodiments, the IL-21 polypeptide comprises an amino acid sequence at least 80% identical to a human IL-21 polypeptide comprising SEQ ID NO: 390. In some embodiments, the IL-21 polypeptide has an isoelectric point that is at least about 0.6 units to about 5 units lower than the isoelectric point of the human IL-21 polypeptide. In some embodiments, the IL-21 polypeptide comprises at least one amino acid substitution that reduces the isoelectric point of the IL-21 polypeptide by about 0.6 units to about 5 units compared to the human IL-21 polypeptide without the amino acid substitution. In some embodiments, the IL-21 polypeptide comprises at least four amino acid substitutions that reduce the isoelectric point of the IL-21 polypeptide by about 0.6 units to about 5 units compared to the human IL-21 polypeptide without the amino acid substitution. In some embodiments, the IL-21 polypeptide comprises up to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions that reduce the isoelectric point. In some embodiments, the human IL-21 polypeptide has an isoelectric point of about 9.42. In some embodiments, the IL-21 polypeptide has an isoelectric point of about 7.12 to about 8.72. In some embodiments, the IL-21 polypeptide provides improved exposure following administration to a subject, measured as an area under the curve (AUC) for the IL-21 polypeptide of at least about 1.5-fold compared to the human IL-21 polypeptide. In some embodiments, the human IL-21 polypeptide comprises a region of about 2 to 20 positively charged amino acid residues, and the IL-21 polypeptide comprises at least one amino acid substitution of one or more positively charged amino acid residues. In some embodiments, the region does not comprise amino acid residues that bind to the IL-21 receptor. In some embodiments, the region comprises amino acid residues S80 to T92 of a human IL-21 polypeptide comprising SEQ ID NO: 390. In some embodiments, the human IL-21 polypeptide comprises at least one positively charged amino acid residue on the surface of the human IL-21 polypeptide in the three-dimensional structure of the human IL-21 polypeptide, does not bind to the IL-21 receptor, and the IL-21 polypeptide comprises at least one amino acid substitution of the at least one positively charged amino acid residue.In some embodiments, the IL-21 polypeptide does not comprise an amino acid substitution at G84 of a human IL-21 polypeptide comprising SEQ ID NO: 390. In some embodiments, the IL-21 polypeptide comprises 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions at positions selected from S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, or T92 of SEQ ID NO: 390. In some embodiments, the IL-21 polypeptide comprises 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions selected from S80G, T81G, N82G, N82E, A83G, A83E, A83S, R85G, R85E, R85S, R86G, R86E, Q87G, Q87E, Q87S, K88G, H89G, H89S, R90G, R90S, R90E, R90A, L91G, L91S, T92G, T92S. In some aspects, the IL-21 polypeptide comprises (a) R85G, R86G, K88G, and R90E, or (b) S80G, T81G, N82E, A83G, R85G, R86G, Q87G, K88G, H89G, R90E, L91G, and T92G. In some aspects, the IL-21 polypeptide comprises the amino acid sequence: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSCFQKAQLKSANTGNNERIINVSIKKLKRKPPX1X2X3X4GX5X6X7X8X9X. 10 X 11 X 12 X1 = G,S; X2 = G,T; X3 = G,E,N; X4 = G,S,E,A; X5 = G,E,S,R; X6 = G,E,R; X7 = S,G,E,Q; X8 = G,K; X9 = G,S,H; X 10 =A,E,S,G,R;X 11 = S, G, L; and X 12 = G, S, T, where X5, X6, X8, X9, and X 10 at least one of which is not the amino acid residue at the same position set forth in SEQ ID NO: 390, and optionally (i) X5=G, X6=G, X8=G, X 10=A、 (ii)X5=G,X6=G,X8=G,X 10 =E、 (iii)X1=G,X2=G,X3=G,X4=S,X5=E,X6=G,X7=S,X8=G,X9=G,X 10 =S、 (iv)X1=G,X2=G,X3=G,X4=S,X5=G,X6=G,X7=S,X8=G,X9=G,X 10 =E、 (v)X5=G,X6=G,X7=S,X8=G,X9=G,X 10 =E、 (vi)X5=G,X6=G,X7=S,X8=G,X9=G,X 10 =S、 (vii)X5=G,X6=G,X7=G,X8=G,X9=G,X 10 =E、 (viii)X5=G,X6=G,X7=G,X8=G,X9=G,X 10 =G、 (ix)X5=G,X6=G,X7=E,X8=G,X9=G,X 10 =G、 (x)X1=G,X2=G,X3=G,X4=S,X5=G,X6=G,X7=S,X8=G,X9=G,X 10 =G,X 11 =S,X 12 =G、 (xi)X1=G,X2=G,X3=G,X4=G,X5=G,X6=G,X7=G,X8=G,X9=G,X 10 =G,X 11 =G,X 12 =G、 (xii)X1=G,X2=G,X3=G,X4=S,X5=G,X6=G,X7=E,X8=G,X9=G,X 10 =G,X 11 =S,X 12 =G、 (xiii)X1=G,X2=G,X3=G,X4=E,X5=G,X6=G,X7=E,X8=G,X9=G,X 10 =G,X 11 =S,X 12 =G、 (xiv)X3=G,X4=G,X5=S,X6=G,X7=G,X8=G,X9=S,X 10 =G,X 11 =G,X 12 =S、 (xv)X3=G,X4=G,X5=E,X6=G,X7=G,X8=G,X9=S,X 10 =G,X 11 =G,X 12 =S、 (xvi)X1=G,X2=G,X3=G,X4=G,X5=E,X6=G,X7=G,X8=G,X9=G,X 10 =G,X 11 =G,X 12 =G、 (xvii)X1=G,X2=G,X3=G,X4=S,X5=G,X6=G,X7=S,X8=G,X9=G,X 10 =E,X 11 =G,X 12 =G、 (xviii)X1=G,X2=G,X3=G,X4=G,X5=G,X6=G,X7=G,X8=G,X9=G,X 10 =E,X 11 =G,X 12 =G、 (xix)X3=G,X4=G,X5=S,X6=G,X7=G,X8=G,X9=S,X 10 =E,X 11 =G,X 12 =S、 (xx)X3=G,X4=G,X5=G,X6=G,X7=G,X8=G,X9=G,X 10 =E,X 11 =G,X 12 =G、 (xxi)X5=G,X6=G,X7=G,X8=G,X9=G,X 10 =E, X11 =G、 (xxii)X3=G,X4=G,X5=G,X6=G,X7=G,X8=G,X9=G,X 10 =E,X 11 =G、 (xxiii)X3=G,X4=G,X5=G,X6=E,X7=G,X8=G,X9=G,X 10 =E,X 11 =G、 (xxiv)X3=G,X4=G,X5=E,X6=G,X7=G,X8=G,X9=G,X 10 =E,X 11 =G、 (xxv)X1=G,X2=G,X3=G,X4=E,X5=G,X6=G,X7=G,X8=G,X9=G,X 10 =E,X 11 =G,X 12 =G、または (xxvi)X1=G,X2=G,X3=E,X4=G,X5=G,X6=G,X7=G,X8=G,X9=G,X 10 =E,X 11 =G,X 12 =Gである。

[0026] In some embodiments, the IL-12 polypeptide comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 391-421. In some embodiments, the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 391-421. In some embodiments, the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO: 421. In some embodiments, the IL-21 polypeptide comprises at least one amino acid substitution that reduces binding to the IL-21 receptor compared to binding by a human IL-21 polypeptide. In some embodiments, the at least one amino acid substitution is a substitution of one or more amino acid residues at positions R5, 18, R9, R11, L13, 114, 116, V17, D18, K72, K73, L74, K75, R76, K77, or K117 of SEQ ID NO: 390. In some embodiments, the at least one amino acid substitution is R5F, R5A, R5E, R5S, R5T, R5N, R5Q, R5V, R5I, R5L, R5Y, and R9A, R9D, R9E, R9H, R9S, R9T, R9N, R9G, R9V, R9I, R9L, R9Y, R11D, R11E, L13F, L13R, I14D, I16A, I16S, I16R, V17I, V17A, D18A, K72A, K72E, K73A, K73E, K75A, K75E, L74I, L74F, L74M, L74V, R76E, R76F, R76A, R76N, R76D, R76S, R76T, R76Q, R76V, R76I, R76L, R76Y, R76M, K77A, K77E, and K117A. In some embodiments, at least one amino acid substitution is R76E or R76Q. In some embodiments, the IL-21 polypeptide comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 422-491. In some embodiments, the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 422-491.

[0027] In any of the preceding or related embodiments, the IL-2 fusion protein comprises: one or two light chains comprising the amino acid sequence of SEQ ID NO: 156, a heavy chain comprising the amino acid sequence of SEQ ID NO: 157, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 158; one or two light chains comprising the amino acid sequence of SEQ ID NO: 159, a heavy chain comprising the amino acid sequence of SEQ ID NO: 160, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 161; one or two light chains comprising the amino acid sequence of SEQ ID NO: 162, a heavy chain comprising the amino acid sequence of SEQ ID NO: 163, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 164; one or two light chains comprising the amino acid sequence of SEQ ID NO: 165, a heavy chain comprising the amino acid sequence of SEQ ID NO: 166, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 167; one or two light chains comprising the amino acid sequence of SEQ ID NO: 168, a heavy chain comprising the amino acid sequence of SEQ ID NO: 169, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 170; one or two light chains comprising the amino acid sequence of SEQ ID NO: 171, a heavy chain comprising the amino acid sequence of SEQ ID NO: 172, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 173; one or two light chains comprising the amino acid sequence of SEQ ID NO: 174, a heavy chain comprising the amino acid sequence of SEQ ID NO: 175, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 176; one or two light chains comprising the amino acid sequence of SEQ ID NO: 187, a heavy chain comprising the amino acid sequence of SEQ ID NO: 188, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 189; one or two light chains comprising the amino acid sequence of SEQ ID NO: 298, a heavy chain comprising the amino acid sequence of SEQ ID NO: 299, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 300; one or two light chains comprising the amino acid sequence of SEQ ID NO: 302, a heavy chain comprising the amino acid sequence of SEQ ID NO: 303, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 304; one or two light chains comprising the amino acid sequence of SEQ ID NO: 306, a heavy chain comprising the amino acid sequence of SEQ ID NO: 307, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 308; one or two light chains comprising the amino acid sequence of SEQ ID NO: 310, a heavy chain comprising the amino acid sequence of SEQ ID NO: 311, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 312; one or two light chains comprising the amino acid sequence of SEQ ID NO: 314, a heavy chain comprising the amino acid sequence of SEQ ID NO: 315, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 316; one or two light chains comprising the amino acid sequence of SEQ ID NO: 318, a heavy chain comprising the amino acid sequence of SEQ ID NO: 319, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 320; one or two light chains comprising the amino acid sequence of SEQ ID NO: 322, a heavy chain comprising the amino acid sequence of SEQ ID NO: 323, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 324; one or two light chains comprising the amino acid sequence of SEQ ID NO: 326, a heavy chain comprising the amino acid sequence of SEQ ID NO: 327, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 328; one or two light chains comprising the amino acid sequence of SEQ ID NO: 330, a heavy chain comprising the amino acid sequence of SEQ ID NO: 331, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 332; one or two light chains comprising the amino acid sequence of SEQ ID NO: 334, a heavy chain comprising the amino acid sequence of SEQ ID NO: 335, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 336; one or two light chains comprising the amino acid sequence of SEQ ID NO: 338, a heavy chain comprising the amino acid sequence of SEQ ID NO: 339, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 340; one or two light chains comprising the amino acid sequence of SEQ ID NO: 342, a heavy chain comprising the amino acid sequence of SEQ ID NO: 343, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 344; one or two light chains comprising the amino acid sequence of SEQ ID NO: 346, a heavy chain comprising the amino acid sequence of SEQ ID NO: 347, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 348; one or two light chains comprising the amino acid sequence of SEQ ID NO: 350, a heavy chain comprising the amino acid sequence of SEQ ID NO: 351, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 352; one or two light chains comprising the amino acid sequence of SEQ ID NO: 156, a heavy chain comprising the amino acid sequence of SEQ ID NO: 157, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 217; one or two light chains comprising the amino acid sequence of SEQ ID NO: 159, a heavy chain comprising the amino acid sequence of SEQ ID NO: 160, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 218; one or two light chains comprising the amino acid sequence of SEQ ID NO: 162, a heavy chain comprising the amino acid sequence of SEQ ID NO: 163, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 219; one or two light chains comprising the amino acid sequence of SEQ ID NO: 165, a heavy chain comprising the amino acid sequence of SEQ ID NO: 166, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 220; one or two light chains comprising the amino acid sequence of SEQ ID NO: 168, a heavy chain comprising the amino acid sequence of SEQ ID NO: 169, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 221; one or two light chains comprising the amino acid sequence of SEQ ID NO: 171, a heavy chain comprising the amino acid sequence of SEQ ID NO: 172, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 222; one or two light chains comprising the amino acid sequence of SEQ ID NO: 174, a heavy chain comprising the amino acid sequence of SEQ ID NO: 175, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 223; one or two light chains comprising the amino acid sequence of SEQ ID NO: 187, a heavy chain comprising the amino acid sequence of SEQ ID NO: 188, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 224; one or two light chains comprising the amino acid sequence of SEQ ID NO: 298, a heavy chain comprising the amino acid sequence of SEQ ID NO: 299, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 301; one or two light chains comprising the amino acid sequence of SEQ ID NO: 302, a heavy chain comprising the amino acid sequence of SEQ ID NO: 303, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 305; one or two light chains comprising the amino acid sequence of SEQ ID NO: 306, a heavy chain comprising the amino acid sequence of SEQ ID NO: 307, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 309; one or two light chains comprising the amino acid sequence of SEQ ID NO: 310, a heavy chain comprising the amino acid sequence of SEQ ID NO: 311, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 313; one or two light chains comprising the amino acid sequence of SEQ ID NO: 314, a heavy chain comprising the amino acid sequence of SEQ ID NO: 315, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 317; one or two light chains comprising the amino acid sequence of SEQ ID NO: 318, a heavy chain comprising the amino acid sequence of SEQ ID NO: 319, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 321; one or two light chains comprising the amino acid sequence of SEQ ID NO: 322, a heavy chain comprising the amino acid sequence of SEQ ID NO: 323, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 325; one or two light chains comprising the amino acid sequence of SEQ ID NO: 326, a heavy chain comprising the amino acid sequence of SEQ ID NO: 327, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 329; one or two light chains comprising the amino acid sequence of SEQ ID NO: 330, a heavy chain comprising the amino acid sequence of SEQ ID NO: 331, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 333; one or two light chains comprising the amino acid sequence of SEQ ID NO: 334, a heavy chain comprising the amino acid sequence of SEQ ID NO: 335, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 337; one or two light chains comprising the amino acid sequence of SEQ ID NO: 338, a heavy chain comprising the amino acid sequence of SEQ ID NO: 339, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 341; one or two light chains comprising the amino acid sequence of SEQ ID NO: 342, a heavy chain comprising the amino acid sequence of SEQ ID NO: 343, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 345; one or two light chains comprising the amino acid sequence of SEQ ID NO: 346, a heavy chain comprising the amino acid sequence of SEQ ID NO: 347, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 349, or It comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 350, a heavy chain comprising the amino acid sequence of SEQ ID NO: 351, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 353.

[0028] In any of the foregoing or related embodiments, the IL-2 fusion protein comprises four polypeptide chains, wherein: the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 334, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 335, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 336, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 334; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 334, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 335, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 337, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 334; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 338, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 339, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 340, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 338; or The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 338, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 339, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 341, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 338.

[0029] In any of the foregoing or related embodiments, the IL-21 fusion protein comprises: one or two light chains comprising the amino acid sequence of SEQ ID NO: 557, a heavy chain comprising the amino acid sequence of SEQ ID NO: 558, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 559; one or two light chains comprising the amino acid sequence of SEQ ID NO: 561, a heavy chain comprising the amino acid sequence of SEQ ID NO: 562, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 563; one or two light chains comprising the amino acid sequence of SEQ ID NO: 565, a heavy chain comprising the amino acid sequence of SEQ ID NO: 566, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 567; one or two light chains comprising the amino acid sequence of SEQ ID NO: 569, a heavy chain comprising the amino acid sequence of SEQ ID NO: 570, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 571; one or two light chains comprising the amino acid sequence of SEQ ID NO: 573, a heavy chain comprising the amino acid sequence of SEQ ID NO: 574, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 575; one or two light chains comprising the amino acid sequence of SEQ ID NO: 577, a heavy chain comprising the amino acid sequence of SEQ ID NO: 578, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 579; one or two light chains comprising the amino acid sequence of SEQ ID NO: 581, a heavy chain comprising the amino acid sequence of SEQ ID NO: 582, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 583; one or two light chains comprising the amino acid sequence of SEQ ID NO: 585, a heavy chain comprising the amino acid sequence of SEQ ID NO: 586, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 587; one or two light chains comprising the amino acid sequence of SEQ ID NO: 589, a heavy chain comprising the amino acid sequence of SEQ ID NO: 590, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 591; one or two light chains comprising the amino acid sequence of SEQ ID NO: 557, a heavy chain comprising the amino acid sequence of SEQ ID NO: 558, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 560; one or two light chains comprising the amino acid sequence of SEQ ID NO: 561, a heavy chain comprising the amino acid sequence of SEQ ID NO: 562, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 564; one or two light chains comprising the amino acid sequence of SEQ ID NO: 565, a heavy chain comprising the amino acid sequence of SEQ ID NO: 566, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 568; one or two light chains comprising the amino acid sequence of SEQ ID NO: 569, a heavy chain comprising the amino acid sequence of SEQ ID NO: 570, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 572; one or two light chains comprising the amino acid sequence of SEQ ID NO: 573, a heavy chain comprising the amino acid sequence of SEQ ID NO: 574, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 576; one or two light chains comprising the amino acid sequence of SEQ ID NO: 577, a heavy chain comprising the amino acid sequence of SEQ ID NO: 578, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 580; one or two light chains comprising the amino acid sequence of SEQ ID NO: 581, a heavy chain comprising the amino acid sequence of SEQ ID NO: 582, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 584; one or two light chains comprising the amino acid sequence of SEQ ID NO: 585, a heavy chain comprising the amino acid sequence of SEQ ID NO: 586, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 588; or It comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 589, a heavy chain comprising the amino acid sequence of SEQ ID NO: 590, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 592.

[0030] In any of the foregoing or related embodiments, the IL-21 fusion protein comprises four polypeptide chains, wherein: the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:557, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:558, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:559, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:557; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:557, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:558, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:560, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:557; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:561, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:562, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:563, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:561; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:561, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:562, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:564, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:561; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 565, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 566, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 567, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 565; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 565, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 566, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 568, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 565; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:569, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:570, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:571, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:569; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:569, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:570, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:572, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:569; or a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 573, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 574, a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 575, and a fourth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 573; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:573, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:574, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:576, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:573; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:577, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:578, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:579, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:577; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:577, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:578, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:580, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:577; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:581, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:582, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:583, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:581; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:581, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:582, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:584, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:581; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:585, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:586, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:587, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:585; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:585, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:586, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:588, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:585; or the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:589, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:590, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:591, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:589; or The first polypeptide chain comprises the amino acid sequence of SEQ ID NO:589, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:590, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:592, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:589.

[0031] In any of the foregoing or related embodiments, the IL-21 fusion protein is administered prior to administration of the IL-2 fusion protein. In other embodiments, the IL-2 fusion protein is administered prior to administration of the IL-21 fusion protein. In some embodiments, the IL-21 fusion protein and the IL-2 fusion protein are administered concurrently, sequentially, or simultaneously.

[0032] In any of the foregoing or related embodiments, the IL-21 fusion protein is formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, the IL-2 fusion protein is formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, the IL-21 fusion protein and the IL-2 fusion protein are formulated in the same pharmaceutical composition. In other embodiments, the IL-21 fusion protein and the IL-2 fusion protein are formulated in separate pharmaceutical compositions. Incorporation by Reference

[0033] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0034] [Figure 1] Schematics of the xmCD8-mIL21v1.1 fusion protein and the xmCD8.1-mIL2v fusion protein are shown. Both fusion proteins contain an anti-mouse CD8 IgG and a single copy of a cytokine (mIL21v1.1 or mIL2v) fused to the C-terminus of one of the heavy chains. In some cases, the Fc region in both schematics contains knobs-in-holes heterodimerization mutations. In some cases, the Fc region in both schematics does not contain knobs-in-holes heterodimerization mutations. [Figure 2A]

[0023] Figure 1 shows an efficacy study of MC38 comparing single agent versus combination of xmCD8-mIL21v1.1 and xmCD8.1-mIL2v administered simultaneously at different dose levels. Single agent versus combination is shown for xmCD8.1-mIL2v at 0.3 mg / kg and xmCD8-mIL21v1.1 at 0.1 mg / kg. [Figure 2B]

[0023] Figure 1 shows an efficacy study of MC38 comparing single agent versus combination of xmCD8-mIL21v1.1 and xmCD8.1-mIL2v administered simultaneously at different dose levels. Single agent versus combination is shown for xmCD8.1-mIL2v at 0.3 mg / kg and xmCD8-mIL21v1.1 at 0.03 mg / kg. [Figure 2C]

[0023] Figure 1 shows an efficacy study of MC38 comparing single agent versus combination of xmCD8-mIL21v1.1 and xmCD8.1-mIL2v administered simultaneously at different dose levels. Single agent versus combination is shown for xmCD8.1-mIL2v at 0.1 mg / kg and xmCD8-mIL21v1.1 at 0.1 mg / kg. [Figure 2D]

[0023] Figure 1 shows an efficacy study of MC38 comparing single agent versus combination of xmCD8-mIL21v1.1 and xmCD8.1-mIL2v administered simultaneously at different dose levels. Single agent versus combination is shown for xmCD8.1-mIL2v at 0.1 mg / kg and xmCD8-mIL21v1.1 at 0.03 mg / kg. [Figure 3] MC38 efficacy study comparing single agent vs. combination of xmCD8-mIL21v1.1 and xmCD8.1-mIL2v administered 1 hour apart at different dose levels. Separate groups of combination treatment are shown, with either xmCD8-mIL21v1.1 administered first or xmCD8.1-mIL2v administered first. Single agent vs. combination of 0.1 mg / kg xmCD8.1-mIL2v and 0.1 mg / kg xmCD8-mIL21v1.1 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present disclosure is based, at least in part, on the discovery that the combination of an IL-2 fusion protein and an IL-21 fusion protein (each fusion protein containing an antibody that specifically binds to human CD8) reduces or suppresses tumor growth in vivo more than administration of either fusion protein alone. Without wishing to be bound by theory, targeting IL-2 and IL-21 to CD8+ T cells using an anti-CD8-specific antibody selectively activates CD8+ T cells to induce an anti-tumor immune response. Because IL-2 activates CD8+ T cells via STAT5 phosphorylation, thereby promoting proliferation and enhancing cytotoxic function, and IL-21 activates CD8+ T cells via STAT3 phosphorylation, maintaining T cell function, preventing exhaustion, promoting a memory phenotype, and enhancing cytotoxic function, targeting these two distinct, complementary, non-redundant pathways is believed to enhance CD8+ T cell activation and, therefore, provide a therapeutic effect.

[0036] In particular, the present disclosure describes methods for treating a disease, such as cancer or an infectious disease, in a mammalian subject. In some embodiments, the treatment comprises administering to the subject a combination of fusion proteins that bind to human CD8. In some embodiments, the fusion proteins comprise IL-21 or IL-2. In some embodiments, the IL-21 fusion protein and the IL-2 fusion protein comprise a human or humanized antibody, antigen-binding fragment, or fusion protein that binds to human CD8.

[0037] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[0038] It should be understood that aspects and embodiments of the present disclosure include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0039] The term "about," as used herein, refers to the normal error range for each value, which would be readily understood by one of ordinary skill in the art. If the usage of the term is not clear to one of ordinary skill in the art given the context in which it is used, "about" means up to ±10% of the particular value. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0040] As used herein, "area under the curve" or "AUC" is a pharmacokinetic parameter that refers to the area under a plot of the plasma concentration of a drug (e.g., an IL-21 polypeptide or fusion protein described herein) versus time after administration. In some embodiments, AUC measures a patient's exposure to a drug and is dependent on dose, bioavailability, and clearance. Methods for measuring AUC are known to those skilled in the art and include, but are not limited to, the rectangular method, the trapezoidal method, and the Simpson method.

[0041] As used herein, "immune cells" refer to cells of the immune system that respond to organisms or other entities that are considered foreign to the host's immune system. They protect the host from foreign pathogens, organisms, and diseases. Immune cells, also known as leukocytes, are involved in both innate and adaptive immune responses to combat pathogens. Innate immune responses occur immediately upon exposure to a pathogen without additional priming or learning processes. Adaptive immune processes require initial priming, followed by the creation of memory, which then leads to enhanced responsiveness upon subsequent encounters with the same pathogen. Innate immune cells include, but are not limited to, monocytes, macrophages, dendritic cells, innate lymphocytes (ILCs) including natural killer (NK) cells, neutrophils, megakaryocytes, eosinophils, and basophils. Adaptive immune cells include B lymphocytes / cells and T lymphocytes / cells. T cell subsets include, but are not limited to, αβCD4+T (naive CD4+, memory CD4+, effector memory CD4+, effector CD4+, regulatory CD4+) and αβCD8+T (naive CD8+, memory CD8+, effector memory CD8+, effector CD8+). B cell subsets include, but are not limited to, naive B cells, memory B cells, and plasma cells. NK T cells and T gamma delta (Tγδ) cells exhibit characteristics of both innate and adaptive lymphocytes.

[0042] "T cells" or "T lymphocytes" are immune cells that play a key role in orchestrating immune responses in health and disease. There are two major T cell subsets with distinct functions and properties: T cells that express the CD8 antigen (CD8 + T cells) are cytotoxic or killer T cells that can lyse target cells using cytotoxic proteins such as granzymes and perforin, and T cells that express the CD4 antigen (CD4 + T cells) are CD8 + They are helper T cells that can regulate the function of many other immune cell types, including T cells, B cells, macrophages, etc. Furthermore, CD4 +T cells are further subdivided into several subsets, such as T regulatory (Treg) cells, which can suppress immune responses, and T helper 1 (Th1), T helper 2 (Th2), and T helper 17 (Th17) cells, which regulate various types of immune responses by secreting immunomodulatory proteins such as cytokines. T cells recognize their targets via the αβ T cell receptor, which binds to unique antigen-specific motifs, and this recognition mechanism is generally required to trigger their cytotoxic and cytokine-secreting functions. "Innate lymphocytes" also include CD8 + and CD4 + They may exhibit T cell characteristics, such as cytotoxic activity or secretion of Th1, Th2, and Th17 cytokines. Some of these innate lymphocyte subsets include NK cells, as well as innate immune-like T cells such as ILC1, ILC2, and ILC3 cells, and Tγδ cells, as well as NK T cells. Typically, these cells can respond rapidly to inflammatory stimuli from infected or damaged tissues, such as immunoregulatory cytokines, but unlike αβ T cells, they can respond without the need to recognize antigen-specific patterns.

[0043] A "cytokine" is a form of immunomodulatory polypeptide that mediates crosstalk between initiating / primary cells and target / effector cells. It can function as a soluble or cell surface receptor associated with target immune cells, activating signal transduction by binding to a "cytokine receptor" on the target immune cell. As used herein, a "cytokine receptor" is a polypeptide on the cell surface that activates intracellular signal transduction upon binding to a cytokine on the extracellular cell surface. Cytokines include, but are not limited to, chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are produced by a variety of cells, including immune cells, endothelial cells, fibroblasts, and stromal cells. A given cytokine can be produced by multiple cell types. Cytokines are pleiotropic, and receptors are expressed on multiple immune cell subsets, so a single cytokine can activate signal pathways in multiple cells. However, depending on the cell type, cytokine signaling events can result in different downstream cellular events, such as activation, proliferation, survival, apoptosis, effector function, and secretion of other immunomodulatory proteins.

[0044] As used herein, "amino acid" refers to naturally occurring carboxy α-amino acids, including alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0045] As used herein, "polypeptide" or "protein" refers to a molecule in which monomers (amino acids) are linearly linked to one another by peptide bonds (also known as amide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids is included within the definition of "polypeptide," and "polypeptide" can be used in place of or synonymously with any of these terms. The term "polypeptide" can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. Polypeptides can be produced by any method, including chemical synthesis. Polypeptides usually, but not necessarily, have a defined three-dimensional structure. Polypeptides of the present disclosure can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids in size. Polypeptides that have a defined three-dimensional structure are referred to as folded, while polypeptides that do not have a defined three-dimensional structure but rather can adopt many different conformations are referred to as unfolded. Polypeptides can also form multimers, such as dimers, trimers, and higher oligomers, i.e., multimers consisting of multiple polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc. can be identical or non-identical. The corresponding higher order structures of such multimers are consequently referred to as homodimers or heterodimers, homotrimers, heterotrimers, etc. The terms "polypeptide" and "protein" also refer to modified polypeptides / proteins that undergo post-expression modifications, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with unnatural amino acids.

[0046] " Identity " as used herein refers to the percentage (%) of sequence identity with respect to reference polypeptide sequence, and is the percentage of amino acid residues in candidate sequence that are identical to the amino acid residues in reference polypeptide sequence, after aligning sequences and introducing gaps as necessary to achieve the maximum percentage of sequence identity, without taking into account any conservative substitutions as part of sequence identity.For the purpose of determining percentage of amino acid sequence identity, alignment can be achieved in various known ways, for example, by using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Appropriate parameters for sequence alignment can be determined, including the algorithm required to achieve maximum alignment across the entire length of the sequence to be compared.However, for the purpose herein, amino acid sequence identity % value is generated using sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington, DC, 20559, and is registered under U.S. Copyright No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are not modified.

[0047] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it may be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 × fraction X / Y, where X is the number of amino acid residues scored as a perfect match by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0048] As used herein, "residue" refers to the position in the protein and the identity of the associated amino acid. For example, Leu 234 (also referred to as Leu234 or L234) is the residue at position 234 in the human antibody IgG1.

[0049] As used herein, "wild-type" refers to an amino acid sequence or nucleotide sequence found in nature, including allelic forms. A wild-type protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0050] A "substitution" refers to a change in a polypeptide or nucleic acid encoding said polypeptide in which an amino acid or nucleotide naturally occurring in the wild-type sequence of the polypeptide or nucleic acid is replaced with another amino acid or nucleotide that does not naturally occur at the same position in the sequence of said polypeptide or nucleic acid. In some embodiments, the substitution(s) are introduced to alter the affinity of the polypeptide for its receptor, thereby changing its activity so that it differs from the affinity and activity of the wild-type cognate polypeptide. In some embodiments, the substitution improves the biophysical properties of the polypeptide. Amino acid substitutions can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is contemplated that methods other than genetic modification, such as chemical modification to change the side chain group of an amino acid, may also be useful.

[0051] "Mutation" refers to an alteration in a polypeptide or nucleic acid encoding said polypeptide in which i) an amino acid and / or nucleotide in the wild-type sequence is substituted with another amino acid or nucleotide, ii) an amino acid and / or nucleotide in the wild-type sequence is deleted, and / or iii) an amino acid and / or nucleotide is inserted.

[0052] As used herein, "CD8" refers to any naturally occurring human CD8. Unless otherwise expressly or contextually indicated, reference to "CD8" refers to CD8aa and / or CD8ab. The amino acid sequence of an exemplary human CD8b, i.e., the beta chain of human CD8, is set forth in UniProt P10966 (CD8B_HUMAN). "CD8a" refers to the alpha chain of human CD8 (e.g., as set forth in UniProt P01732 (CD8A_HUMAN)). "CD8aa" refers to the homodimer of CD8a. "CD8ab" refers to the heterodimer of CD8a and CD8b. "CD8," "CD8a," "CD8b," "CD8aa," and "CD8ab" encompass unprocessed forms as well as mature forms resulting from intracellular processing. "CD8," "CD8a," "CD8b," "CD8aa," and "CD8ab" also include, but are not limited to, naturally occurring variants, such as allelic or splice variants, or variants.

[0053] As used herein, "interleukin-2" or "IL-2" refers to any naturally occurring human IL-2 or variants thereof, unless otherwise indicated. In some cases, "IL-2" encompasses unprocessed IL-2 as well as "mature IL-2," which is the form of IL-2 that results from processing within a cell. An exemplary form of unprocessed naturally occurring human IL-2 contains an additional N-terminal amino acid signal peptide attached to mature IL-2. "IL-2" also includes, but is not limited to, naturally occurring variants of IL-2, such as allelic or splice variants, or variants. The amino acid sequence of an exemplary naturally occurring human IL-2 is set forth in UniProt P60568 (IL2_HUMAN).

[0054] "Affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). Affinity is generally measured by the dissociation constant (K D ), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Thus, equivalent affinities may involve different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) technology (e.g., BIAcore), BioLayer Interferometry (BLI) technology (e.g., Octet), and other conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0055] As used herein, "binding" or "specific binding" refers to the ability of a polypeptide or antigen-binding molecule to selectively interact with a polypeptide or target antigen receptor, respectively, and this specific interaction can be distinguished from non-target, undesired, or non-specific interactions. Examples of specific binding include, but are not limited to, the binding of IL-2 cytokine to specific receptors (e.g., IL-2Rα, IL-2Rβ, and IL-2Rγ) and the binding of an antigen-binding molecule to a specific antigen (e.g., CD8 or PD-1).

[0056] A "mutant IL-2 polypeptide" refers to an IL-2 polypeptide that has reduced affinity for its receptor; such reduced affinity results in reduced biological activity of the mutant. Reduced affinity, and therefore activity, can be achieved by introducing a small number of amino acid mutations or substitutions. Mutant IL-2 polypeptides can also have other modifications to the peptide backbone, including, but not limited to, amino acid deletions, substitutions, cyclization, disulfide bonds, or post-translational modifications (e.g., glycosylation or carbohydrate alterations), chemical or enzymatic modifications to the polypeptide (e.g., attachment of PEG to the polypeptide backbone), addition of peptide tags or labels, or fusion to proteins or protein domains, to generate a final construct with desired properties, such as reduced affinity for IL-2Rβγ. The desired activity can also include improved biophysical properties compared to wild-type IL-2 polypeptides. Multiple modifications may be combined to achieve desired activity modifications, such as reduced affinity or improved biophysical properties. As a non-limiting example, an amino acid sequence for consensus N-linked glycosylation may be incorporated into the polypeptide to allow for glycosylation. In another non-limiting example, lysines may be incorporated onto the polypeptide to allow for PEGylation. Preferably, a mutation(s) is introduced into the polypeptide to alter its activity.

[0057] In some cases, a "mutant IL-21 polypeptide" refers to an IL-21 polypeptide that has reduced affinity for its receptor, where such reduced affinity results in reduced biological activity of the variant. Reduced affinity, and therefore activity, can be achieved by introducing a small number of amino acid mutations or substitutions. Mutant IL-21 polypeptides can also have other modifications to the peptide backbone, including, but not limited to, amino acid deletions, substitutions, cyclization, disulfide bonds, or post-translational modifications (e.g., glycosylation or carbohydrate changes) of the polypeptide, chemical or enzymatic modifications to the polypeptide (e.g., attachment of PEG to the polypeptide backbone), addition of peptide tags or labels, or fusion to proteins or protein domains, to generate a final construct with desired properties, such as reduced affinity for IL-21R. The desired activity can also include improved biophysical properties compared to the wild-type IL-21 polypeptide. Multiple modifications may be combined to achieve desired activity modifications, such as reduced affinity or improved biophysical properties. As a non-limiting example, an amino acid sequence for consensus N-linked glycosylation may be incorporated into the polypeptide to allow for glycosylation. In another non-limiting example, lysines may be incorporated onto the polypeptide to allow for PEGylation. Preferably, a mutation(s) is introduced into the polypeptide to alter its activity.

[0058] The terms "antibody" and "immunoglobulin" are used interchangeably and are used herein in the broadest sense to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and single domain antibodies (as described in more detail herein), so long as they exhibit the desired antigen-binding activity.

[0059] An antibody (immunoglobulin) refers to a protein having a structure substantially similar to that of a natural antibody. A "natural antibody" refers to a naturally occurring immunoglobulin molecule with a diverse structure. For example, natural immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also referred to as heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also referred to as a variable light domain or light chain variable domain, followed by a constant light (CL) domain, also referred to as a light chain constant region. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al., 2000, Cellular and Mol, and Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). Antibodies (immunoglobulins) are assigned to different classes depending on the amino acid sequence of the heavy chain constant domain. There are five major classes of antibodies: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain. Immunoglobulins essentially consist of two Fab molecules and an Fc domain linked via an immunoglobulin hinge region.

[0060] As used herein, "Fc" or "Fc region" or "Fc domain" refers to the C-terminal region of an antibody heavy chain containing at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. Fc can refer to the last two constant region immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and optionally all or part of the flexible hinge at the N-terminus of these domains. In the case of IgA and IgM, Fc can include the J chain. An IgG Fc region includes the IgG CH2 domain and the IgG CH3 domain, and in some cases, the hinge. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The "hinge" region typically extends from about amino acid residue 216 to about amino acid residue 230. The hinge region herein may be a native hinge domain or a variant hinge domain. The "CH2 domain" of a human IgG Fc region typically extends from about amino acid residue 231 to about amino acid residue 340. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises the sequence of residues from the C-terminus of the Fc region to the CH2 domain, i.e., from about amino acid residue 341 to about amino acid residue 447 of IgG. The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "protrusion" ("knob") introduced into one of its chains and a corresponding "cavity" ("hole") introduced into the other chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference).Thus, the definition of "Fc domain" includes both amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or fragments thereof. An "Fc fragment" in this context may contain fewer amino acids from either or both the N- and C-termini, but still retain the ability to dimerize with another Fc domain or Fc fragment, generally as can be detected using standard size-based methods (e.g., non-denaturing chromatography, size-exclusion chromatography, etc.). A human IgG Fc domain is particularly used in the present disclosure and may be an Fc domain derived from human IgG1, IgG2, or IgG4.

[0061] A "variant Fc domain" or "Fc variant" or "variant Fc" contains amino acid modifications (e.g., substitutions, additions, and deletions) compared to a parent Fc domain. The term also includes naturally occurring allelic variants of the Fc region of an immunoglobulin. Generally, a variant Fc domain has at least about 80, 85, 90, 95, 97, 98, or 99 percent identity (using an identity algorithm discussed below, and in one embodiment utilizing the BLAST algorithm known in the art, using default parameters) to a corresponding parent human IgG Fc domain. Alternatively, a variant Fc domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid alterations compared to the parent Fc domain. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Furthermore, as discussed herein, the variant Fc domains herein still retain the ability to form dimers with another Fc domain, as determined using known techniques described herein, such as non-denaturing gel electrophoresis.

[0062] As used herein, "Fc gamma receptor," "FcγR," or "Fc gamma R" refers to any member of a family of proteins that bind to the Fc region of an IgG antibody and are encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, the entire contents of which are incorporated by reference), as well as any undiscovered human FcγR or FcγR isoform or allotype. FcγRs can be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any unidentified mouse FcγR or FcγR isoform or allotype.

[0063] As used herein, "effector function" refers to a biochemical event resulting from the interaction of the Fc region of an antibody with an Fc receptor or ligand, which varies depending on the antibody isotype. Effector functions include, but are not limited to, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. "Antibody-dependent cellular cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcR (such as natural killer (NK) cells, neutrophils, and macrophages) recognize antibody-bound on target cells and subsequently cause lysis of the target cells. ADCC correlates with binding to FcγRIIIa, and increased binding to FcγRIIIa results in increased ADCC activity. To assess 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 5,821,337 may be performed. As used herein, "ADCP" or antibody-dependent cellular phagocytosis refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express FcγRs bind to antibodies on target cells, resulting in the subsequent phagocytosis of the target cells.

[0064] "Fc null" and "Fc null variant" are used interchangeably herein to describe a modified Fc with reduced or abolished effector function. Such Fc null or Fc null variants have reduced or abolished binding to FcγR and / or complement receptors. Preferably, such Fc null or Fc null variants have abolished effector function. Exemplary methods for modification include, but are not limited to, chemical modification, amino acid residue substitution, insertion, and deletion. Exemplary amino acid positions of an Fc molecule (numbering according to the EU numbering scheme) in which one or more modifications have been introduced at positions i) IgG1: C220, C226, C229, E233, L234, L235, G237, P238, S239, D265, S267, N297, L328, P331, K322, A327, and P329, ii) IgG2: V234, G237, D265, H268, N297, V309, A330, A331, K322, and iii) IgG4: L235, G237, D265, and E318 to reduce the effector function of the resulting variant.Exemplary Fc molecules with reduced effector function include those with one or more of the following substitutions: i) IgG1: N297A, N297Q, N297G, D265A / N297A, D265A / N297Q, C220S / C226S / C229S / P238S, S267E / L328F, C226S / C229S / E233P / L234V / L235A, L 234F / L235E / P331S,L234A / L235A,L234A / L235A / G237A,L234A / L235A / G237A / K322A,L234A / L235A / G 237A / A330S / A331S,L234A / L235A / P329G,E233P / L234V / L235A / G236del / S239K,E233P / L234V / L235A / G236del / S267K,E233P / L234V / L235A / G236del / S239K / A327G,E233P / L234V / L235A / G236del / S267K / A327G and E233P / L234V / L235A / G236del,L234A / L235A / G237 deletion, ii) IgG2:A330S / A331S,V234A / G237A , V234A / G237A / D265A, D265A / A330S / A331S, V234A / G237A / D265A / A330S / A331S, and H268Q / V309L / A330S / A331S, iii) IgG4: L235A / G237A / E318A, D265A, L235A / G237A / D265A and L235A / G237A / D265A / E318A.

[0065] As used herein, "epitope" refers to a determinant capable of specific binding to the variable region of an antibody molecule, known as the paratope. An epitope is a group of molecules, such as amino acids or sugar side chains, that typically possess specific structural and charge characteristics. A single antigen may have multiple epitopes. An epitope may include amino acid residues directly involved in binding as well as other amino acid residues not directly involved in binding, such as those effectively blocked by the antigen-binding peptide (i.e., those within the footprint of the antigen-binding peptide). Epitopes may be either conformational or linear. An epitope typically contains at least three amino acids, more commonly at least five or eight to ten amino acids. Antibodies that recognize the same epitope can be identified by simple immunoassays, such as "binning," which demonstrate the ability of one antibody to block the binding of another antibody to a target antigen.

[0066] As used herein, "linker" refers to a molecule that connects two polypeptide chains. The linker may be a polypeptide linker or a synthetic chemical linker (see, for example, the linkers disclosed in Protein Engineering, 9(3), 299-305, 1996). The length and sequence of the polypeptide linker are not particularly limited and can be selected by those skilled in the art according to the purpose. The polypeptide linker comprises one or more amino acids. In some embodiments, the polypeptide linker is a peptide at least 5 amino acids long, preferably 5 to 100 amino acids long, and more preferably 10 to 50 amino acids long. In one embodiment, the peptide linker is G, S, GS, SG, SGG, GGS, or GSG (G = glycine and S = serine). In another embodiment, the peptide linker is (GGGS)xGn (SEQ ID NO:74) or (GGGGS)xGn (SEQ ID NO:75) or (GGGGGS)xGn (SEQ ID NO:76) or S(GGGS)xGn (SEQ ID NO:386) or S(GGGGS)xGn (SEQ ID NO:387) or S(GGGGGS)xGn (SEQ ID NO:388) where x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 and n=0, 1, 2, or 3. Preferably, the linker is (GGGGS)xGn where x=2, 3, or 4 and n=0 (SEQ ID NO:77), more preferably, the linker is (GGGGS)xGn where x=3 and n=0 (SEQ ID NO:78). In some embodiments, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 79) or SGGGGSGGGGSGGGGS (SEQ ID NO: 389).Synthetic chemical linkers include cross-linkers routinely used for cross-linking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(succinimidyl) suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(succinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0067] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA), encoding a polypeptide of the present disclosure. A polynucleotide may contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds such as those found in peptide nucleic acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. In some embodiments, one or more vectors (particularly expression vectors) containing such nucleic acids are provided. In one embodiment, a method of producing a polypeptide of the present disclosure is provided, comprising culturing a host cell containing a nucleic acid encoding the polypeptide under conditions suitable for expression of the polypeptide and recovering the polypeptide from the host cell. "Recombinant" means that the protein is produced in an exogenous host cell using recombinant nucleic acid techniques. Recombinantly produced proteins expressed in a host cell are considered isolated for purposes of the present disclosure, as are naturally occurring or recombinant proteins that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0068] "Isolated," when used to describe various polypeptides disclosed herein, refers to a polypeptide that has been identified, separated, and / or recovered from the cell or cell culture in which the polypeptide was expressed. Typically, an isolated polypeptide has been purified by at least one purification step. No required level of purity exists; "purified" or "purified" refers to an increase in the concentration of a target protein relative to the concentration of contaminants in a composition compared to the starting material. As used herein, "isolated protein" refers to a target protein that is substantially free of other proteins with different binding specificities.

[0069] The term "cancer" refers to a physiological condition in mammals that is usually characterized by uncontrolled and abnormal cell growth, which may invade or metastasize to other parts of the body. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancer include lung cancer, small cell lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar lung cancer, squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, head and neck cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, thyroid cancer, uterine cancer, gastrointestinal cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, endometrial cancer, uterine cancer, fallopian tube cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, These include soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic cancer, mesothelioma, bladder cancer, liver cancer, hepatocellular carcinoma, cervical cancer, salivary gland cancer, bile duct cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma (including refractory versions of any of the above cancers, or a combination of one or more of the above cancers).

[0070] In some cases, the term "combination treatment" or "combination therapy" refers to the administration of two or more active agents or therapies in a regimen that produces a beneficial effect of the combination, including co-administration of the active agents or therapies in a substantially simultaneous manner, e.g., in a single formulation having a fixed ratio of active agents, or co-administration in a sequential manner, e.g., in multiple separate formulations of each active agent or therapy. Combination therapy also includes combinations in which the individual components (e.g., active ingredients or therapies) may be administered at different times and / or by different routes, but which act in combination to exhibit a beneficial effect or pharmacokinetic and pharmacodynamic effect of the combined agents or therapeutic approaches.

[0071] fusion proteins Provided herein are IL-2 and IL-21 fusion proteins comprising any one of the anti-CD8 antibodies, or antigen-binding domains, or antibody fragments disclosed herein. In some embodiments, the fusion proteins of the present disclosure comprise a first portion comprising a human or humanized antibody or antigen-binding fragment thereof that specifically binds to CD8 (e.g., any one of the anti-CD8 antibodies described below), and a second portion comprising IL-2 or IL-21. In some embodiments, the first portion is fused directly to the second portion. In some embodiments, the first portion is fused to the second portion via a linker.

[0072] In some embodiments, the fusion proteins described herein induce activation of cells expressing human CD8ab heterodimers at least 2-fold, at least 5-fold, or at least 10-fold more potently than activation of cells expressing human CD8aa homodimers. In some embodiments, the fusion proteins induce activation of CD8+ T cells at least 2-fold, at least 5-fold, or at least 10-fold more potently than activation of NK cells. In some embodiments, activation potency is measured by EC50 as assessed by cell proliferation. Exemplary assays are further described herein.

[0073] Fusion protein format In some embodiments, the first portion comprises an antibody (e.g., an anti-CD8 antibody of the present disclosure). In some embodiments, the first portion comprises an antibody fragment (e.g., an anti-CD8 antibody fragment of the present disclosure). In some embodiments, the first portion comprises a single chain antibody or a single chain variable fragment (scFv). In some embodiments, the first portion comprises a VHH antibody. In some embodiments, the first portion comprises one or two antibody heavy chain polypeptides and one or two antibody light chain polypeptides (e.g., of an anti-CD8 antibody of the present disclosure). In some embodiments, the first portion comprises three heavy chain CDRs and / or three light chain CDRs of a single anti-CD8 antibody of the present disclosure, e.g., as shown in Tables 1-3. In some embodiments, the first portion comprises the VH and / or VL domain(s) of a single anti-CD8 antibody of the present disclosure, e.g., as shown in Table 3. In some embodiments, the first portion further comprises one or two human IgG Fc domains. In some embodiments, one or two human IgG Fc domains are IgG1, IgG2, IgG3, or IgG4 Fc domains. In some embodiments, one or two human IgG Fc domains do not have a C-terminal lysine residue. In some embodiments, one or two human IgG Fc domains comprise an amino acid modification (e.g., substitution, deletion, addition, etc.). In some embodiments, the Fc domain modification promotes heterodimer formation (e.g., as shown in Table 4). In some embodiments, one or two Fc domains comprise an Fcγ null mutation.

[0074] In some embodiments, the first portion comprises two antibody heavy chain polypeptides comprising, from N-terminus to C-terminus, a structure according to formula [I]: VH-CH1-hinge-CH2-CH3 [I] and two antibody light chain polypeptides comprising, from N-terminus to C-terminus, a structure according to formula [II]: VL-CL [II], where VH is a VH domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2-CH3 are antibody Fc domains, VL is a VL domain, and CL is an antibody constant light chain domain. In some embodiments, the N-terminus of the second portion is fused to the C-terminus of one of the two CH3 domains.

[0075] In some embodiments, the first portion is a first antibody heavy chain polypeptide comprising, from N-terminus to C-terminus, a structure according to formula [I]: VH-CH1-hinge-CH2-CH3 [I], An antibody light chain polypeptide comprising, from N-terminus to C-terminus, a structure according to formula [II]: VL-CL [II], and a second antibody heavy chain polypeptide comprising, from N-terminus to C-terminus, a structure according to formula [III]: Hinge-CH2-CH3 [III], wherein VH is a VH domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2-CH3 are antibody Fc domains, VL is a VL domain, and CL is an antibody constant light chain domain. In some embodiments, the N-terminus of the second portion is fused to the C-terminus of the CH3 domain of the second antibody heavy chain polypeptide. In some embodiments, the N-terminus of the second portion is fused to the C-terminus of the CH3 domain of the first antibody heavy chain polypeptide.

[0076] In some embodiments, the first portion is a first antibody heavy chain polypeptide comprising, from N-terminus to C-terminus, a structure according to formula [I]: VH-CH1-hinge-CH2-CH3 [I], An antibody light chain polypeptide comprising, from N-terminus to C-terminus, a structure according to formula [II]: VL-CL [II], and a second antibody heavy chain polypeptide comprising, from N-terminus to C-terminus, a structure according to formula [III]: Hinge-CH2-CH3 [III], wherein VH is a VH domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2-CH3 are antibody Fc domains, VL is a VL domain, and CL is an antibody constant light chain domain. In some embodiments, the C-terminus of the second portion is fused to the N-terminus of the hinge domain of a second antibody heavy chain polypeptide.

[0077] In some embodiments, the anti-CD8 antibodies of the present disclosure are multispecific (e.g., bispecific) antibodies or antibody fragments. For example, in some embodiments, a multispecific antibody (e.g., a bispecific antibody) comprises a first antigen-binding domain (e.g., as described above) that binds to CD8 and a second antigen-binding domain that binds to a target of interest. In some embodiments, a bispecific antibody can be generated by fusing additional binding sites to either the heavy or light chain of an immunoglobulin. Examples of additional binding sites include, but are not limited to, variable regions, scFvs, Fabs, VHHs, and peptides.

[0078] In some embodiments, the recombinant bispecific antibodies disclosed herein can be broadly classified into two categories: i) formats resulting from the combination of only variable regions, and ii) formats combining variable regions and an Fc domain. Representatives of the first category are tandem scFvs (taFvs), diabodies (Dbs), DARTs, single-chain diabodies (scDbs), Fab-Fcs, tandem Fabs, dual variable region Fabs, and tandem dAb / VHHs. The two variable regions can be linked together via covalent or non-covalent interactions.

[0079] Non-covalent interactions can include pairing variable regions using heterodimerization modules such as leucine zippers, dock-and-lock methods using the regulatory subunit of cAMP-dependent protein kinase (PKA) and anchor domains of A-kinase anchoring proteins (AKAPs), or knob-into-hole CH3 domains (U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001)).

[0080] In some embodiments, bispecific antibodies are generated on the natural immunoglobulin structure, which contains two pairs of heavy and light chains, each pair having a distinct binding specificity. Homodimerization of the two heavy chains in IgG is mediated by CH3 interactions. To promote heterodimer formation, genetic modifications are introduced into the two respective CH3 regions. Thus, heterodimerization mutations often involve steric repulsion, charge steering interactions, or interchain disulfide bond formation. Exemplary, non-limiting Fc modifications to promote heterodimerization include:

[0081] [Table 1-1]

[0082] [Table 1-2]

[0083] In some embodiments, bispecific antibodies can be generated by post-production assembly of half antibodies, thereby overcoming the problem of mispairing of heavy and light chains. These antibodies often contain modifications to promote heterodimerization of the half antibodies. Exemplary systems include, but are not limited to, the knob-into-hole IgG1 (EEE-RRR), IgG2 (EEE-RRRR) (Strop et al. J Mol Biol (2012)), and DuoBody (F405L-K409R) systems listed in Table 4. In such cases, the half antibodies are individually produced in separate cell lines and purified. The purified antibodies are then subjected to mild reduction to obtain the half antibodies, which are then assembled into bispecific antibodies. The heterodimeric bispecific antibodies are then purified from the mixture using conventional purification methods.

[0084] In some embodiments, strategies for generating bispecific antibodies that do not rely on preferential chain pairing can also be employed. These strategies typically involve introducing genetic modifications into antibodies in such a way that heterodimers have different biochemical or biophysical properties than homodimers, thus allowing the heterodimers to be selectively purified from homodimers after assembly or expression. One example was introducing H435R / Y436F into the CH3 domain of IgG1 to abolish Fc binding to Protein A resin, and then co-expressing the H435R / Y436F variant with wild-type Fc. The resulting homodimeric antibody containing two copies of H435R / Y436F was unable to bind to a Protein A column, while a heterodimeric antibody containing one copy of the H435R / Y436F mutation exhibited reduced affinity for Protein A compared to the strong interaction of the homodimeric wild-type antibody (Tustian et al., 2016). Other examples include kappa / lambda antibodies (Fischer et al., Nature Communication 2015) and the introduction of different charges (E357Q, S267K, or N208D / Q295E / N384D / Q418E / N421D) in each chain (US2018 / 0142040A1; (Strop et al. J Mol Biol (2012))).

[0085] In some embodiments, bispecific antibodies can be generated by fusing additional binding sites to either the heavy or light chain of an immunoglobulin, including, but not limited to, variable regions, scFv, Fab, VHH, and peptides.

[0086] Fusion protein Fc region In some embodiments, an antibody or fusion protein of the present disclosure comprises an Fc region. In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate FcγR binding and / or effector function. In some embodiments, the Fc region (e.g., an IgG1 Fc region) comprises substitutions at one or more of the following positions: C220, C226, C229, E233, L234, L235, G237, P238, S239, D265, S267, N297, L328, P331, K322, A327, and P329. In some embodiments, the Fc region (e.g., an IgG2 Fc region) comprises substitutions at one or more of the following positions: V234, G237, D265, H268, N297, V309, A330, A331, K322. In some embodiments, the Fc region (eg, an IgG4 Fc region) comprises substitutions at one or more of the following positions: L235, G237, D265, and E318. In some embodiments, the Fc region (e.g., an IgG1 Fc region) comprises one or more of the following mutations or mutations: N297A, N297Q, N297G, D265A / N297A, D265A / N297Q, C220S / C226S / C229S / P238S, S267E / L328F, C226S / C229S / E233P / L234V / L235A, L234F / L235E / P331S, L234A / L235A, L234A / L235A / G237A, L234A / L235A / G237A / K322A, L234A / L235A / G237A / A330S / A331S, L234A / L235A / P329G, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, and E233P / L234V / L235A / G236del, L234A / L235A / G237 deletions.In some embodiments, the Fc region (e.g., an IgG2 Fc region) comprises one or more of the following mutations or groups of mutations: A330S / A331S, V234A / G237A, V234A / G237A / D265A, D265A / A330S / A331S, V234A / G237A / D265A / A330S / A331S, and H268Q / V309L / A330S / A331S. In some embodiments, the Fc region (e.g., an IgG4 Fc region) comprises one or more of the following mutations or groups of mutations: L235A / G237A / E318A, D265A, L235A / G237A / D265A, and L235A / G237A / D265A / E318A. In some embodiments, the Fc region comprises one, two, three, or all of the following mutations, as numbered according to the EU index: L234A, L235A, G237A, and K322A. In some embodiments, the Fc region comprises one, two, or all of the following mutations, as numbered according to the EU index: L234A, L235A, and G237A.

[0087] In some embodiments, the first and second Fc domains of the fusion protein comprise one or more of the following Fc mutations that reduce effector function: L234A, L235A, G237A, and K322A (according to EU numbering). In some embodiments, the first and second Fc domains of the fusion protein comprise the following Fc mutations that reduce effector function: L234A, L235A, and G237A (according to EU numbering). In some embodiments, the first and second Fc domains of the fusion protein comprise the following Fc mutations that reduce effector function: L234A, L235A, G237A, and K322A (according to EU numbering). In some embodiments, the first and second Fc domains of the fusion protein comprise the following amino acid substitutions that promote heterodimer formation: Y349C / T366W (knob) and S354C, T366S, L368A, and Y407V (hole).

[0088] In some embodiments, heterodimerization and / or mutations to alter Fcγ receptor binding reduced the stability of the Fc. Therefore, additional mutation(s) were added to the Fc region to enhance its stability. For example, one or more disulfide bond pairs, such as A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C, were introduced into the Fc region. Another example is the introduction of S228P into an IgG4-based bispecific antibody to stabilize the hinge disulfide. Additional examples include the introduction of K338I, A339K, and K340S mutations to enhance Fc stability and aggregation resistance (Geo et al., 2019 Mol Pharm. 2019;16:3647).

[0089] Antibodies and antigen-binding domains In some embodiments, the fusion proteins described herein comprise an antibody, antibody fragment, or antigen-binding domain that specifically binds to human CD8b and / or human CD8ab. Any of the anti-CD8 antibodies of the present disclosure (e.g., that specifically bind to human CD8b and / or human CD8ab) can be utilized in the fusion proteins, methods, and uses disclosed herein.

[0090] In some embodiments, an anti-CD8 antibody of the disclosure specifically binds to human CD8b and / or human CD8ab with at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 200-fold greater affinity than its binding to human CD8a and / or human CD8aa, e.g., expressed on natural killer (NK) cells (e.g., human NK cells). In some embodiments, an anti-CD8 antibody of the disclosure specifically binds to human CD8b and / or human CD8ab with at least 10-fold greater affinity than its binding to human CD8a and / or human CD8aa, e.g., expressed on natural killer (NK) cells. In some embodiments, human CD8b and / or human CD8ab are expressed on the surface of human cells, e.g., human T cells.

[0091] In some embodiments, anti-CD8 antibodies of the present disclosure specifically bind to cells expressing human CD8ab heterodimers on their surface (e.g., human T cells) with an EC50 of less than 1000 nM. In some embodiments, anti-CD8 antibodies of the present disclosure specifically bind to human CD8+ T cells.

[0092] In some embodiments, an anti-CD8 antibody of the present disclosure is a human antibody or antibody fragment. In some embodiments, the human antibody or antibody fragment comprises human-derived CDR and framework sequences in the variable domain, e.g., isolated from humans or generated using a library containing human antibody sequences (e.g., CDR sequences). In some embodiments, the anti-CD8 antibody of the present disclosure is a humanized antibody or antibody fragment. In some embodiments, the humanized antibody or antibody fragment comprises non-human-derived CDRs (e.g., derived from mouse, rabbit, goat, etc.) and human-derived framework sequences in the variable domain. In some embodiments, the human antibody or humanized antibody further comprises a human Fc region. In some embodiments, the human Fc region further comprises one or more Fc mutations, e.g., as disclosed herein. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0093] There are several definitions for the CDR sequences of antibody variable domains known in the art. Unless otherwise specified, CDR sequences are described herein according to the definition of Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3). However, other definitions are known and are intended for use. For example, in some embodiments, CDR sequences can be described according to the definition of Chothia (see, for example, Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). The exact framework sequences may vary depending on the particular CDR definition used, but as is known in the art, the first framework sequence (FW-1) refers to the sequence from the N-terminus of the VH or VL domain to the start of CDR-H1 / -L1, the second framework sequence (FW-2) refers to the sequence from the end of CDR-H1 / -L1 to the start of CDR-H2 / -L2, the third framework sequence (FW-3) refers to the sequence from the end of CDR-H2 / -L2 to the start of CDR-H3 / -L3, and the fourth framework sequence (FW-4) refers to the sequence from the end of CDR-H3 / -L3 to the C-terminal boundary of the VH or VL domain.

[0094] Other numbering systems are known in the art and are suitable for use in numbering the CDRs of the VH and / or VL amino acid sequences described herein. For example, IMGT (INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM) variable region numbering is the numbering of residues in an immunoglobulin variable heavy or light chain according to the IMGT method, as described in Lefranc, M.-P., "The IMGT unique numbering for immunoglobulins, T cell receptors and Ig-like domains," The Immunologist, 7, 132-136 (1999), which is expressly incorporated herein by reference in its entirety. As used herein, "IMGT sequence numbering" or "IMGT numbering" refers to numbering sequences encoding variable regions according to IMGT. For the heavy chain variable domain, when numbered according to IMGT, the hypervariable regions span amino acids 27-38 for CDR1, 56-65 for CDR2, and 105-117 for CDR3. For the light chain variable domain, when numbered according to IMGT, the hypervariable regions span amino acids 27-38 for CDR1, 56-65 for CDR2, and 105-117 for CDR3. When the CDRs are numbered according to IMGT, heavy chain framework (FR) residues are located at approximately residues 1-26 (HC-FR1), 39-55 (HC-FR2), 66-104 (HC-FR3), and 118-128 (HC-FR4) in the heavy chain, and light chain FR residues are located at approximately residues 1-26 (LC-FR1), 39-55 (LC-FR2), 66-104 (LC-FR3), and 118-128 (LC-FR4).

[0095] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, an anti-CD8 antibody of the disclosure comprises antibody xhCD8v1 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v1 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO: 58, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO: 59.

[0096] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 177, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 178, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 179, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 180, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 181, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 182. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v8 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v8 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO: 185, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO: 186.

[0097] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 62, and / or the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 63. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v2 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v2 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO: 62, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO: 63.

[0098] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 64, and / or the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 65. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v3 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v3 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO: 64, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO: 65.

[0099] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 66, and / or the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 67. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v4 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v4 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO: 66, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO: 67.

[0100] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 68, and / or the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 69. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v5 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v5 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO: 68, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO: 69.

[0101] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 70, and / or the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 71. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v6 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v6 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is human. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO:70, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO:71.

[0102] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 72, and / or the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 73. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v7 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v7 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is human. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO: 72, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO: 73.

[0103] In some embodiments, an anti-CD8 antibody of the disclosure comprises a CDR-H1 (SEQ ID NO: 259) comprising the amino acid sequence of X1X2AIS (wherein X1 is S, K, G, N, R, D, T, or G and X2 is Y, L, H, or F), X1X2X3PX4X5X6X7X8X9YX 10 QKFX 11 G (X1 is G or H, X2 is I or F, X3 is I, N, or M, X4 is G, N, H, S, R, I, or A, X5 is A, N, H, S, T, F, or Y, X6 is A, D, or G, X7 is T, E, K, V, Q, or A, X8 is A or T, X9 is N or K, and X 10 is A or N, and X 11X1 is D or A, X2 is A, G, E, R, Y, K, N, Q, L, or F, X3 is A, L, P, or Y, X4 is I or L, X5 is R, A, Q, or S, X6 is A or D, and X7 is D, E, A, or S), and CDR-H3 (SEQ ID NO: 261) comprising the amino acid sequence of X1X2SX3X4IX5GX6LN (X1 is R or G, X2 is A or T, X3 is Q or E, X4 is E, N, T, S, A, K, D, G, R, or Q, X5 is Y or S, and X X1 is A or S, X2 is T, S, E, Q, or D, X3 is N, R, A, E, or H, X4 is Q or A, and X5 is S or D); and CDR-L3 (SEQ ID NO: 264) comprising the amino acid sequence of QX1X2X3X4X5PWT (X1 is S, N, D, Q, A, or E, X2 is T, I, or S, X3 is Y, L, or F, X4 is D, G, T, E, Q, A, or Y, and X5 is A, T, R, S, K, or Y). In some embodiments, the VH domain further comprises an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 274), an FW-2 comprising the sequence WVRQAPGQGLEWMG (SEQ ID NO: 275), an FW-3 comprising the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 276), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277).In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0104] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 225, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 226, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 227, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 245, and / or the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 246. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v9 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v9 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO:245, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO:246. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 274), an FW-2 comprising the sequence WVRQAPGQGLEWMG (SEQ ID NO: 275), an FW-3 comprising the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 276), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277).In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0105] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 225, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 232, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 234, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 235, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 236. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 251, and / or the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 252. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 251, and the VL domain comprises the amino acid sequence of SEQ ID NO: 252. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v12 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v12 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO: 251, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO: 252. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 274), an FW-2 comprising the sequence WVRQAPGQGLEWMG (SEQ ID NO: 275), an FW-3 comprising the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 276), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277).In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0106] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 225, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 232, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 253, and / or the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 254. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v13 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v13 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO: 253, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO: 254. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 274), an FW-2 comprising the sequence WVRQAPGQGLEWMG (SEQ ID NO: 275), an FW-3 comprising the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 276), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277).In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0107] In some embodiments, an anti-CD8 antibody of the disclosure comprises a CDR-H1 (SEQ ID NO: 268) comprising the amino acid sequence of X1YX2MS (X1 is S, D, E, A, or Q, and X2 is A, G, or T); a CDR-H2 (SEQ ID NO: 269) comprising the amino acid sequence of DIX1X2X3GX4X5TX6YADSVKG (X1 is T, N, S, Q, E, H, R, or A, X2 is Y, W, F, or H, X3 is A, S, Q, E, or T, X4 is G or E, X5 is S or I, and X6 is A or G); and a CDR-H3 (SEQ ID NO: 269) comprising the amino acid sequence of X1X2X3YX4WX5X6AX7DX8 (X1 is S or X2 is N, H, A, D, L, Q, Y, or R, X3 is A, N, S, or G, X4 is A, V, R, E, or S, X5 is D or S, X6 is D, N, Q, E, S, T, or L, X7 is L, F, or M, and X8 is I, Y, or V), and a VL domain comprising CDR-L1 (SEQ ID NO: 40) comprising the amino acid sequence of RASQSVSSNLA, CDR-L2 (SEQ ID NO: 41) comprising the amino acid sequence of GASSRAT, and CDR-L3 (SEQ ID NO: 42) comprising the amino acid sequence of QQYGSSPPVT. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:281), an FW-2 comprising the sequence WVRQAPGKGLEWVS (SEQ ID NO:282), an FW-3 comprising the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:283), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO:284) or WGQGTLVTVSS (SEQ ID NO:285). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO:293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO:294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO:296).

[0108] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 229, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 230, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 231, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 247, and / or the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 248. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v10 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v10 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO:247, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO:248. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 281), an FW-2 comprising the sequence WVRQAPGKGLEWVS (SEQ ID NO: 282), an FW-3 comprising the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 283), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285).In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0109] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 229, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 230, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 231, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 249, and / or the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 250. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 249, and the VL domain comprises the amino acid sequence of SEQ ID NO: 250. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v11 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v11 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO: 249, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO: 250. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 281), an FW-2 comprising the sequence WVRQAPGKGLEWVS (SEQ ID NO: 282), an FW-3 comprising the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 283), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285).In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0110] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 229, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 237, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 231, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 255, and / or the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 256. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v14 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v14 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO:255, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO:256. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 281), an FW-2 comprising the sequence WVRQAPGKGLEWVS (SEQ ID NO: 282), an FW-3 comprising the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 283), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285).In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0111] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 229, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 237, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 231, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 257, and / or the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 258. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v15 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v15 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 derived from the sequence of SEQ ID NO:257, and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 derived from the sequence of SEQ ID NO:258. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 281), an FW-2 comprising the sequence WVRQAPGKGLEWVS (SEQ ID NO: 282), an FW-3 comprising the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 283), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285).In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0112] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 54, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 36.In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 183, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 184, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 179, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 180, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 181, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 182.

[0113] In some embodiments, an anti-CD8 antibody of the disclosure comprises a CDR-H1 (SEQ ID NO: 265) comprising the amino acid sequence of GX1X2FX3X4X5 (wherein X1 is G, Y, S, or A; X2 is T, S, G, R, N, or H; X3 is S, T, R, H, Y, G, or P; X4 is S, K, G, N, R, D, T, or G; and X5 is Y, L, H, or F); X1 is D or A, X2 is A, G, E, R, Y, K, N, Q, L, or F, X3 is A, L, P, or Y, X4 is I or L, X5 is R, A, Q, or S, X6 is A or D, and X7 is D, E, A, or X1 is R or G, X2 is A or T, X3 is Q or E, X4 is E, N, T, S, A, K, D, G, R, or Q, X5 is Y or S, and X6 is A or V), CDR-L1 (SEQ ID NO: 262) comprising the amino acid sequence of GX1X2X3LX4X5 (X1 is A or S, X2 is T, S, E, Q, or D, and X3 is , N, R, A, E, or H, X4 is Q or A, and X5 is S or D), and CDR-L3 (SEQ ID NO: 264) having the amino acid sequence of QX1X2X3X4X5PWT (X1 is S, N, D, Q, A, or E, X2 is T, I, or S, X3 is Y, L, or F, X4 is D, G, T, E, Q, A, or Y, and X5 is A, T, R, S, K, or Y).In some embodiments, the VH domain further comprises FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO:278), FW-2 comprising the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO:279), FW-3 comprising the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:280), and / or FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO:277). In some embodiments, the VL domain further comprises FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:289), FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO:290), FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:291), and / or FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO:292).

[0114] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 238, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 239, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO: 278), an FW-2 comprising the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO: 279), an FW-3 comprising the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 280), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0115] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 238, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 234, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 235, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 236. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO: 278), an FW-2 comprising the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO: 279), an FW-3 comprising the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 280), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0116] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 238, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO: 278), an FW-2 comprising the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO: 279), an FW-3 comprising the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 280), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0117] In some embodiments, an anti-CD8 antibody of the disclosure comprises a CDR-H1 (SEQ ID NO: 271) comprising the amino acid sequence of GFTFX1X2Y (X1 is S, D, E, Q, S, or A, and X2 is S, D, E, A, or Q); a CDR-H2 (SEQ ID NO: 272) comprising the amino acid sequence of X1X2X3GX4X5 (X1 is T, N, S, Q, E, H, R, or A, X2 is Y, W, F, or H, X3 is A, S, Q, E, or T, X4 is G or E, and X5 is S or I); and a CDR-H3 (SEQ ID NO: 273) comprising the amino acid sequence of X1X2X3YX4WX5X6AX7DX8 (X1 is S or A, and X2 is , N, H, A, D, L, Q, Y, or R, X3 is A, N, S, or G, X4 is A, V, R, E, or S, X5 is D or S, X6 is D, N, Q, E, S, T, or L, X7 is L, F, or M, and X8 is I, Y, or V), and a VL domain comprising CDR-L1 (SEQ ID NO: 40) comprising the amino acid sequence of RASQSVSSNLA, CDR-L2 (SEQ ID NO: 41) comprising the amino acid sequence of GASSRAT, and CDR-L3 (SEQ ID NO: 42) comprising the amino acid sequence of QQYGSSPPVT. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:286), an FW-2 comprising the sequence AMSWVRQAPGKGLEWVSDI (SEQ ID NO:287), an FW-3 comprising the sequence TAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:288), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO:284) or WGQGTLVTVSS (SEQ ID NO:285). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO:293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO:294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO:296).

[0118] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 240, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 241, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 242, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 286), an FW-2 comprising the sequence AMSWVRQAPGKGLEWVSDI (SEQ ID NO: 287), an FW-3 comprising the sequence TAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 288), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0119] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 240, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 244, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 242, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 286), an FW-2 comprising the sequence AMSWVRQAPGKGLEWVSDI (SEQ ID NO: 287), an FW-3 comprising the sequence TAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 288), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO: 296). In some embodiments, the disclosure provides an anti-CD8 antibody comprising a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of a single antibody listed in Table 1, and a VL domain comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of a single antibody listed in Table 1. For example, the anti-CD8 antibody comprises the six CDRs of antibody xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​xhCD8v15, the V9 family, or the V11 family, as shown in Table 1.In some embodiments, the present disclosure provides an anti-CD8 antibody comprising a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequence of a single antibody listed in Table 2, and a VL domain comprising the CDR-L1, CDR-L2, and CDR-L3 sequence of a single antibody listed in Table 2. For example, the anti-CD8 antibody comprises the six CDRs of antibody xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​xhCD8v15, V9 family, or V11 family, as shown in Table 2. In some embodiments, the disclosure provides fusion proteins comprising an anti-CD8 antibody comprising a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequence of a single antibody listed in Table 1, and a VL domain comprising the CDR-L1, CDR-L2, and CDR-L3 sequence of a single antibody listed in Table 1. For example, the anti-CD8 antibody of the fusion protein comprises the six CDRs of antibody xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​xhCD8v15, V9 family, or V11 family, as shown in Table 1. In some embodiments, the present disclosure provides fusion proteins comprising an anti-CD8 antibody comprising a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequence of a single antibody listed in Table 2, and a VL domain comprising the CDR-L1, CDR-L2, and CDR-L3 sequence of a single antibody listed in Table 2. For example, the anti-CD8 antibody of the fusion protein comprises the six CDRs of antibody xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​xhCD8v15, V9 family, or V11 family, as shown in Table 2.In some embodiments, the disclosure provides an anti-CD8 antibody comprising a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of the VH domain listed in Table 3, and a VL domain comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of the VL domain listed in Table 3 (in some embodiments, the VH and VL domains are derived from the same single antibody listed in Table 3). For example, the anti-CD8 antibody comprises the VH and VL of antibody xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​or xhCD8v15 shown in Table 3. In some embodiments, the disclosure provides fusion proteins comprising an anti-CD8 antibody comprising a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of the VH domain listed in Table 3, and a VL domain comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of the VL domain listed in Table 3 (in some embodiments, the VH and VL domains are derived from the same single antibody listed in Table 3). In some embodiments, the disclosure provides anti-CD8 antibodies comprising the VH domain sequence and VL domain sequence of a single antibody listed in Table 3. In some embodiments, the disclosure provides fusion proteins comprising an anti-CD8 antibody comprising the VH domain sequence and VL domain sequence of a single antibody listed in Table 3. For example, the anti-CD8 antibody of the fusion protein comprises the VH and VL of antibody xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​or xhCD8v15 shown in Table 3.

[0120] [Table 2-1]

[0121] [Table 2-2]

[0122] [Table 2-3]

[0123] [Table 2-4]

[0124] [Table 3-1]

[0125] [Table 3-2]

[0126] [Table 3-3]

[0127] [Table 3-4]

[0128] [Table 4-1]

[0129] [Table 4-2]

[0130] [Table 4-3]

[0131] Fusion Protein Cytokines In some embodiments, the second portion induces activation of CD8+ T cells. In some embodiments, the second portion comprises a polypeptide that induces signaling through IL-2Rβγ. In some embodiments, the second portion comprises a polypeptide that induces signaling through IL-21R. In some embodiments, the second portion comprises an IL-21 polypeptide (e.g., a human IL-21 polypeptide or a derivative thereof). In some embodiments, the second portion comprises an IL-2 polypeptide (e.g., a human IL-2 polypeptide or a derivative thereof).

[0132] IL-2 polypeptide In some embodiments, the second portion comprises an IL-2 polypeptide (eg, a human IL-2 polypeptide or a derivative thereof).

[0133] In some embodiments, the IL-2 polypeptide has a binding affinity for IL-2Rα that is 50% or more less than the binding affinity for IL-2Rα of a wild-type IL-2 polypeptide comprising the sequence of SEQ ID NO: 81. In some embodiments, "wild-type IL-2" and "human IL-2" are used interchangeably and refer to a polypeptide having the amino acid sequence of SEQ ID NO: 81. In some embodiments, the IL-2 polypeptide has a binding affinity for IL-2Rβ that is 50% or more less than the binding affinity for IL-2Rβ of a wild-type IL-2 polypeptide comprising the sequence of SEQ ID NO: 81. In some embodiments, the IL-2 polypeptide has a binding affinity for IL-2Rγ that is 50% or more less than the binding affinity for IL-2Rγ of a wild-type IL-2 polypeptide comprising the sequence of SEQ ID NO: 81. The difference in binding affinity between wild-type and disclosed polypeptides for IL-2Rα and IL-2Rβ can be measured, for example, in standard surface plasmon resonance (SPR) assays that measure the affinity of protein-protein interactions, as are well known to those of skill in the art. Because the affinity of wild-type IL-2 polypeptides for IL-2Rγ is so low, differences in binding affinity between wild-type and disclosed polypeptides for IL-2Rγ cannot be reliably measured by SPR assays. Instead, reduced affinity for IL-2Rγ can be inferred by measuring pSTAT5 and performing in vitro assays that compare the activity of IL-2 polypeptides with and without substitutions that reduce IL-2Rγ affinity on IL-2R-expressing cells. Exemplary sequences for IL-2Rα, IL-2Rβ, and IL-2Rγ are shown below.

[0134] IL-2Rα:

[0135] MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI (SEQ ID NO: 82)

[0136] IL-2Rβ:

[0137] MAAPALSWRLPLLILLLPLATSWASAAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 83)

[0138] IL-2Rγ:

[0139] MLKPSLPFTSLLFLQLPLLGVGLNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET (SEQ ID NO: 84)

[0140] In some embodiments, the IL-2 polypeptide is an IL-2 polypeptide that comprises one or more mutations compared to a human IL-2 polypeptide comprising the sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 81). In some embodiments, the IL-2 polypeptide of the present disclosure has one or more, two or more, or three or more affinity-reducing amino acid substitutions compared to, for example, a wild-type mature IL-2 polypeptide having the amino acid sequence of SEQ ID NO: 81. In some embodiments, the one or more, two or more, or three or more substituted residues are selected from the group consisting of Q11, H16, L18, L19, D20, D84, S87, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, 192, T123, Q126, S127, 1129, and S130. A reduced affinity for IL-2Rα can be obtained by substituting one or more of the following residues in the sequence of the wild-type mature IL-2 polypeptide: R38, F42, K43, Y45, E62, P65, E68, V69, and L72. Reduced affinity for IL-2Rβ can be obtained by substituting one or more of the following residues: E15, H16, L19, D20, D84, S87, N88, V91, and 192. Reduced affinity for IL-2Rγ can be obtained by substituting one or more of the following residues in the sequence of the wild-type mature IL-2 polypeptide: Q11, L18, Q22, T123, Q126, S127, 1129, and S130.

[0141] In some embodiments, the IL-2 polypeptide comprises an F42A or F42K amino acid substitution compared to the wild-type mature IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide comprises an F42A or F42K amino acid substitution and an R38A, R38D, R38E, E62Q, E68A, E68Q, E68K, or E68R amino acid substitution compared to the wild-type mature IL-2 amino acid sequence. For example, in some embodiments, the IL-2 polypeptide comprises F42A, R38A and F42A, R38D and F42A, R38E and F42A, F42A and E62Q, F42A and E68A, F42A and E68Q, F42A and E68K, F42A and E68R, or R38A and F42K amino acid substitution(s) compared to the wild-type mature IL-2 amino acid sequence set forth, e.g., in SEQ ID NO:81. In some embodiments, the IL-2 polypeptide comprises an R38E and an F42A amino acid substitution relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide comprises an R38D and an F42A amino acid substitution relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide comprises an F42A and an E62Q amino acid substitution relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide comprises an R38A and an F42K amino acid substitution relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide comprises an R38D and an F42A amino acid substitution relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide comprises an R38A and an F42K amino acid substitution relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide comprises an F42A and an E62Q amino acid substitution relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide has any of the following amino acids compared to the wild-type IL-2 amino acid sequence: H16E, H16D, D20N, M23A, M23R, M23K, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, S87K, S87A, N88A, N88D, N88G, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, T123A, T123B, T123C, T123D, T123E, T123F, T123G, T123H, T123I ...In some embodiments, the IL-2 polypeptide comprises a T123E, T123K, T123Q, Q126S, Q126T, Q126E, S127A, S127E, S127K, or S127Q amino acid substitution(s) compared to the wild-type mature IL-2 amino acid sequence, including F42A, R38A and F42A, R38D and F42A, R38E and F42A, F42A and E62Q, F42A and E68A, F42A and E68Q, F42A and E68K, F42A and E68R, or R38A and F42K amino acid substitution(s) compared to the wild-type mature IL-2 amino acid sequence (e.g., as set forth in SEQ ID NO: 81). and including a 0N, M23A, M23R, M23K, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, S87K, S87A, N88A, N88D, N88G, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, T123A, T123E, T123K, T123Q, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K, or S127Q amino acid substitution. For example, in some embodiments, an IL-2 polypeptide comprises R38E, F42A, and H16E amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, an IL-2 polypeptide comprises R38E, F42A, and H16D amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, an IL-2 polypeptide comprises R38E, F42A, and D84K amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, an IL-2 polypeptide comprises R38E, F42A, and D84R amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, an IL-2 polypeptide comprises R38E, F42A, and N88S amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, an IL-2 polypeptide comprises R38E, F42A, and N88A amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide comprises R38E, F42A, and N88G amino acid substitutions relative to the wild-type IL-2 amino acid sequence.The IL-2 polypeptide comprises R38E, F42A, and N88R amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide comprises R38E, F42A, and N88T amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide comprises R38E, F42A, and N88D amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide comprises R38E, F42A, and V91E amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide comprises R38E, F42A, and Q126S amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 81, which comprises one of the following sets of amino acid substitutions (relative to the sequence of SEQ ID NO: 81): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A, and N88S; R38E, F42A, and N88A; R38E, F42A , and N88G;R38E, F42A, and N88R;R38E, F42A, and N88T;R38E, F42A, and N88D;R38E, F42A, and V91E;R38E, F42A, and D84H;R38E, F42A, and D84K;R38E, F42A, and D84R;H16D, R38E, and F42A;H16E, R38E, and F42A;R38E, F42 A and Q126S; R38D, F42A and N88S; R38D, F42A and N88A; R38D, F42A and N88G; R38D, F42A and N88R; R38D, F42A and N88T; R38D, F42A and N88D; R38D, F42A and V91E; R38D, F42A, and D84H; R38D, F42A, and D84K; R38D, F42A, and and D84R; H16D, R38D and F42A; H16E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S; R38A, F42K, and N88A; R38A, F42K, and N88G; R38A, F42K, and N88R; R38A, F42K, and N88T; R38A, F42K, and N88D; R38A, F42K,and V91E;R38A, F42K, and D84H;R38A, F42K, and D84K;R38A, F42K, and D84R;H16D, R38A, and F42K;H16E, R38A, and F42K;R38A, F42K, and Q126S;F42A, E62Q, and N88S;F42A, E62Q, and N88A;F42A, E62Q, and N88G;F42A, E62Q, and N88R;F42A, E62Q, and N88T;F42A, E62Q, and N88D;F42A, E62Q, and V91E;F42A, E62Q, and D84H;F4 2A, E62Q, and D84K; F42A, E62Q, and D84R; H16D, F42A, and E62Q; H16E, F42A, and E62Q; F42A, E62Q, and Q126S; R38E, F42A, and C125A; R38D, F42A, and C125A; F42A, E62Q, and C125A; R38A, F42K, and C125A; R38E, F42A, N88S, and C125A; R38E, F42A, N88A, and C125A; R38E, F42A, N88G, and C125A; R38E, F42A, N88R, and C125A; R 38E, F42A, N88T, and C125A; R38E, F42A, N88D, and C125A; R38E, F42A, V91E, and C125A; R38E, F42A, D84H, and C125A; R38E, F42A, D84K, and C125A; R38E, F42A, D84R, and C125A; H16D, R38E, F42A, and C125A; H16E, R38E, F42A, and C125A; R38E, F42A, C125A, and Q126S; R38D, F42A, N88S, and C125A; R38D, F42A, N88A, and C12 5A;R38D, F42A, N88G, and C125A;R38D, F42A, N88R, and C125A;R38D, F42A, N88T, and C125A;R38D, F42A, N88D, and C125A;R38D, F42A, V91E, and C125A;R38D, F42A, D84H, and C125A;R38D, F42A, D84K, and C125A;R38D, F42A, D84R, and C125A;H16D, R38D, F42A, and C125A;H16E, R38D, F42A, and C125A;R38D, F42A, C125A,and Q126S; R38A, F42K, N88S, and C125A; R38A, F42K, N88A, and C125A; R38A, F42K, N88G, and C125A; R38A, F42K, N88R, and C125A; R38A, F42K, N88T, and C125A; R38A, F42K, N88D, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H, and C125A; R38A, F42K, D84K, and C12 5A;R38A, F42K, D84R, and C125A;H16D, R38A, F42K, and C125A;H16E, R38A, F42K, and C125A;R38A, F42K, C125A, and Q126S;F42A, E62Q, N88S, and C125A;F42A, E62Q, N88A, and C125A;F42A, E62Q, N88G, and C125A;F42A, E62Q, N88R, and C125A;F42A, E62Q, N88T, and C125A;F42 A, E62Q, N88D, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A; F42A, E62Q, and D84K, and C125A; F42A, E62Q, and D84R, and C125A; H16D, F42A, and E62Q, and C125A; H16E, F42A, E62Q, and C125A; F42A, E62Q, C125A, and Q126S; F42A, N88S, and C125A; F42A, N88A and C125A; F42A, N88G, and C125A; F42A, N88R, and C125A; F42A, N88T, and C125A; F42A, N88D, and C125A; F42A, V91E, and C125A; F42A, D84H, and C125A; F42A, D84K, and C125A; F42A, D84R, and C125A; H16D, F42A, and C125A; H16E, F42A, and C125A; and F42A, C125A and Q126S. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 having one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 81, wherein the one, two, three, four, or five substitution(s) are selected from the group consisting of Q11, H16, L18, L19, D20, Q22, R38, F42, K43, Q50, Q60, Q70, Q80, Q90, Q11, Q22, R38, F42, K43, Q50, Q62, Q74, Q76, Q78, Q79, Q81, Q92, Q93, Q94, Q95, Q96, Q97, Q98, Q99, Q100, Q101, Q102, Q103, Q104, Q105, Q106, Q107, Q108, Q110, Q111, Q112, Q113, Q114, Q115, Q116, Q118, Q119, Q120, Q121, Q122, Q123, Q124, Q125, Q126, Q127, Q128, Q129, Q130, Q131, Q132, Q133, Q134, Q135, Q136, Q137, Q138, Q139, Q140, Q141, Q142, Q143, Q144, Q145, Q146, Q147, Q148, Q149, Q150, Q151, Q152, Q153, Q154, Q155, Q156, Q157, Q158, Q159, Q159, Q160, Q161In some embodiments, the IL-2 polypeptide comprises a substitution(s) at a position selected from the group consisting of Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, 192, T123, Q126, S127, 1129, and S130. In some embodiments, the IL-2 polypeptide comprises the following amino acid substitutions (sequence of SEQ ID NO: 81): Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, 192, T123, Q126, S127, 1129, and S130. and N88R; R38E, F42A, and N88T; R38E, F42A, and N88D; R38E, F42A, and V91E; R38E, F42A, and D84H; R38E, F42A, and D84H. 84K;R38E, F42A, and D84R;H16D, R38E, and F42A;H16E, R38E, and F42A;R38E, F42A, and Q126S;R38D, F42A, and N88S;R38D, F42A, and N88A;R38D, F42A, and N88G;R38D, F42A, and N88R;R38D, F42A, and N88T;R38D, F42A, and N88D;R38D, F42A, and V91E;R38D, F42A, and D84H;R38D, F42A, and D84K;R38D, F42A, and D84R; H16D, R38D and F42A; H16E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S; R38A, F42K, and N88A; R38A, F42K, and N88G; R38A, F42K, and N88R; R38A, F42K, and N88T; R38A, F42K, and N88D; R38A, F42K, and V91E; R38A, F42K, and D84H; R38A, F42K, and D84K; R38A, F42K, and D84R; H16D, R38A, and F42K; H16E, R38A, and F42K; R38A, F42K, and Q126S; F42A, E62Q, and N88S; F42A, E62Q, and N88A; F42A, E62Q, and N88G; F42A, E62Q, and N88R; F42A, E62Q, and N88T; F42A, E62Q, and N88D; F42A, E62Q, and V91E; F42A, E62Q, and D84H; F42A, E62Q, and D84K; and F42A, E62Q, and D84R. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, and N88S.In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, and N88A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, and N88G. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, and N88R. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, and N88T. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, and N88D. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, and V91E. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, and D84H. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, and D84K. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, and D84R. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: H16D, R38D, and F42A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: H16E, R38D, and F42A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, and Q126S.In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, and N88S. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, and N88A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, and N88G. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, and N88R. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, and N88T. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, and N88D. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, and V91E. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, and D84H. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, and D84K. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, and D84R. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: H16D, R38A, and F42K. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: H16E, R38A, and F42K.In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, and Q126S. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, and N88S. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, and N88A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, and N88G. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, and N88R. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, and N88T. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, and N88D. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, and V91E. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, and D84H. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, and D84K. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, and D84R.

[0142] In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81, which has an additional amino acid substitution at position C125 compared to SEQ ID NO: 81. In some aspects, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with one of the following sets of amino acid substitutions (relative to the sequence of SEQ ID NO: 81): R38E, F42A, and C125A; R38D, F42A, and C125A; F42A, E62Q, and C125A; R38A, F42K, and C125A; R38E, F42A, N88S, and C125A; R38E, F42A, N88A, and C125A; R38E, F42A, N88G, and C125A; R38E, F42A, N88R, and and C125A; R38E, F42A, N88D, and C125A; R38E, F42A, N88T, and C125A; R38E, F42A, V91E, and C125A; R38E, F42A, D84H, and C125A; R38E, F42A, D84K, and C125A; R38E, F42A, D84R, and C125A; H16D, R38E, F42A, and C125A; H16E, R38E, F42A, and C125A; R38E, F42A, C125A and Q126S; R38D, F42A, N88S, and and C125A; R38D, F42A, N88A, and C125A; R38D, F42A, N88G, and C125A; R38D, F42A, N88R, and C125A; R38D, F42A, N88T, and C125A; R38D, F42A, N88D, and C125A; R38D, F42A, V91E, and C125A; R38D, F42A, D84H, and C125A; R38D, F42A, D84K, and C125A; R38D, F42A, D84R, and C125A; H16D, R38D, F42A, and and C125A; H16E, R38D, F42A, and C125A; R38D, F42A, C125A, and Q126S; R38A, F42K, N88S, and C125A; R38A, F42K, N88G, and C125A; R38A, F42K, N88R, and C125A; R38A, F42K, N88T, and C125A; R38A, F42K, N88D, and C125A; R38A, F42K, N88A, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H,and C125A; R38A, F42K, D84K, and C125A; R38A, F42K, D84R, and C125A; H16D, R38A, F42K, and C125A; H16E, R38A, F42K, and C125A; R38A, F42K, C125A and Q126S; F42A, E62Q, N88S, and C125A; F42A, E62Q, N88A, and C125 A; F42A, E62Q, N88G, and C125A; F42A, E62Q, N88R, and C125A; F42A, E62Q, N88T, and C125A; F42A, E62Q, N88D, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A; F42A, E62Q, and D84K, and C125A; F42A, E62Q, and D84R, and C125A; H16D, F42A, and E62Q, and C125A; H16E, F42A, E62Q, and C125A; F42A, E62Q, C125A, and Q126S; F42A, N88S, and C125A; F42A, N88A, and C125A; F42A, N88G, and C125A; F42A, N88R, and C125A In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 having the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38E, F42A, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 having the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38E, F42A, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 having the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A,In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38E, F42A, N88S, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38E, F42A, N88A, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38E, F42A, N88G, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38E, F42A, N88R, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38E, F42A, N88D, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38E, F42A, N88T, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38E, F42A, V91E, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38E, F42A, D84H, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38E, F42A, D84K, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38E, F42A, D84R, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: H16D, R38E,In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: H16E, R38E, F42A, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38E, F42A, C125A, and Q126S. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, N88S, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, N88A, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, N88G, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, N88R, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, N88T, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, N88D, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, V91E, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, D84H, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D,In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, D84R, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: H16D, R38D, F42A, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: H16E, R38D, F42A, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38D, F42A, C125A, and Q126S. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, N88S, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, N88G, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, N88R, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, N88T, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, N88D, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, N88A ...In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions: R38A, F42K, V91E, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, D84H, and C125A. In some embodiments, the IL-2 polypeptide comprises In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, D84K, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, D84R, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: H16D, R38A, F42K, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: H16E, R38A, F42K, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: R38A, F42K, C125A, and Q126S. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, N88S, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, N88A, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, N88G, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, N88R, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, N88T, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, N88D, and C125A.In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, V91E, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, and D84H, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, and D84K, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, and D84R, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: H16D, F42A, and E62Q, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: H16E, F42A, E62Q, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, E62Q, C125A, and Q126S. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, N88S, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, N88A, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, N88G, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, N88R, and C125A.In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, N88T, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, N88D, and C125A; F42A, V91E, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, D84H, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, D84K, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, D84R, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: H16D, F42A, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: H16E, F42A, and C125A. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with the following set of amino acid substitutions relative to the sequence of SEQ ID NO: 81: F42A, C125A, and Q126S.

[0143] In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 80). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 85). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 86). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 87). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 88).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 89). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 90). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 91). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 92). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 93).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 94). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 95). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 96). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 97). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 98).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 99). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 100). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 101). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 102). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 103).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 104). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 105). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 106). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 107). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 108).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 109). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 110). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 111). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 112). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 113). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 114). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 115). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 116). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 117).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 118). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 119). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 120). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 121). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 122).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 123). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 124). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 125). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 126). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 127).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 128). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 129). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 130). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 131). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 132).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 133). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 134). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 135). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 136). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 137).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 138). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 139). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 140). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 141). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 142). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 143). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 144). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 145). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 146).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 147). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 148). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 149). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 150). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 151).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 152). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 153). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 154). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 155). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 190).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 191). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 192). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 193). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 194). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 195).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 196). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 197). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 198). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 199). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 200).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 201). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 202). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 203). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTL. In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 205). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 206). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 207). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 208). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 209).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 210). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 211). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 212). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 213). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 214).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 215). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 216). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 297). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 354). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 355).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 356). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 357). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 358). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 359). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 360).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 361). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 362). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 363). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 364). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 365).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 366). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 367). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 368). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNR. In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 369). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 370). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 371). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 372). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 373). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 374).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 375). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 376). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 377). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 378). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 379).In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 380). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 381). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 382). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 383). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of an IL-2 polypeptide listed in Table 6.

[0144] In some embodiments, the IL-2 polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 80, 85-155, 190-216, 297, or 354-383. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence having at least 80% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 80, 85-155, 190-216, 297, or 354-383. In some embodiments, an IL-2 polypeptide comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 80, 85-155, 190-216, 297, or 354-383. In some embodiments, an IL-2 polypeptide comprises an amino acid sequence having at least 90% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 80, 85-155, 190-216, 297, or 354-383. In some embodiments, an IL-2 polypeptide comprises an amino acid sequence having at least 95% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 80, 85-155, 190-216, 297, or 354-383. In some embodiments, an IL-2 polypeptide comprises an amino acid sequence having at least 96% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 80, 85-155, 190-216, 297, or 354-383. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence having at least 97% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 80, 85-155, 190-216, 297, or 354-383. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence having at least 98% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 80, 85-155, 190-216, 297, or 354-383.In some embodiments, the IL-2 polypeptide comprises an amino acid sequence having at least 99% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 80, 85-155, 190-216, 297, or 354-383. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence having 100% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 80, 85-155, 190-216, 297, or 354-383.

[0145] [Table 5]

[0146] In some embodiments, the IL-2 polypeptides of the present disclosure also contain other modifications, including, but not limited to, mutations and deletions, that provide additional advantages, such as improved biophysical properties. Improved biophysical properties include, but are not limited to, improved thermostability, aggregation tendency, acid reversibility, viscosity, and production in mammalian, bacterial, or yeast cells. For example, residue C125 may be replaced with a neutral amino acid, such as serine, alanine, threonine, or valine, or the N-terminal A1 residue may be deleted, all of which are described in U.S. Patent No. 4,518,584. IL-2 polypeptides may also contain a mutation at residue M104, e.g., M104A, as described in U.S. Patent No. 5,206,344. Thus, in certain embodiments, the IL-2 polypeptides of the present disclosure contain the amino acid substitution C125A. In other embodiments, one, two, or three N-terminal residues are deleted.

[0147] Exemplary IL-2 Fusion Proteins In some embodiments, a fusion protein of the disclosure comprises one, two, or all three polypeptides shown for a single fusion protein in Table 5. In some embodiments, a fusion protein of the disclosure comprises two light chains, a heavy chain that comprises an IL-2 fusion, and a heavy chain that does not comprise an IL-2 fusion for a single fusion protein in Table 5. As is known in the art, the C-terminal lysine of some antibody heavy chain species may be cleaved in some portions of the molecule. Thus, in some embodiments, a fusion protein of the disclosure comprises, for a single fusion protein of Table 5, two light chains, a heavy chain that comprises an IL-2 fusion, and a heavy chain that does not comprise an IL-2 fusion, wherein the heavy chain that does not comprise the IL-2 fusion comprises the sequence of SEQ ID NO: 158, 161, 164, 167, 170, 173, 176, 189, 300, 304, 308, 312, 326, 320, 324, 328, 332, 336, 340, 344, 348, or 352. In some embodiments, a fusion protein of the disclosure comprises, for a single fusion protein of Table 5, two light chains, a heavy chain that comprises an IL-2 fusion, and a heavy chain that does not comprise an IL-2 fusion, wherein the heavy chain that does not comprise the IL-2 fusion comprises the sequence of SEQ ID NO: 217-224, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, or 353.In some embodiments, the disclosure provides a plurality of fusion proteins of the disclosure (e.g., in a mixture), wherein each fusion protein of the plurality comprises, for a single fusion protein of Table 5, two light chains, a heavy chain comprising an IL-2 fusion, and a heavy chain without an IL-2 fusion, wherein the heavy chain without an IL-2 fusion comprises the sequence of SEQ ID NO: 158, 161, 164, 167, 170, 173, 176, 189, 300, 304, 308, 312, 326, 320, 324, 328, 332, 336, 340, 344, 348, or 352 for each fusion protein, or the sequence of SEQ ID NO: 217-224, 301, 305, 309, 313, 317, 318, 329, 336, 340, 344, 348, or 352 for each fusion protein. 21, 325, 329, 333, 337, 341, 345, 349, or 353, or a plurality thereof includes a mixture of species representing truncated (e.g., comprising the sequence of SEQ ID NO: 158, 161, 164, 167, 170, 173, 176, 189, 300, 304, 308, 312, 326, 320, 324, 328, 332, 336, 340, 344, 348, or 352 for each fusion protein) and uncleaved (e.g., comprising the sequence of SEQ ID NO: 217-224, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, or 353 for each fusion protein) species.

[0148] For example, in some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 156, a heavy chain comprising the amino acid sequence of SEQ ID NO: 157, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 158. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 159, a heavy chain comprising the amino acid sequence of SEQ ID NO: 160, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 161. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 162, a heavy chain comprising the amino acid sequence of SEQ ID NO: 163, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 164. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 165, a heavy chain comprising the amino acid sequence of SEQ ID NO: 166, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 167. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 168, a heavy chain comprising the amino acid sequence of SEQ ID NO: 169, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 170. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 171, a heavy chain comprising the amino acid sequence of SEQ ID NO: 172, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 173. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 174, a heavy chain comprising the amino acid sequence of SEQ ID NO: 175, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 176. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 187, a heavy chain comprising the amino acid sequence of SEQ ID NO: 188, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 189. In some embodiments, a fusion protein of the disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 298, a heavy chain comprising the amino acid sequence of SEQ ID NO: 299, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 300. In some embodiments, a fusion protein of the disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 302, a heavy chain comprising the amino acid sequence of SEQ ID NO: 303, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 304.In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 306, a heavy chain comprising the amino acid sequence of SEQ ID NO: 307, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 308. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 310, a heavy chain comprising the amino acid sequence of SEQ ID NO: 311, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 312. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 314, a heavy chain comprising the amino acid sequence of SEQ ID NO: 315, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 316. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 318, a heavy chain comprising the amino acid sequence of SEQ ID NO: 319, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 320. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 322, a heavy chain comprising the amino acid sequence of SEQ ID NO: 323, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 324. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 326, a heavy chain comprising the amino acid sequence of SEQ ID NO: 327, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 328. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 334, a heavy chain comprising the amino acid sequence of SEQ ID NO: 335, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 336. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 338, a heavy chain comprising the amino acid sequence of SEQ ID NO: 339, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 340. In some embodiments, a fusion protein of the disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 342, a heavy chain comprising the amino acid sequence of SEQ ID NO: 343, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 344. In some embodiments, a fusion protein of the disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 346, a heavy chain comprising the amino acid sequence of SEQ ID NO: 347, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 348.In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 350, a heavy chain comprising the amino acid sequence of SEQ ID NO: 351, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 352.

[0149] In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 156, a heavy chain comprising the amino acid sequence of SEQ ID NO: 157, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 217. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 159, a heavy chain comprising the amino acid sequence of SEQ ID NO: 160, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 218. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 162, a heavy chain comprising the amino acid sequence of SEQ ID NO: 163, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 219. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 165, a heavy chain comprising the amino acid sequence of SEQ ID NO: 166, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 220. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 168, a heavy chain comprising the amino acid sequence of SEQ ID NO: 169, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 221. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 171, a heavy chain comprising the amino acid sequence of SEQ ID NO: 172, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 222. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 174, a heavy chain comprising the amino acid sequence of SEQ ID NO: 175, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 223. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 187, a heavy chain comprising the amino acid sequence of SEQ ID NO: 188, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 224. In some embodiments, a fusion protein of the disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 298, a heavy chain comprising the amino acid sequence of SEQ ID NO: 299, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 301. In some embodiments, a fusion protein of the disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 302, a heavy chain comprising the amino acid sequence of SEQ ID NO: 303, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 305.In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 306, a heavy chain comprising the amino acid sequence of SEQ ID NO: 307, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 309. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 310, a heavy chain comprising the amino acid sequence of SEQ ID NO: 311, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 313. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 314, a heavy chain comprising the amino acid sequence of SEQ ID NO: 315, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 317. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 318, a heavy chain comprising the amino acid sequence of SEQ ID NO: 319, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 321. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 322, a heavy chain comprising the amino acid sequence of SEQ ID NO: 323, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 325. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 326, a heavy chain comprising the amino acid sequence of SEQ ID NO: 327, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 329. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 330, a heavy chain comprising the amino acid sequence of SEQ ID NO: 331, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 333. In some embodiments, a fusion protein of the present disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 334, a heavy chain comprising the amino acid sequence of SEQ ID NO: 335, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 337. In some embodiments, a fusion protein of the disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 338, a heavy chain comprising the amino acid sequence of SEQ ID NO: 339, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 341. In some embodiments, a fusion protein of the disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 342, a heavy chain comprising the amino acid sequence of SEQ ID NO: 343, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 345.In some embodiments, a fusion protein of the disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 346, a heavy chain comprising the amino acid sequence of SEQ ID NO: 347, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 349. In some embodiments, a fusion protein of the disclosure comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 350, a heavy chain comprising the amino acid sequence of SEQ ID NO: 351, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 353.

[0150] In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 156, a heavy chain comprising the amino acid sequence of SEQ ID NO: 157, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 158 or 217. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 159, a heavy chain comprising the amino acid sequence of SEQ ID NO: 160, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 161 or 218. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 162, a heavy chain comprising the amino acid sequence of SEQ ID NO: 163, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 164 or 219. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 165, a heavy chain comprising the amino acid sequence of SEQ ID NO: 166, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 167 or 220. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 168, a heavy chain comprising the amino acid sequence of SEQ ID NO: 169, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 170 or 221. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 171, a heavy chain comprising the amino acid sequence of SEQ ID NO: 172, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 173 or 222. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 174, a heavy chain comprising the amino acid sequence of SEQ ID NO: 175, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 176 or 223. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 187, a heavy chain comprising the amino acid sequence of SEQ ID NO: 188, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 189 or 224.In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 298, a heavy chain comprising the amino acid sequence of SEQ ID NO: 299, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 300 or 301. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 302, a heavy chain comprising the amino acid sequence of SEQ ID NO: 303, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 304 or 305. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 306, a heavy chain comprising the amino acid sequence of SEQ ID NO: 307, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 308 or 309. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 310, a heavy chain comprising the amino acid sequence of SEQ ID NO: 311, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 312 or 313. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 314, a heavy chain comprising the amino acid sequence of SEQ ID NO: 315, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 316 or 317. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 318, a heavy chain comprising the amino acid sequence of SEQ ID NO: 319, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 320 or 321. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 322, a heavy chain comprising the amino acid sequence of SEQ ID NO: 323, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 324 or 325. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 326, a heavy chain comprising the amino acid sequence of SEQ ID NO: 327, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 328 or 329.In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 330, a heavy chain comprising the amino acid sequence of SEQ ID NO: 331, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 332 or 333. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 334, a heavy chain comprising the amino acid sequence of SEQ ID NO: 335, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 336 or 337. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 338, a heavy chain comprising the amino acid sequence of SEQ ID NO: 339, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 340 or 341. In some embodiments, the disclosure provides a mixture of fusion protein species, each species comprising one or two light chains comprising the amino acid sequence of SEQ ID NO: 342, a heavy chain comprising the amino acid sequence of SEQ ID NO: 343, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 344 or 345. In some embodiments, the disclosure provides a mixture of fusion protein species, each of which comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 346, a heavy chain comprising the amino acid sequence of SEQ ID NO: 347, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 348 or 349. In some embodiments, the disclosure provides a mixture of fusion protein species, each of which comprises one or two light chains comprising the amino acid sequence of SEQ ID NO: 350, a heavy chain comprising the amino acid sequence of SEQ ID NO: 351, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 352 or 353.

[0151] In some embodiments, the disclosure provides a fusion protein comprising two heavy chain sequences and two light chain sequences of a single fusion protein listed in Table 5, where one heavy chain sequence has an IL2 fusion and the other heavy chain sequence does not have an IL2 fusion, and the two light chain sequences are identical. In some embodiments, the heavy chain sequence without the IL2 fusion comprises a C-terminal lysine. In some embodiments, the fusion protein is a Form A protein shown in Figure 7. For example, in some embodiments, the fusion protein comprises four polypeptide chains, where (1) a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 334, a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 335, a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 336, and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 334, or (2) a first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 334, a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 335, a third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 337, and a fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 338. (3) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 338, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 339, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 340, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 338; or (4) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 338, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 339, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 341, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 338.

[0152] [Table 6-1]

[0153] [Table 6-2]

[0154] [Table 6-3]

[0155]

Table 6-4

[0156]

Table 6-5

[0157]

Table 6-6

[0158]

Table 6-7

[0159]

Table 6-8

[0160]

Table 6-9

[0161]

Table 6-10

[0162]

Table 6-11

[0163]

Table 6-12

[0164]

Table 6-13

[0165] [Table 6-14]

[0166] [Table 6-15]

[0167] [Table 6-16]

[0168] [Table 6-17]

[0169] [Table 6-18]

[0170] [Table 6-19]

[0171] [Table 6-20]

[0172] [Table 6-21]

[0173] [Table 6-22]

[0174] Properties of IL-2 fusion proteins The preferential activity of a targeted IL-2 fusion protein containing an IL-2 polypeptide on antigen-expressing cells is demonstrated in assays involving antigen-expressing cells that also express IL-2Rβγ or IL-2Rαβγ and non-antigen-expressing cells. One such assay is an in vitro assay that measures the phosphorylation of STAT5 (pSTAT5) and / or the expression of the proliferation marker Ki-67 in human immune cells, such as human peripheral blood and / or tumor-infiltrating immune cells, upon exposure to an IL-2 polypeptide. In one format of the assay, the activity of the targeted IL-2 fusion protein is measured on antigen-expressing and non-expressing cells to demonstrate selectivity for antigen-expressing cells. In another format of the assay, the activity of a targeted IL-2 fusion protein containing an IL-2 polypeptide on antigen-expressing cells is compared with the activity of a non-targeted IL-2 fusion protein containing the same IL-2 polypeptide and a control antibody that does not recognize either antigen on antigen-expressing cells to demonstrate the magnitude of rescue in signal transduction of the IL-2 polypeptide when fused to an antigen-binding molecule.

[0175] In some embodiments, fusion proteins of the present disclosure containing a CD8b antigen binding molecule activate CD8b+IL-2Rβ+ cells at least 10-fold, at least 50-fold, or at least 100-fold compared to CD8b-IL-2Rβ+ cells. In some embodiments, the fusion proteins activate CD8b+IL-2Rβ+ cells 50-fold, 100-fold, or 200-fold more than a fusion molecule comprising the IL-2 variant polypeptide and a control antibody that does not bind to any antigen expressed on the cells. Activation of the cells by the IL-2 fusion protein is determined by measuring the expression of pSTAT5 or the cell proliferation marker Ki67 in the cells after treatment with the IL-2 fusion protein.

[0176] In some embodiments, a fusion protein of the present disclosure exhibits one or more of the following: binds to human CD8, does not block the interaction of CD8 with MHC class I, and activates CD8+ T cells with at least 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, or 1000-fold greater potency than, for example, NK cell activation. In some embodiments, whether an anti-CD8 antibody or fusion protein of the present disclosure blocks the interaction of CD8 with MHC class I can be analyzed, for example, by assaying the activation status of CD8+ T cells (e.g., upon antigen stimulation) in the presence or absence of the anti-CD8 antibody or fusion protein. In some embodiments, CD8+ T cell and / or NK cell activation is measured, for example, by assaying one or more markers of proliferation (e.g., Ki67), IL-2Rβ / γ downstream signaling, and / or STAT5 downstream signaling (e.g., the percentage of treated cells expressing one or more markers).

[0177] IL-21 polypeptide Interleukin-21 (IL-21) is a cytokine that can be expressed by T cells, B cells, NK cells, and myeloid cells, and it regulates the activity of both innate and adaptive immune cells, improving T cell survival and effector function. Several Phase I and Phase II clinical trials have included IL-21 as an investigational product for the treatment of cancer, inflammatory diseases, and autoimmune diseases, including melanoma, renal cell carcinoma, acute myeloid leukemia, non-Hodgkin's lymphoma, ovarian cancer, colorectal cancer, systemic lupus erythematosus, Crohn's disease, and rheumatoid arthritis.

[0178] IL-21 has a four-helix bundle structure and exists as a monomer. In humans, two isoforms of IL-21 are known, each derived from a precursor molecule. The first IL-21 isoform contains 162 amino acids (aa), of which the first 29 residues constitute a signal peptide. The second IL-21 isoform contains 153 aa, of which the first 29 residues constitute a signal peptide, similar to the first isoform.

[0179] IL-21 binds to the heterodimeric IL-21 receptor complex, which consists of the IL-21 receptor (IL-21R) and the common gamma chain (γc). The IL-21 receptor complex is expressed on the surface of T cells, B cells, and NK cells. The IL-21 receptor complex is structurally similar to the IL-2 receptor complex in that each of these cytokine receptor complexes contains γc.

[0180] Binding of IL-21 to the IL-21 receptor complex activates the JAK / STAT signaling pathway, resulting in target gene activation. IL-21-induced signaling may be therapeutically desirable, but careful consideration of the timing and location of signaling is required given the broad expression profile of IL-21 and its ability to enhance CD8+ T cell responses and suppress antigen presentation and T cell priming.

[0181] In some embodiments, the present disclosure provides IL-21 polypeptides comprising at least one amino acid substitution compared to the human IL-21 amino acid sequence set forth herein as SEQ ID NO: 390. In some embodiments, "wild-type IL-21" and "human IL-21" are used interchangeably and refer to the amino acid sequence of SEQ ID NO: 390. Such IL-21 polypeptides comprising at least one amino acid substitution compared to SEQ ID NO: 390 are also referred to herein as IL-21 muteins. In some embodiments, the IL-21 polypeptides described herein comprise at least one, and no more than X, amino acid substitutions, where X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or more. In some embodiments, the IL-21 polypeptides described herein comprise at least 35 amino acid substitutions compared to SEQ ID NO: 390. In some embodiments, the IL-21 polypeptides described herein comprise an amino acid sequence that differs by 10 amino acids, 15 amino acids, 20 amino acids, or 25 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 390). In some embodiments, the IL-21 polypeptides described herein comprise an amino acid sequence that differs by 16 amino acids or less from the amino acid sequence of human IL-21 (SEQ ID NO: 390). In some embodiments, the IL-21 polypeptides described herein comprise an amino acid sequence that differs by 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 390). In some embodiments, the IL-21 polypeptides described herein comprise an amino acid sequence that differs by 7 amino acids or less or 5 amino acids or less from the amino acid sequence of human IL-21 (SEQ ID NO: 390). In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof described herein comprises an amino acid sequence that differs by 10, 11, 12, 13, 14, 15, or 16 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 390).In some embodiments, the IL-21 polypeptides described herein comprise an amino acid sequence that differs by 3, 4, 5, or 6 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 390). In some embodiments, the IL-21 polypeptides described herein comprise an amino acid sequence that differs by 5-16 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 390). In some embodiments, the IL-21 polypeptides described herein comprise an amino acid sequence that differs by 3-6 amino acids or 1-5 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 390). In some embodiments, the IL-21 described herein comprises an amino acid sequence that differs by 1 or 2 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 390).

[0182] Changes in isoelectric point or charge distribution In some embodiments, the IL-21 polypeptide comprises at least one mutation that alters its isoelectric point, with or without affecting binding to IL-21R. In some embodiments, the IL-21 polypeptide comprises at least one mutation that alters its isoelectric point without affecting binding to IL-21R. A change in the isoelectric point of a protein alters the surface charge distribution, which can thereby affect the protein yield, nonspecific binding, and serum or circulating half-life of a given protein. In some embodiments, the mutation in the IL-21 polypeptide lowers the theoretical isoelectric point by a given unit as measured by the public database ProtParam (SwissProt), incorporated herein by reference.

[0183] In some embodiments, the IL-21 polypeptide has an isoelectric point difference of about 0.6 to about 5.0 units compared to the isoelectric point of SEQ ID NO: 390, which has an isoelectric point of about 9.42. In some embodiments, the IL-21 polypeptide has an isoelectric point of about 6.0 to about 9.0. In some embodiments, the IL-21 polypeptide has a theoretical isoelectric point of about 6 to about 9. In some embodiments, the IL-21 polypeptide has a theoretical isoelectric point of at least about 6. In some embodiments, the IL-21 polypeptide comprises a theoretical isoelectric point of at most about 9.In some embodiments, the IL-21 polypeptide is from about 9 to about 8.9, from about 9 to about 8.8, from about 9 to about 8.7, from about 9 to about 8.5, from about 9 to about 8.3, from about 9 to about 8.1, from about 9 to about 7.9, from about 9 to about 7.7, from about 9 to about 7.5, from about 9 to about 7.3, from about 9 to about 7, from about 9 to about 6.5, from about 9 to about 6.0, from about 8.9 to about 8.8, from about 8.9 to about 8.7, from about 8.9 to about 8.5, from about 8.9 to about 8.3, from about 8.9 to about 8.1, from about 8.9 to about 7.9, from about 8.9 to about 7.7, from about 8.9 to about 7.5, from about 8.9 to about 7.3, from about 8.9 to about 7, about 8.9 to about 6.5, about 8.9 to about 6.0, about 8.8 to about 8.7, about 8.8 to about 8.5, about 8.8 to about 8.3, about 8.8 to about 8.1, about 8.8 to about 7.9, about 8.8 to about 7.7, about 8.8 to about 7.5, about 8.8 to about 7.3, about 8.8 to about 7, about 8.8 to about 6.5, about 8.8 to about 6.0, about 8.7 to about 8.6, about 8.7 to about 8.5, about 8.7 to about 8.3, about 8.7 to about 8.1, about 8.7 to about 7.9, about 8.7 to about 7.7, about 8.7 to 7.5, about 8.7 to about 7.3, about 8.7 to about 7, about 8.7 to about 6.5, about 8.7 to about 6.0, about 8.5 to about 8.3, about 8.5 to about 8.4, about 8.5 to about 8.39, about 8.5 to about 8.1, about 8.5 to about 7.9, about 8.5 to about 7.7, about 8.5 to about 7.5, about 8.5 to about 7.3, about 8.5 to about 7, about 8.5 to about 6.5, about 8.5 to about 6.0, about 8.39, about 8.3 to about 8.1, about 8.3 to about 7.9, about 8.3 to about 7.7, about 8.3 to about 7.5, about 8.3 to about 7.3, about 8.3 to about 7, about 8.3 to about 6.5, about 8.3 to about 6.0, about 8.1 to about 7.9, about 8.1 to about 7.7, about 8 The theoretical isoelectric point is about 0.1 to about 7.5, about 8.1 to about 7.3, about 8.1 to about 7, about 8.1 to about 6.5, about 8.1 to about 6.0, about 7.9 to about 7.7, about 7.9 to about 7.5, about 7.9 to about 7.3, about 7.9 to about 7, about 7.9 to about 6.5, about 7.9 to about 6.0, about 7.7 to about 7.5, about 7.7 to about 7.3, about 7.7 to about 7, about 7.5 to about 7.3, about 7.5 to about 7.3, about 7.5 to about 7, or about 7.3 to about 7, about 7.3 to about 6.5, about 7.3 to about 6.0, about 7.0 to about 6.5, about 7.0 to about 6.0, or about 6.5 to about 6.0.In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof has a theoretical isoelectric point of about 9, about 8.9, about 8.8, about 8.79, about 8.7, about 8.5, about 8.39, about 8.3, about 8.1, about 7.9, about 7.7, about 7.5, about 7.3, about 7, about 6.5, or about 6.0. In some embodiments, an IL-21 polypeptide has a theoretical isoelectric point of less than 9.0. In some embodiments, an IL-21 polypeptide has a theoretical isoelectric point of less than 8.9, less than 8.8, less than 8.7, less than 8.5, less than 8.3, less than 8.1, less than 7.9, less than 7.7, less than 7.5, less than 7.3, less than 7.2, less than 7.0, less than 6.5, or less than 6.2.

[0184] In some examples, the shift in isoelectric point can increase protein yield of the IL-21 polypeptide during purification compared to human IL-21. In some embodiments, the increase in protein yield is at least 5% greater than human IL-21. In some embodiments, the increased protein yield is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% greater than wild-type IL-21. In some embodiments, the increased protein yield is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% greater than human IL-21. In some embodiments, the increased protein yield is between 5% and 10%, between 5% and 15%, between 5% and 20%, between 5% and 25%, between 5% and 50%, between 5% and 75%, between 5% and 100%, between 5% and 125%, between 5% and 150%, between 5% and 175%, between 5% and 200%, between 5% and 225%, between 5% and 250%, between 5% and 275%, between 5% and 300%, between 10% and 15%, between 10% and 20%, between 10% and 25% %, 10%~50%, 10%~75%, 10%~100%, 10%~125%, 10%~150%, 10%~175%, 10%~200%, 10%~225%, 10%~250%, 10%~275%, 10%~300%, 15%~20%, 15%~25%, 15%~50%, 15%~75%, 15%~100%, 15%~125%, 15%~150%, 15%~ 175%, 15%~200%, 15%~225%, 15%~250%, 15%~275%, 15%~300%, 20%~25%, 20%~50%, 20%~75%, 20%~100%, 20%~125%, 20%~150%, 20%~175%, 20%~200%, 20%~225%, 20%~250%, 20%~275%, 20%~300%, 25%~50 %, 25%~75%, 25%~100%, 25%~125%, 25%~150%, 25%~175%, 25%~200%, 25%~225%, 25%~250%, 25%~275%, 25%~300%, 50%~75%, 50%~100%, 50%~125%, 50%~150%, 50%~175%, 50%~200%, 75%~100%, 75%~125%,75%~150%, 75%~175%, 75%~200%, 75%~225%, 75%~250%, 75%~275%, 75%~300%, 100%~125%, 100%~150%, 100%~175%, 100%~200%, 100%~225%, 100%~250%, 100%~275%, 100%~300%, 125%~150%, 125%~175%, 125%~200%, 125%~225%, 125%~250%, 1 25% to 275%, 125% to 300%, 150% to 175%, 150% to 200%, 175% to 200%, 175% to 225%, 175% to 250%, 175% to 275%, 175% to 300%, 200% to 225%, 200% to 250%, 200% to 275%, 200% to 300%, 225% to 250%, 225% to 275%, 225% to 300%, 250% to 275%, 250% to 300%, or 275% to 300%. In some embodiments, the increased protein yield is about 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% greater than human IL-21.

[0185] In some embodiments, human IL-21 comprises a region of 2 to 20 positively charged amino acid residues. In some embodiments, the region is 2 to 3, 2 to 4, 2 to 5, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 11, 3 to 12, 3 to 13, 3 to 14, 3 to 15, 3 to 16, 3 to 17, 3 to 18, 3 to 19, 3 to 20, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 4 to 11, 4 to 12, 4 to 13, 4 to 14, 4 to 15, 4 to 16 , 4-17, 4-18, 4-19, 4-20, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 6-20, 7-8, 7-9, 7-10, 7-11, 7-12, 7-13, 7-14, 7-15, 7-16, 7-17, 7-18, 7-19, 7-20, 8-9, 8- 10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 12-13, and 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 14-15, 14-16, 14-17, 14-18, 14-19, 14-20, 15-16, 15-17, 15-18, 15-19, 15-20, 16-17, 16-18, 16-19, 16-20, 17-18, 17-19, 17-20, 18-19, 18-20, or 19-20 positively charged amino acid residues.In some embodiments, human IL-21 comprises a region of 12 positively charged amino acid residues, hi some embodiments, human IL-21 comprises a region of 2-20 amino acid residues in which at least one amino acid is positively charged.In some embodiments, the regions are 2-3, 2-4, 2-5, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-14 , 4-15, 4-16, 4-17, 4-18, 4-19, 4-20, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 6-20, 7-8, 7-9, 7-10, 7-11, 7-12, 7-13, 7-14, 7-15, 7-16, 7-17, 7- 18, 7-19, 7-20, 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 11-12, 11-13, 11-14, 11-15, 11-16, 1 1-17, 11-18, 11-19, 11-20, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 14-15, 14-16, 14-17, 3-18, 14-19, 14-20, 15-16, 15-17, 15-18, 15-19, 15-20, 16-17, 16-18, 16-19, 16-20, 17-18, 17-19, 17-20 It comprises 18-19, 18-20, or 19-20 amino acid residues, and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues are positively charged.In some embodiments, human IL-21 comprises a region of 12 amino acid residues, in which 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues are positively charged. In some embodiments, human IL-21 comprises a region of 12 amino acid residues, in which 2, 3, 4, 5, or 6 amino acid residues are positively charged.

[0186] In some embodiments, the amino acids within the region do not bind to the human IL-21 receptor. In some embodiments, the region includes amino acid residues S80 through T92 of SEQ ID NO: 390. In some embodiments, the region includes positively charged amino acid residues R85, R86, K88, H89, and R90. In some embodiments, the IL-21 polypeptide includes at least one amino acid substitution within the region of human IL-21. In some embodiments, the IL-21 polypeptide includes at least one amino acid substitution at S80 through T92. In some embodiments, the IL-21 polypeptide includes at least four amino acid substitutions at S80 through T92. In some embodiments, the IL-21 polypeptide includes at least four amino acid substitutions at S80 through T92, except that G84 is not substituted. In some embodiments, the IL-21 polypeptide includes at least one amino acid substitution at R85, R86, K88, H89, or R90. In some embodiments, the IL-21 polypeptide comprises an amino acid substitution at one or more of S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, and T92. In some embodiments, the IL-21 polypeptide comprises an amino acid substitution at S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, and T92.

[0187] In some embodiments, an IL-21 polypeptide of the disclosure has one or more, two or more, or three or more charge-reducing amino acid substitutions compared to a wild-type mature IL-21 polypeptide having, for example, the amino acid sequence of SEQ ID NO: 390. In some embodiments, the one or more, two or more, or three or more substituted residues are selected from the following group: K56, S80, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92.

[0188] In some embodiments, an IL-21 polypeptide (SEQ ID NO: 390) having an altered isoelectric point compared to human IL-21 comprises a mutation at a position selected from the group consisting of K56, S80, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92 of SEQ ID NO: 390. In some embodiments, an IL-21 polypeptide (SEQ ID NO: 390) having an altered isoelectric point compared to human IL-21 comprises a mutation at a position selected from the group consisting of S80, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92 of SEQ ID NO: 390. In some embodiments, the IL-21 polypeptide (SEQ ID NO: 390) having an altered isoelectric point compared to human IL-21 comprises a mutation at a position selected from the group consisting of S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, and T92 of SEQ ID NO: 390.

[0189] In some embodiments, the IL-21 polypeptide comprises a mutation at position K56 of SEQ ID NO: 390. In some embodiments, the mutation comprises K56G, K56S, K56E, K56D, or K56A. In some embodiments, the IL-21 polypeptide comprises a mutation at position S80. In some embodiments, the mutation comprises S80G, S80A, S80D, or S80E. In some embodiments, the IL-21 polypeptide comprises a mutation at position T81 of SEQ ID NO: 390. In some embodiments, the mutation comprises T81G, T81S, T81E, T81D, or T81A. In some embodiments, the IL-21 polypeptide comprises a mutation at position N82 of SEQ ID NO: 390. In some embodiments, the mutation comprises N82G, N82S, N82E, N82D, or N82A. In some embodiments, the IL-21 polypeptide comprises a mutation at position A83 of SEQ ID NO: 390. In some embodiments, the mutation comprises A83G, A83S, A83E, or A83D. In some embodiments, the IL-21 polypeptide comprises a mutation at position G84 of SEQ ID NO: 390. In some embodiments, the mutation comprises G84A, G84S, G84E, or G84D. In some embodiments, G84 is not mutated. In some embodiments, the IL-21 polypeptide comprises a mutation at position R85 ​​of SEQ ID NO: 390. In some embodiments, the mutation comprises R85G, R85S, R85E, R85D, or R85A. In some embodiments, the IL-21 polypeptide comprises a mutation at position R86 of SEQ ID NO: 390. In some embodiments, the mutation comprises R86G, R86S, R86E, R86D, or R86A. In some embodiments, the IL-21 polypeptide comprises a mutation at position Q87 of SEQ ID NO: 390. In some embodiments, the mutation comprises Q87G, Q87S, Q87E, Q87D, or Q87A. In some embodiments, the IL-21 polypeptide comprises a mutation at position K88 of SEQ ID NO: 390. In some instances, the mutation comprises K88G, K88S, K88E, K88D, or K88A. In some embodiments, the IL-21 polypeptide comprises a mutation at position H89 of SEQ ID NO: 390. In some embodiments, the mutation comprises H89G, H89S, H89E, H89D, or H89A.In some embodiments, the IL-21 polypeptide comprises a mutation at position R90 of SEQ ID NO: 390. In some embodiments, the mutation comprises R90G, R90S, R90E, R90D, or R90A. In some embodiments, the IL-21 polypeptide comprises a mutation at position L91 of SEQ ID NO: 390. In some embodiments, the mutation comprises L91G, L91S, L91E, L91D, or L91A. In some embodiments, the IL-21 polypeptide comprises a mutation at position T92 of SEQ ID NO: 390. In some embodiments, the mutation comprises T92G, T92S, T92E, T92D, or T92A.

[0190] In some embodiments, the IL-21 polypeptide comprises SEQ ID NO: 390, which comprises one of the following sets of amino acid substitutions (relative to SEQ ID NO: 390): G85, G86, G88, and A90; G85, G86, G88, and E90; A56, A75, G85, G86, G88, and E90; A56, E75, G85, G86, G88, and A90; E56, A75, G85, G86, G88, and A90; G80, G81, G82, S83, E85, G86, S87, G88, G89, and S90; G80, G81, G82, S83, G85, G86, S87, G88, G89, S90; G85, G86, S87, G88, G89, and E90; G85, G86, S87, G88, G89, and S90; G85, G86, G87, G88, G89, and E90; G85, G86, G87, G88, G89, and G90; G85, G86, E87, G88, G89, and G90; G80, G81, G82, S83, G85, G86, S87, G88, G89, G90, S91 and G92; G80, G81, G82, G83, G85, G86, G87, G88, G89, G90, G91 and G92; G80, G81, G82, S83, G85, G86, E87, G88, G89, G90, S91 and G92; G80, G81, G82, E83, G85, G86, E87, G88, G89, G90, S91 and G92; G82, G83, S85, G86, G87, G88, S89, G90, G91 and S92; G82, G83, E85, G86, G87, G88, S89, G90, G91 and S92; G80, G81, G82, G83, E85, G86, G87, G88, G89, G90, G91 and G92; G80, G 81, G82, S83, G85, G86, S87, G88, G89, E90, G91 and G92; G80, G81, G82, G83, G85, G86, G87, G88, G89, E90, G91 and G92; G82, G83, S85, G86, G87, G88, S89, E90, G91 and S92; G82, G83, G85, G86, G87, G88, G89, E90, G91 and G92; G85, G86, G87, G88, G89, E90 and G91; G82, G83, G85, G86, G87, G88, G89, E90 and G91;G82, G83, G85, E86, G87, G88, G89, E90, and G91; G82, G83, E85, G86, G87, G88, G89, E90, and G91; G80, G81, G82, G83, E84, G85, G86, G87, G88, G89, E90, G91, and G92; G80, G81, G82, E83, G85, G86, G87, G88, G89, E90, G91, and G92; or G80, G81, E82, G83, G85, G86, G87, G88, G89, E90, G91, and G92.

[0191] In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids A56, A75, G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids A56, A75, G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids A56, E75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids A56, E75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids A56, A75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids A56, A75, G85, G86, G88, and A90.In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids E56, A75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids E56, A75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, S83, E85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, S83, E85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, S90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises the amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, S90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises the amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, E90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, E90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, S87, G88, G89, and E90.In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, G87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, G87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, G87, G88, G89, and G90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, G87, G88, G89, and G90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, E87, G88, G89, and G90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, E87, G88, G89, and G90.

[0192] In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, S83, G85, G86, E87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, S83, G85, G86, E87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, E83, G85, G86, E87, G88, G89, G90, S91, and G92.In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, E83, G85, G86, E87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G82, G83, S85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G82, G83, S85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G82, G83, E85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G82, G83, E85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, G83, E85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, G83, E85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, E90, G91, and G92.In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G82, G83, S85, G86, G87, G88, S89, E90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G82, G83, S85, G86, G87, G88, S89, E90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, G87, G88, G89, E90, and G91.In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G82, G83, G85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G82, G83, G85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G82, G83, G85, E86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G82, G83, G85, E86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G82, G83, E85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G82, G83, E85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, G83, E84, G85, G86, G87, G88, G89, E90, G91, and G92.In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, G83, E84, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, E83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, G82, E83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, E82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 390 and comprises amino acids G80, G81, E82, G83, G85, G86, G87, G88, G89, E90, G91, and G92.

[0193] In some embodiments, a charge variant IL-21 polypeptide or functional fragment or variant thereof is provided, comprising a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 391-421.

[0194] In some embodiments, the IL-21 polypeptide has from about 75% sequence identity to about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 391-421. In some embodiments, the IL-21 polypeptide has at least about 75% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 391-421. In some embodiments, the IL-21 polypeptide has up to about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 391-421. In some embodiments, the IL-21 polypeptide has between about 75% sequence identity and about 80% sequence identity, between about 75% sequence identity and about 85% sequence identity, about 75% sequence identity and about 85% sequence identity, about 75% sequence identity or less to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. Sequence identity: about 90% to about 95%; about 75% to about 96%; about 75% to about 97%; about 75% to about 98%; about 75% to about 99%; about 75% to about 100%; about 80% to about 85%; about 80% to about 90% , about 80% sequence identity to about 95% sequence identity, about 80% sequence identity to about 96% sequence identity, about 80% sequence identity to about 97% sequence identity, about 80% sequence identity to about 98% sequence identity, about 80% sequence identity to about 99% sequence identity, about 80% sequence identity to about 100% sequence identity, about 85% sequence identity to about 90% sequence identity, about 85% sequence identity to about 95% sequence identity, about 85% sequence identity up to about 96% sequence identity, about 85% sequence identity to about 97% sequence identity, about 85% sequence identity to about 98% sequence identity, about 85% sequence identity to about 99% sequence identity, about 85% sequence identity to about 100% sequence identity, about 90% sequence identity to about 95% sequence identity, about 90% sequence identity to about 96% sequence identity, about 90% sequence identity to about 97% sequence identity, about 90% sequence identity to about 98% sequence identity,Approximately 90% to 99% sequence identity, approximately 90% to 100% sequence identity, approximately 95% to 96% sequence identity, approximately 95% to 97% sequence identity, approximately 95% to 98% sequence identity, approximately 95% to 99% sequence identity, approximately 95% to 100% sequence identity, approximately 96% to 97% sequence identity, approximately 96% to 98% sequence identity The sequence has sequence identity, about 96% to about 99% sequence identity, about 96% to about 100% sequence identity, about 97% to about 98% sequence identity, about 97% to about 99% sequence identity, about 97% to about 100% sequence identity, a...

Claims

1. IL-21 fusion protein and IL-2 fusion protein for use in the treatment of subjects with disease, (a) The IL-21 fusion protein comprises (i) a first antibody or its antigen-binding fragment that specifically binds to human CD8, and (ii) an IL-21 polypeptide. (b) The IL-2 fusion protein comprises (i) a second antibody or antigen-binding fragment that specifically binds to human CD8, and (ii) an IL-2 polypeptide, comprising an IL-21 fusion protein and an IL-2 fusion protein.

2. The IL-21 fusion protein and IL-2 fusion protein according to claim 1, wherein the disease includes cancer or an infectious disease.

3. The IL-21 fusion protein and IL-2 fusion protein according to claim 1 or 2, wherein the first antibody or its antigen-binding fragment and the second antibody or its antigen-binding fragment are the same.

4. The first antibody or antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable (VH) domain, the light chain comprises a light chain variable (VL) domain, and in the first antibody or antigen-binding fragment, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 13, CDR-H2 containing the amino acid sequence of SEQ ID NO: 14, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 15, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 24, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 27, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 30, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 31, CDR-H2 containing the amino acid sequence of SEQ ID NO: 32, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 33, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 34, CDR-L2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 36, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 37, CDR-H2 containing the amino acid sequence of SEQ ID NO: 38, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 177, CDR-H2 containing the amino acid sequence of SEQ ID NO: 178, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 179, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 180, CDR-L2 containing the amino acid sequence of SEQ ID NO: 181, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 182, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 226, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 227, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 230, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 231, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 232, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 234, CDR-L2 containing the amino acid sequence of SEQ ID NO: 235, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 236, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 232, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 237, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 231, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 15, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 53, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 24, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 27, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 30, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 54, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 33, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 34, CDR-L2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 36, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 55, CDR-H2 containing the amino acid sequence of SEQ ID NO: 56, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 55, CDR-H2 containing the amino acid sequence of SEQ ID NO: 57, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, CDR-H2 containing the amino acid sequence of SEQ ID NO: 50, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 183, CDR-H2 containing the amino acid sequence of SEQ ID NO: 184, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 179, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 180, CDR-L2 containing the amino acid sequence of SEQ ID NO: 181, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 182, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 239, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 240, CDR-H2 containing the amino acid sequence of SEQ ID NO: 241, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 242, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 234, CDR-L2 containing the amino acid sequence of SEQ ID NO: 235, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 236, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228, or The IL-21 fusion protein and IL-2 fusion protein according to claim 1 or 2, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 240, CDR-H2 containing the amino acid sequence of SEQ ID NO: 244, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 242, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

42.

5. The first antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable (VH) domain, and the light chain comprises a light chain variable (VL) domain, and the VH and VL domains of the first antibody or its antigen-binding fragment are (a) Sequence IDs 58 and 59, respectively (b) Sequence IDs 247 and 248, respectively (c) Sequence IDs 62 and 63, respectively (d) Sequence IDs 64 and 65, respectively (e) Sequence IDs 66 and 67, respectively (f) Sequence IDs 68 and 69, respectively (g) Sequence IDs 70 and 71, respectively (h) Sequence IDs 72 and 73, respectively (i) Sequence IDs 185 and 186, respectively (j) Sequence IDs 245 and 246, respectively (k) Sequence IDs 249 and 250, respectively (l) Sequence IDs 251 and 252, respectively (m) Sequence numbers 253 and 254, respectively (n) Sequence IDs 255 and 256, respectively, or (o) The IL-21 fusion protein and the IL-2 fusion protein according to claim 1 or 2, each comprising the amino acid sequences of SEQ ID NOs. 257 and 258, respectively.

6. The second antibody or antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable (VH) domain, and the light chain comprises a light chain variable (VL) domain, and in the second antibody or antigen-binding fragment, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 13, CDR-H2 containing the amino acid sequence of SEQ ID NO: 14, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 15, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 24, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 27, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 30, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 31, CDR-H2 containing the amino acid sequence of SEQ ID NO: 32, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 33, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 34, CDR-L2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 36, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 37, CDR-H2 containing the amino acid sequence of SEQ ID NO: 38, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 177, CDR-H2 containing the amino acid sequence of SEQ ID NO: 178, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 179, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 180, CDR-L2 containing the amino acid sequence of SEQ ID NO: 181, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 182, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 226, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 227, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 230, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 231, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 232, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 234, CDR-L2 containing the amino acid sequence of SEQ ID NO: 235, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 236, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 232, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 237, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 231, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 15, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 53, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 24, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 27, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 30, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 54, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 33, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 34, CDR-L2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 36, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 55, CDR-H2 containing the amino acid sequence of SEQ ID NO: 56, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 55, CDR-H2 containing the amino acid sequence of SEQ ID NO: 57, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, CDR-H2 containing the amino acid sequence of SEQ ID NO: 50, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 183, CDR-H2 containing the amino acid sequence of SEQ ID NO: 184, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 179, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 180, CDR-L2 containing the amino acid sequence of SEQ ID NO: 181, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 182, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 239, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 240, CDR-H2 containing the amino acid sequence of SEQ ID NO: 241, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 242, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 234, CDR-L2 containing the amino acid sequence of SEQ ID NO: 235, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 236, The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228, or The IL-21 fusion protein and IL-2 fusion protein according to claim 1 or 2, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 240, CDR-H2 containing the amino acid sequence of SEQ ID NO: 244, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 242, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

42.

7. The second antibody or antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable (VH) domain, and the light chain comprises a light chain variable (VL) domain, and the VH and VL domains of the second antibody or antigen-binding fragment are (a) Sequence IDs 58 and 59, respectively (b) Sequence IDs 247 and 248, respectively (c) Sequence IDs 62 and 63, respectively (d) Sequence IDs 64 and 65, respectively (e) Sequence IDs 66 and 67, respectively (f) Sequence IDs 68 and 69, respectively (g) Sequence IDs 70 and 71, respectively (h) Sequence IDs 72 and 73, respectively (i) Sequence IDs 185 and 186, respectively (j) Sequence IDs 245 and 246, respectively (k) Sequence IDs 249 and 250, respectively (l) Sequence IDs 251 and 252, respectively (m) Sequence numbers 253 and 254, respectively (n) Sequence IDs 255 and 256, respectively, or (o) The IL-21 fusion protein and the IL-2 fusion protein according to claim 1 or 2, each comprising the amino acid sequences of SEQ ID NOs. 257 and 258, respectively.

8. The IL-21 fusion protein and IL-2 fusion protein according to claim 1 or 2, wherein the IL-2 polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 80, 85-155, 190-216, 297, and 354-383.

9. The IL-21 fusion protein and IL-2 fusion protein according to claim 1 or 2, wherein the IL-21 polypeptide comprises an amino acid sequence that is at least 80% identical to the human IL-21 polypeptide comprising SEQ ID NO: 390, the IL-21 polypeptide comprises at least one amino acid substitution that reduces the isoelectric point of the IL-21 polypeptide by about 0.6 units to about 5 units compared to the human IL-21 polypeptide without amino acid substitution, and the at least one amino acid substitution is located in the region of amino acid residues located S80 to T92, numbered according to SEQ ID NO:

390.

10. The IL-21 fusion protein and IL-2 fusion protein according to claim 1 or 2, wherein the IL-21 polypeptide comprises 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions selected from the group consisting of S80G, T81G, N82G, N82E, A83G, A83E, A83S, R85G, R85E, R85S, R86G, R86E, Q87G, Q87E, Q87S, K88G, H89G, H89S, R90G, R90S, R90E, R90A, L91G, L91S, T92G, and T92S, numbered according to Sequence ID No.

390.

11. The IL-21 polypeptide comprises at least one amino acid substitution that reduces binding to the IL-21 receptor compared to binding by human IL-21 polypeptide, wherein the at least one amino acid substitution is a substitution of one or more amino acid residues at positions R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, or K117 of SEQ ID NO: 390, and optionally the at least one amino acid substitution is R5F, R5A, R5E, R5S, R5T, R5N, R5Q, R5V, R5I, R5L, R5Y, I8E, R9A, R9D, R9E, IL-21 fusion protein and IL-2 fusion protein according to claim 1 or 2, selected from R9H, R9S, R9T, R9N, R9G, R9V, R9I, R9L, R9Y, R11D, R11E, L13F, L13R, I14D, I16A, I16S, I16R, V17I, V17A, D18A, K72A, K72E, K73A, K73E, K75A, K75E, L74I, L74F, L74M, L74V, R76E, R76F, R76A, R76N, R76D, R76S, R76T, R76Q, R76V, R76I, R76L, R76Y, R76M, K77A, K77E, and K117A.

12. The IL-21 fusion protein and IL-2 fusion protein according to claim 1 or 2, wherein the IL-21 polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs. 422 to 491, and optionally, the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs. 422 to 491.

13. The IL-2 fusion protein comprises four polypeptide chains, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 334, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 335, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 336, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 334, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 334, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 335, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 337, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 334, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 338, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 339, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 340, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 338, or The IL-21 fusion protein and IL-2 fusion protein according to claim 1 or 2, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 338, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 339, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 341, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

338.

14. The IL-21 fusion protein comprises four polypeptide chains, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 557, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 558, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 559, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 557, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 557, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 558, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 560, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 557, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 561, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 562, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 563, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 561, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 561, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 562, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 564, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 561, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 565, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 566, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 567, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 565, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 565, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 566, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 568, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 565, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 569, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 570, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 571, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 569, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 569, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 570, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 572, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 569, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 573, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 574, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 575, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 573, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 573, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 574, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 576, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 573, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 577, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 578, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 579, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 577, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 577, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 578, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 580, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 577, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 581, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 582, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 583, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 581, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 581, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 582, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 584, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 581, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 585, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 586, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 587, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 585, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 585, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 586, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 588, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 585, The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 589, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 590, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 591, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 589, or The IL-21 fusion protein and IL-2 fusion protein according to claim 1 or 2, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 589, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 590, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 592, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

589.

15. The IL-21 fusion protein and IL-2 fusion protein according to claim 1 or 2, wherein the first antibody or its antigen-binding fragment and the second antibody or its antigen-binding fragment each target CD8ab or CD8b.

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