Combination of cytokine fusion proteins and CD8 antigen binding molecules

Abstract

Provided herein are anti-CD8 antigen binding molecules and fusion polypeptides having IL-2 or IL-21 comprising a CD8 antigen binding molecule for selectively modulating the function of CD8 + T cells relative to other immune cells. The present disclosure provides methods of using IL-2 and IL-21 fusion proteins in combination to treat disease.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] 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 Art

[0003] CD8+ T cells expressing the αβ T cell receptor constitute a large subset of major histocompatibility (MHC) class I restricted T cells that mediate adaptive immunity against various pathogens and cancers. They can also be pathogenic and contribute to disease in certain autoimmune and inflammatory disorders. Regulating CD8+ T cell function, either by activating it in cases of infection and cancer or by inhibiting it in certain autoimmune diseases, can offer therapeutic benefits.

[0004] CD8+ T cell activation and differentiation are largely controlled by soluble immunomodulatory proteins such as cytokines. The biological activity of cytokines is mediated by their binding, typically with very high affinity, to their corresponding cytokine receptors on the cell surface, thereby effectively stimulating downstream signal transduction and triggering various cellular processes that regulate immune cell phenotype and function. Cytokines often exhibit pleiotropic effects, inducing multiple downstream cellular events such as activation, proliferation, survival, apoptosis, and the secretion of other immunomodulatory proteins. Furthermore, because their receptors are expressed on multiple subsets of immune cells, cytokines act not only on CD8+ T cells but also on other immune and non-immune cells that express their receptors.

[0005] The challenge of cytokine immunotherapy lies in the fact that, in some cases, while activating immune cells to enhance the immune response, the same cytokines can also activate counter-regulatory pathways, as exemplified by IL-2 and IFNγ. These counter-regulatory pathways can activate both regulatory T cell responses and repressive pathways. Therefore, there remains a need for improved therapies that target CD8+ T cells without activating regulatory T cell responses and repressive pathways. Summary of the Invention

[0006] In some aspects, this disclosure provides a method for treating a disease in a subject, the method comprising administering to the subject an effective amount of the following:

[0007] (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

[0008] (b) 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.

[0009] This is used to treat the subject.

[0010] In some aspects, this disclosure provides a method for treating a disease in a subject who has received or is receiving treatment with an IL-21 fusion protein comprising an antibody that specifically binds to human CD8 or an antigen-binding fragment thereof and an IL-21 peptide, the method comprising: administering to the subject an effective amount of an IL-2 fusion protein comprising (i) an antibody that specifically binds to human CD8 or an antigen-binding fragment thereof; and (ii) an IL-2 peptide, thereby treating the subject.

[0011] In some aspects, this disclosure provides a method for treating a disease in a subject who has received or is receiving treatment with an IL-2 fusion protein comprising an antibody that specifically binds to human CD8 or an antigen-binding fragment thereof and an IL-2 peptide, the method comprising: administering to the subject an effective amount of an IL-2 fusion protein comprising (i) an antibody that specifically binds to human CD8 or an antigen-binding fragment thereof; and (ii) an IL-21 peptide, thereby treating the subject.

[0012] In some aspects, this disclosure provides a kit comprising one or more containers containing: (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) 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; and optionally a pharmaceutically acceptable carrier; and instructions for administering the IL-21 and IL-2 fusion proteins to a subject in need in parallel, sequentially, or simultaneously.

[0013] In some aspects, this disclosure provides a kit comprising: a container containing an antibody or antigen-binding fragment thereof that specifically binds to human CD8; and an IL-21 fusion protein of an IL-21 peptide; and optionally a pharmaceutically acceptable carrier; and instructions for administering the IL-21 fusion protein to a subject in need of treatment who has received or is receiving treatment with an IL-2 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds to human CD8; and an IL-2 peptide, thereby treating the subject.

[0014] In some aspects, this disclosure provides a kit comprising: a container containing an antibody or antigen-binding fragment thereof that specifically binds to human CD8; and an IL-2 fusion protein of an IL-2 peptide; and optionally a pharmaceutically acceptable carrier; and instructions for administering the IL-2 fusion protein to a subject in need of treatment who has received or is receiving treatment with an IL-21 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds to human CD8; and an IL-21 peptide, thereby treating the subject.

[0015] In some aspects, this disclosure provides the use of an IL-21 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds to human CD8 and an IL-21 peptide for administration to a subject in need, said subject having received or being received treatment with an IL-2 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds to human CD8; and an IL-2 peptide.

[0016] In some aspects, this disclosure provides the use of an IL-2 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds to human CD8 and an IL-21 peptide for administration to a subject in need, said subject having received or currently receiving treatment with an IL-21 fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds to human CD8; and an IL-21 peptide.

[0017] In any of the foregoing or related aspects, the subject in need suffers from a disease. In some aspects, the disease is cancer. In some aspects, the disease is an infectious disease.

[0018] In any of the foregoing or related aspects, the treatment induces an immune response in the subject. In some aspects, the immune response is a T-cell response.

[0019] In any of the foregoing or related aspects, the treatment delays cancer progression in the subject. In some aspects, the treatment reduces tumor volume in the subject. In some aspects, the treatment reduces or inhibits tumor growth in the subject. In some aspects, the treatment delays cancer progression, reduces tumor volume, and / or reduces or inhibits tumor growth in the subject more than treatment with either the IL-2 fusion protein or the IL-21 fusion protein alone.

[0020] In any of the foregoing or related aspects, the antibodies or antigen-binding fragments of the IL-21 and IL-2 fusion proteins are identical. In some aspects, the antibodies or antigen-binding fragments of the IL-21 and IL-2 fusion proteins are different. In some aspects, the antibodies or antigen-binding fragments of the IL-21 and IL-2 fusion proteins bind to cells expressing human CD8ab heterodimers on their surfaces at an EC50 of less than 1000 nM. In some aspects, the antibodies or antigen-binding fragments of the IL-21 and IL-2 fusion proteins bind to human CD8+ T cells.

[0021] In some aspects, the antibody or antigen-binding fragment of the IL-21 and IL-2 fusion protein binds to different epitopes on CD8ab or CD8b. In some aspects, the antibody or antigen-binding fragment of the IL-21 and IL-2 fusion protein does not compete for binding to CD8ab or CD8b. In some aspects, the IL-21 fusion protein comprises a first antibody or antigen-binding fragment that specifically binds to human CD8, and the IL-2 fusion protein comprises a second antibody or antigen-binding fragment that specifically binds to human CD8, wherein the first antibody and the second antibody bind to different epitopes on CD8ab or CD8b. In some aspects, the first antibody and the second antibody do not cross-competitively bind to CD8ab or CD8b.

[0022] In some aspects, the antibody or antigen-binding fragment of the IL-21 and IL-2 fusion protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein:

[0023] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:13; CDR-H2, which contains the amino acid sequence of SEQ ID NO:14; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:15; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:16; CDR-L2, which contains the amino acid sequence of SEQ ID NO:17; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:18;

[0024] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:19; CDR-H2, which contains the amino acid sequence of SEQ ID NO:20; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:21; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:22; CDR-L2, which contains the amino acid sequence of SEQ ID NO:23; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:24;

[0025] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:25; CDR-H2, which contains the amino acid sequence of SEQ ID NO:26; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:27; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:28; CDR-L2, which contains the amino acid sequence of SEQ ID NO:29; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:30;

[0026] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:31; CDR-H2, which contains the amino acid sequence of SEQ ID NO:32; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:33; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:34; CDR-L2, which contains the amino acid sequence of SEQ ID NO:35; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:36;

[0027] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:37; CDR-H2, which contains the amino acid sequence of SEQ ID NO:38; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:39; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:40; CDR-L2, which contains the amino acid sequence of SEQ ID NO:41; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:42;

[0028] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:43; CDR-H2, which contains the amino acid sequence of SEQ ID NO:44; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:45; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:46; CDR-L2, which contains the amino acid sequence of SEQ ID NO:47; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:48;

[0029] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:1; CDR-H2, which contains the amino acid sequence of SEQ ID NO:2; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:3; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:4; CDR-L2, which contains the amino acid sequence of SEQ ID NO:5; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:6;

[0030] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:177; CDR-H2, which contains the amino acid sequence of SEQ ID NO:178; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:179; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:180; CDR-L2, which contains the amino acid sequence of SEQ ID NO:181; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:182;

[0031] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:225; CDR-H2, which contains the amino acid sequence of SEQ ID NO:226; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:227; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:16; CDR-L2, which contains the amino acid sequence of SEQ ID NO:17; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:228;

[0032] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:229; CDR-H2, which contains the amino acid sequence of SEQ ID NO:230; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:231; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:40; CDR-L2, which contains the amino acid sequence of SEQ ID NO:41; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:42;

[0033] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:225; CDR-H2, which contains the amino acid sequence of SEQ ID NO:232; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:233; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:234; CDR-L2, which contains the amino acid sequence of SEQ ID NO:235; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:236;

[0034] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:225; CDR-H2, which contains the amino acid sequence of SEQ ID NO:232; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:233; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:16; CDR-L2, which contains the amino acid sequence of SEQ ID NO:17; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:228; or

[0035] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:229; CDR-H2, which contains the amino acid sequence of SEQ ID NO:237; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:231; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:40; CDR-L2, which contains the amino acid sequence of SEQ ID NO:41; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:42.

[0036] In some aspects, the antibody or antigen-binding fragment of the IL-21 and IL-2 fusion protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein:

[0037] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:51; CDR-H2, which contains the amino acid sequence of SEQ ID NO:52; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:15; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:16; CDR-L2, which contains the amino acid sequence of SEQ ID NO:17; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:18;

[0038] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:53; CDR-H2, which contains the amino acid sequence of SEQ ID NO:52; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:21; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:22; CDR-L2, which contains the amino acid sequence of SEQ ID NO:23; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:24;

[0039] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:49; CDR-H2, which contains the amino acid sequence of SEQ ID NO:52; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:27; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:28; CDR-L2, which contains the amino acid sequence of SEQ ID NO:29; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:30;

[0040] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:54; CDR-H2, which contains the amino acid sequence of SEQ ID NO:52; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:33; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:34; CDR-L2, which contains the amino acid sequence of SEQ ID NO:35; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:36;

[0041] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:55; CDR-H2, which contains the amino acid sequence of SEQ ID NO:56; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:39; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:40; CDR-L2, which contains the amino acid sequence of SEQ ID NO:41; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:42;

[0042] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:55; CDR-H2, which contains the amino acid sequence of SEQ ID NO:57; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:45; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:46; CDR-L2, which contains the amino acid sequence of SEQ ID NO:47; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:48;

[0043] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:49; CDR-H2, which contains the amino acid sequence of SEQ ID NO:50; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:3; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:4; CDR-L2, which contains the amino acid sequence of SEQ ID NO:5; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:6;

[0044] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:183; CDR-H2, which contains the amino acid sequence of SEQ ID NO:184; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:179; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:180; CDR-L2, which contains the amino acid sequence of SEQ ID NO:181; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:182;

[0045] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:238; CDR-H2, which contains the amino acid sequence of SEQ ID NO:239; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:233; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:16; CDR-L2, which contains the amino acid sequence of SEQ ID NO:17; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:228;

[0046] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:240; CDR-H2, which contains the amino acid sequence of SEQ ID NO:241; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:242; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:40; CDR-L2, which contains the amino acid sequence of SEQ ID NO:41; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:42;

[0047] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:238; CDR-H2, which contains the amino acid sequence of SEQ ID NO:243; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:233; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:234; CDR-L2, which contains the amino acid sequence of SEQ ID NO:235; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:236;

[0048] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:238; CDR-H2, which contains the amino acid sequence of SEQ ID NO:243; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:233; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:16; CDR-L2, which contains the amino acid sequence of SEQ ID NO:17; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:228; or

[0049] The VH domain comprises: CDR-H1, which contains the amino acid sequence of SEQ ID NO:240; CDR-H2, which contains the amino acid sequence of SEQ ID NO:244; and CDR-H3, which contains the amino acid sequence of SEQ ID NO:242; and wherein the VL domain comprises: CDR-L1, which contains the amino acid sequence of SEQ ID NO:40; CDR-L2, which contains the amino acid sequence of SEQ ID NO:41; and CDR-L3, which contains the amino acid sequence of SEQ ID NO:42.

[0050] In some respects, the VH and VL domains comprise amino acid sequences selected from the group consisting of:

[0051] (a) SEQ ID NO:58 and 59 respectively;

[0052] (b) SEQ ID NO:247 and 248 respectively;

[0053] (c) SEQ ID NO:62 and 63 respectively;

[0054] (d) SEQ ID NO:64 and 65 respectively;

[0055] (e) SEQ ID NO:66 and 67 respectively;

[0056] (f) SEQ ID NO: 68 and 69 respectively;

[0057] (g) SEQ ID NO:70 and 71 respectively;

[0058] (h) SEQ ID NO:72 and 73 respectively;

[0059] (i) SEQ ID NO: 185 and 186 respectively;

[0060] (j) SEQ ID NO:245 and 246 respectively;

[0061] (k) SEQ ID NO: 249 and 250 respectively;

[0062] (l) SEQ ID NO:251 and 252 respectively;

[0063] (m) are SEQ ID NO: 253 and 254 respectively;

[0064] (n) respectively SEQ ID NO:255 and 256; and

[0065] (o) SEQ ID NO:257 and 258 respectively.

[0066] In any of the foregoing or related aspects, (i) the IL-2 peptide is directly linked to or linked to an antibody or antigen-binding fragment via a adapter, and / or (ii) the IL-2 peptide is directly linked to or linked to an antibody or antigen-binding fragment via a adapter. In some aspects, (i) the IL-2 peptide is linked to an antibody or antigen-binding fragment via a Gly-Ser adapter, and / or (ii) the IL-2 peptide is linked to an antibody or antigen-binding fragment via a Gly-Ser adapter.

[0067] In any of the foregoing or related aspects, the IL-2 fusion protein, the IL-21 fusion protein, or both the IL-2 and IL-21 fusion protein comprise:

[0068] Two antibody heavy chain polypeptides, which contain the structure according to formula [I] from the N-terminus to the C-terminus:

[0069] VH-CH1-Hinge-CH2-CH3 [I]

[0070] And two antibody light chain polypeptides, which contain the structure according to formula [II] from the N-terminus to the C-terminus:

[0071] VL-CL [II]

[0072] Where VH is the VH domain, CH1 is the antibody CH1 domain, the hinge is the antibody hinge domain, CH2-CH3 are the antibody Fc domains, VL is the VL domain, and CL is the antibody light chain constant domain; and

[0073] The N-terminus of the IL-2 polypeptide or the IL-21 polypeptide is fused to the C-terminus of one of the two CH3 domains.

[0074] In some aspects, according to the EU index number, one or both of the antibody heavy chain peptides contain the following amino acid substitutions: L234A, L235A, and G237A. In some aspects, according to the EU index number, the first of the antibody heavy chain peptides contains the amino acid substitutions Y349C and T366W, and the second of the antibody heavy chain peptides contains the amino acid substitutions S354C, T366S, L368A, and Y407V.

[0075] In any of the foregoing or related aspects, the binding affinity of the IL-2 peptide to IL-2Rα is reduced by 50% or more compared to the binding affinity of the human IL-2 peptide comprising SEQ ID NO:81. In some aspects, the IL-2 peptide comprises one or more amino acid mutations that reduce the binding affinity of the IL-2 peptide compared to the human IL-2 peptide comprising SEQ ID NO:81. In some aspects, the IL-2 polypeptide comprises, relative to SEQ ID NO:81, the amino acid sequence of SEQ ID NO:81 having one, two, three, four, or five amino acid substitutions, and wherein said one, two, three, four, or five substitutions comprise substitutions at positions in SEQ ID NO:81 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, I92, T123, Q126, S127, I129, and S130. In some aspects, the IL-2 polypeptide (relative to the sequence of SEQ ID NO:81) comprises SEQ ID NO:81 having one of the following amino acid substitution groups. Sequence of 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; R 38E, 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, 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 aspects, the IL-2 polypeptide comprises, relative to SEQ ID NO:81, the sequence of SEQ ID NO:81 having another amino acid substitution at position C125. In some aspects, the IL-2 polypeptide (relative to the sequence of SEQ ID NO:81) comprises the sequence of SEQ ID NO:81 having one of the following amino acid substitution groups: 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; R38 E, 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 C1 25A; 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, F42 A, 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 C 125A; 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; 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; 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 aspects, the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO:80 or SEQ ID NO:297. In some aspects, the IL-2 polypeptide comprises sequences selected from the group consisting of: SEQ ID NO: 80, 85-155, 190-216, 297, and 354-383.

[0076] In any of the foregoing or related aspects, the IL-21 polypeptide comprises an amino acid sequence that is at least 80% identical to that of the human IL-21 polypeptide comprising SEQ ID NO:390. In some aspects, the isoelectric point of the IL-21 polypeptide is at least about 0.6 units to about 5 units lower than that of the human IL-21 polypeptide. In some aspects, the IL-21 polypeptide comprises at least one amino acid substitution that lowers the isoelectric point of the IL-21 polypeptide by about 0.6 units to about 5 units relative to the unsubstituted human IL-21 polypeptide. In some aspects, the IL-21 polypeptide comprises at least four amino acid substitutions that lower the isoelectric point of the IL-21 polypeptide by about 0.6 units to about 5 units relative to the unsubstituted human IL-21 polypeptide. In some aspects, the IL-21 polypeptide comprises up to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions that lower the isoelectric point. In some aspects, the isoelectric point of the human IL-21 peptide is about 9.42. In some aspects, the isoelectric point of the IL-21 peptide is from about 7.12 to about 8.72. In some aspects, the IL-21 peptide, when administered to a subject, results in improved exposure relative to the human IL-21 peptide, as measured by an area under the curve (AUC) at least about 1.5 times higher. In some aspects, the human IL-21 peptide comprises a region of about 2 to 20 positively charged amino acid residues, and wherein the IL-21 peptide comprises at least one amino acid substitution having one or more of the positively charged amino acid residues. In some aspects, the region does not include amino acid residues that bind to the IL-21 receptor. In some aspects, the region comprises amino acid residues S80 to T92 of the human IL-21 peptide containing SEQ ID NO:390. In some aspects, the human IL-21 polypeptide comprises at least one positively charged amino acid residue on the surface of the human IL-21 polypeptide having a three-dimensional structure and does not bind to the IL-21 receptor, wherein the IL-21 polypeptide comprises at least one amino acid substitution having said at least one positively charged amino acid residue. In some aspects, the IL-21 polypeptide does not contain an amino acid substitution at G84 of the human IL-21 polypeptide of SEQ ID NO:390. In some aspects, 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 aspects, the IL-21 polypeptide comprises 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions selected from the following: 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: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPX1X2X3X4GX5X6X7X8X9X. 10 X 11 X 12 CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ IDNO: 554), where X1=G, S; X2=G, T; X3=G, E, N; 10 =A, E, S, G, R; X 11 =S, G, L; and X 12 =G,S,T, with the condition X5, X6, X8, X9 and X 10 At least one of them is not an amino acid residue at the same position as described in SEQ ID NO:390, optionally wherein:

[0077] (i)X5=G,X6=G,X8=G,X 10 =A;

[0078] (ii)X5=G, X6=G, X8=G,X 10 =E;

[0079] (iii) X1=G, X2=G, X3=G, X4=S, X5=E, X6=G, X7=S, X8=G, X9=G, 10 =S;

[0080] (iv)X1=G,X2=G,X3=G,X4=S,X5=G,X6=G,X7=S,X8=G,X9=G,X 10 =E;

[0081] (v)X5=G,X6=G,X7=S,X8=G,X9=G,X 10 =E;

[0082] (vi)X5=G,X6=G,X7=S,X8=G,X9=G,X 10 =S;

[0083] (vii)X5=G,X6=G,X7=G,X8=G,X9=G,X 10 =E;

[0084] (viii)X5=G,X6=G,X7=G,X8=G,X9=G,X 10 =G;

[0085] (ix)X5=G,X6=G,X7=E,X8=G,X9=G,X 10 =G;

[0086] (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;

[0087] (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;

[0088] (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;

[0089] (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;

[0090] (xiv)X3=G,X4=G,X5=S,X6=G,X7=G,X8=G,X9=S,X10 =G,X 11 =G,X 12 =S;

[0091] (xv)X3=G,X4=G,X5=E,X6=G,X7=G,X8=G,X9=S,X 10 =G,X 11 =G,X 12 =S;

[0092] (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;

[0093] (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;

[0094] (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;

[0095] (xix)X3=G,X4=G,X5=S,X6=G,X7=G,X8=G,X9=S,X 10 =E,X 11 =G,X 12 =S;

[0096] (xx)X3=G,X4=G,X5=G,X6=G,X7=G,X8=G,X9=G,X 10 =E,X 11 =G,X 12 =G;

[0097] (xxi)X5=G,X6=G,X7=G,X8=G,X9=G,X 10 =E,X 11 =G;

[0098] (xxii)X3=G,X4=G,X5=G,X6=G,X7=G,X8=G,X9=G,X 10 =E,X 11 =G;

[0099] (xxiii) X3 = G, X4 = G, X5 = G, X6 = E, X7 = G, X8 = G, X9 = G, X 10 = E, X 11 = G;

[0100] (xxiv) X3 = G, X4 = G, X5 = E, X6 = G, X7 = G, X8 = G, X9 = G, X 10 = E, X 11 = G;

[0101] (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; or

[0102] (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.

[0103] In some aspects, the IL-12 polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence selected from SEQ ID NO:391-421. In some aspects, the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NO:391-421. In some aspects, the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO:421. In some aspects, the IL-21 polypeptide comprises at least one amino acid substitution that reduces binding to the IL-21 receptor compared to binding with the human IL-21 polypeptide. In some aspects, the at least one amino acid substitution is at one or more amino acid residues at position R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, or K117 of SEQ ID NO:390. In some aspects, the at least one amino acid substitution is selected from: R5F, R5A, R5E, R5S, R5T, R5N, R5Q, R5V, R5I, R5L, R5Y, I8E, R9A, R9D, R9E, R9H, R9S, R9T, R9N, R9G, R9V, R9I, R9L, R9Y, R11D, R11E, L13F, L13R, I14D, I16A, I16S, The amino acids are: 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 aspects, the at least one amino acid is substituted with R76E or R76Q. In some aspects, the IL-21 polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequences selected from SEQ ID NO:422-491. In some aspects, the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NO:422-491.

[0104] In any of the foregoing or related aspects, the IL-2 fusion protein comprises:

[0105] One or two light chains containing the amino acid sequence of SEQ ID NO:156, heavy chains containing the amino acid sequence of SEQ ID NO:157, and heavy chains containing the amino acid sequence of SEQ ID NO:158.

[0106] One or two light chains containing the amino acid sequence of SEQ ID NO:159, heavy chains containing the amino acid sequence of SEQ ID NO:160, and heavy chains containing the amino acid sequence of SEQ ID NO:161.

[0107] One or two light chains containing the amino acid sequence of SEQ ID NO:162, heavy chains containing the amino acid sequence of SEQ ID NO:163, and heavy chains containing the amino acid sequence of SEQ ID NO:164.

[0108] One or two light chains containing the amino acid sequence of SEQ ID NO:165, heavy chains containing the amino acid sequence of SEQ ID NO:166, and heavy chains containing the amino acid sequence of SEQ ID NO:167.

[0109] One or two light chains containing the amino acid sequence of SEQ ID NO:168, heavy chains containing the amino acid sequence of SEQ ID NO:169, and heavy chains containing the amino acid sequence of SEQ ID NO:170.

[0110] One or two light chains containing the amino acid sequence of SEQ ID NO:171, heavy chains containing the amino acid sequence of SEQ ID NO:172, and heavy chains containing the amino acid sequence of SEQ ID NO:173.

[0111] One or two light chains containing the amino acid sequence of SEQ ID NO:174, heavy chains containing the amino acid sequence of SEQ ID NO:175, and heavy chains containing the amino acid sequence of SEQ ID NO:176.

[0112] One or two light chains containing the amino acid sequence of SEQ ID NO:187, heavy chains containing the amino acid sequence of SEQ ID NO:188, and heavy chains containing the amino acid sequence of SEQ ID NO:189.

[0113] One or two light chains containing the amino acid sequence of SEQ ID NO:298, heavy chains containing the amino acid sequence of SEQ ID NO:299, and heavy chains containing the amino acid sequence of SEQ ID NO:300;

[0114] One or two light chains containing the amino acid sequence of SEQ ID NO:302, heavy chains containing the amino acid sequence of SEQ ID NO:303, and heavy chains containing the amino acid sequence of SEQ ID NO:304;

[0115] One or two light chains containing the amino acid sequence of SEQ ID NO:306, heavy chains containing the amino acid sequence of SEQ ID NO:307, and heavy chains containing the amino acid sequence of SEQ ID NO:308.

[0116] One or two light chains containing the amino acid sequence of SEQ ID NO:310, heavy chains containing the amino acid sequence of SEQ ID NO:311, and heavy chains containing the amino acid sequence of SEQ ID NO:312.

[0117] One or two light chains containing the amino acid sequence of SEQ ID NO:314, heavy chains containing the amino acid sequence of SEQ ID NO:315, and heavy chains containing the amino acid sequence of SEQ ID NO:316.

[0118] One or two light chains containing the amino acid sequence of SEQ ID NO:318, heavy chains containing the amino acid sequence of SEQ ID NO:319, and heavy chains containing the amino acid sequence of SEQ ID NO:320.

[0119] One or two light chains containing the amino acid sequence of SEQ ID NO:322, heavy chains containing the amino acid sequence of SEQ ID NO:323, and heavy chains containing the amino acid sequence of SEQ ID NO:324;

[0120] One or two light chains containing the amino acid sequence of SEQ ID NO:326, heavy chains containing the amino acid sequence of SEQ ID NO:327, and heavy chains containing the amino acid sequence of SEQ ID NO:328;

[0121] One or two light chains containing the amino acid sequence of SEQ ID NO:330, heavy chains containing the amino acid sequence of SEQ ID NO:331, and heavy chains containing the amino acid sequence of SEQ ID NO:332;

[0122] One or two light chains containing the amino acid sequence of SEQ ID NO:334, heavy chains containing the amino acid sequence of SEQ ID NO:335, and heavy chains containing the amino acid sequence of SEQ ID NO:336.

[0123] One or two light chains containing the amino acid sequence of SEQ ID NO:338, heavy chains containing the amino acid sequence of SEQ ID NO:339, and heavy chains containing the amino acid sequence of SEQ ID NO:340;

[0124] One or two light chains containing the amino acid sequence of SEQ ID NO:342, heavy chains containing the amino acid sequence of SEQ ID NO:343, and heavy chains containing the amino acid sequence of SEQ ID NO:344;

[0125] One or two light chains containing the amino acid sequence of SEQ ID NO:346, heavy chains containing the amino acid sequence of SEQ ID NO:347, and heavy chains containing the amino acid sequence of SEQ ID NO:348.

[0126] One or two light chains containing the amino acid sequence of SEQ ID NO:350, heavy chains containing the amino acid sequence of SEQ ID NO:351, and heavy chains containing the amino acid sequence of SEQ ID NO:352.

[0127] One or two light chains containing the amino acid sequence of SEQ ID NO:156, heavy chains containing the amino acid sequence of SEQ ID NO:157, and heavy chains containing the amino acid sequence of SEQ ID NO:217.

[0128] One or two light chains containing the amino acid sequence of SEQ ID NO:159, heavy chains containing the amino acid sequence of SEQ ID NO:160, and heavy chains containing the amino acid sequence of SEQ ID NO:218.

[0129] One or two light chains containing the amino acid sequence of SEQ ID NO:162, heavy chains containing the amino acid sequence of SEQ ID NO:163, and heavy chains containing the amino acid sequence of SEQ ID NO:219.

[0130] One or two light chains containing the amino acid sequence of SEQ ID NO:165, heavy chains containing the amino acid sequence of SEQ ID NO:166, and heavy chains containing the amino acid sequence of SEQ ID NO:220.

[0131] One or two light chains containing the amino acid sequence of SEQ ID NO:168, heavy chains containing the amino acid sequence of SEQ ID NO:169, and heavy chains containing the amino acid sequence of SEQ ID NO:221.

[0132] One or two light chains containing the amino acid sequence of SEQ ID NO:171, heavy chains containing the amino acid sequence of SEQ ID NO:172, and heavy chains containing the amino acid sequence of SEQ ID NO:222;

[0133] One or two light chains containing the amino acid sequence of SEQ ID NO:174, heavy chains containing the amino acid sequence of SEQ ID NO:175, and heavy chains containing the amino acid sequence of SEQ ID NO:223;

[0134] One or two light chains containing the amino acid sequence of SEQ ID NO:187, heavy chains containing the amino acid sequence of SEQ ID NO:188, and heavy chains containing the amino acid sequence of SEQ ID NO:224;

[0135] One or two light chains containing the amino acid sequence of SEQ ID NO:298, heavy chains containing the amino acid sequence of SEQ ID NO:299, and heavy chains containing the amino acid sequence of SEQ ID NO:301;

[0136] One or two light chains containing the amino acid sequence of SEQ ID NO:302, heavy chains containing the amino acid sequence of SEQ ID NO:303, and heavy chains containing the amino acid sequence of SEQ ID NO:305;

[0137] One or two light chains containing the amino acid sequence of SEQ ID NO:306, heavy chains containing the amino acid sequence of SEQ ID NO:307, and heavy chains containing the amino acid sequence of SEQ ID NO:309.

[0138] One or two light chains containing the amino acid sequence of SEQ ID NO:310, heavy chains containing the amino acid sequence of SEQ ID NO:311, and heavy chains containing the amino acid sequence of SEQ ID NO:313.

[0139] One or two light chains containing the amino acid sequence of SEQ ID NO:314, heavy chains containing the amino acid sequence of SEQ ID NO:315, and heavy chains containing the amino acid sequence of SEQ ID NO:317.

[0140] One or two light chains containing the amino acid sequence of SEQ ID NO:318, heavy chains containing the amino acid sequence of SEQ ID NO:319, and heavy chains containing the amino acid sequence of SEQ ID NO:321.

[0141] One or two light chains containing the amino acid sequence of SEQ ID NO:322, heavy chains containing the amino acid sequence of SEQ ID NO:323, and heavy chains containing the amino acid sequence of SEQ ID NO:325;

[0142] One or two light chains containing the amino acid sequence of SEQ ID NO:326, heavy chains containing the amino acid sequence of SEQ ID NO:327, and heavy chains containing the amino acid sequence of SEQ ID NO:329;

[0143] One or two light chains containing the amino acid sequence of SEQ ID NO:330, heavy chains containing the amino acid sequence of SEQ ID NO:331, and heavy chains containing the amino acid sequence of SEQ ID NO:333.

[0144] One or two light chains containing the amino acid sequence of SEQ ID NO:334, heavy chains containing the amino acid sequence of SEQ ID NO:335, and heavy chains containing the amino acid sequence of SEQ ID NO:337;

[0145] One or two light chains containing the amino acid sequence of SEQ ID NO:338, heavy chains containing the amino acid sequence of SEQ ID NO:339, and heavy chains containing the amino acid sequence of SEQ ID NO:341;

[0146] One or two light chains containing the amino acid sequence of SEQ ID NO:342, heavy chains containing the amino acid sequence of SEQ ID NO:343, and heavy chains containing the amino acid sequence of SEQ ID NO:345.

[0147] One or two light chains containing the amino acid sequence of SEQ ID NO:346, heavy chains containing the amino acid sequence of SEQ ID NO:347, and heavy chains containing the amino acid sequence of SEQ ID NO:349; or

[0148] One or two light chains containing the amino acid sequence of SEQ ID NO:350, heavy chains containing the amino acid sequence of SEQ ID NO:351, and heavy chains containing the amino acid sequence of SEQ ID NO:353.

[0149] In any of the foregoing or related aspects, the IL-2 fusion protein comprises four polypeptide chains, wherein:

[0150] 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.

[0151] 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.

[0152] 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

[0153] 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:341, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO:338.

[0154] In any of the foregoing or related aspects, the IL-21 fusion protein comprises:

[0155] One or two light chains containing the amino acid sequence of SEQ ID NO:557, heavy chains containing the amino acid sequence of SEQ ID NO:558, and heavy chains containing the amino acid sequence of SEQ ID NO:559.

[0156] One or two light chains containing the amino acid sequence of SEQ ID NO:561, heavy chains containing the amino acid sequence of SEQ ID NO:562, and heavy chains containing the amino acid sequence of SEQ ID NO:563.

[0157] One or two light chains containing the amino acid sequence of SEQ ID NO:565, heavy chains containing the amino acid sequence of SEQ ID NO:566, and heavy chains containing the amino acid sequence of SEQ ID NO:567.

[0158] One or two light chains containing the amino acid sequence of SEQ ID NO:569, heavy chains containing the amino acid sequence of SEQ ID NO:570, and heavy chains containing the amino acid sequence of SEQ ID NO:571.

[0159] One or two light chains containing the amino acid sequence of SEQ ID NO:573, heavy chains containing the amino acid sequence of SEQ ID NO:574, and heavy chains containing the amino acid sequence of SEQ ID NO:575.

[0160] One or two light chains containing the amino acid sequence of SEQ ID NO:577, heavy chains containing the amino acid sequence of SEQ ID NO:578, and heavy chains containing the amino acid sequence of SEQ ID NO:579.

[0161] One or two light chains containing the amino acid sequence of SEQ ID NO:581, heavy chains containing the amino acid sequence of SEQ ID NO:582, and heavy chains containing the amino acid sequence of SEQ ID NO:583.

[0162] One or two light chains containing the amino acid sequence of SEQ ID NO:585, heavy chains containing the amino acid sequence of SEQ ID NO:586, and heavy chains containing the amino acid sequence of SEQ ID NO:587.

[0163] One or two light chains containing the amino acid sequence of SEQ ID NO:589, heavy chains containing the amino acid sequence of SEQ ID NO:590, and heavy chains containing the amino acid sequence of SEQ ID NO:591.

[0164] One or two light chains containing the amino acid sequence of SEQ ID NO:557, heavy chains containing the amino acid sequence of SEQ ID NO:558, and heavy chains containing the amino acid sequence of SEQ ID NO:560.

[0165] One or two light chains containing the amino acid sequence of SEQ ID NO:561, heavy chains containing the amino acid sequence of SEQ ID NO:562, and heavy chains containing the amino acid sequence of SEQ ID NO:564.

[0166] One or two light chains containing the amino acid sequence of SEQ ID NO:565, heavy chains containing the amino acid sequence of SEQ ID NO:566, and heavy chains containing the amino acid sequence of SEQ ID NO:568.

[0167] One or two light chains containing the amino acid sequence of SEQ ID NO:569, heavy chains containing the amino acid sequence of SEQ ID NO:570, and heavy chains containing the amino acid sequence of SEQ ID NO:572.

[0168] One or two light chains containing the amino acid sequence of SEQ ID NO:573, heavy chains containing the amino acid sequence of SEQ ID NO:574, and heavy chains containing the amino acid sequence of SEQ ID NO:576.

[0169] One or two light chains containing the amino acid sequence of SEQ ID NO:577, heavy chains containing the amino acid sequence of SEQ ID NO:578, and heavy chains containing the amino acid sequence of SEQ ID NO:580.

[0170] One or two light chains containing the amino acid sequence of SEQ ID NO:581, heavy chains containing the amino acid sequence of SEQ ID NO:582, and heavy chains containing the amino acid sequence of SEQ ID NO:584.

[0171] One or two light chains containing the amino acid sequence of SEQ ID NO:585, heavy chains containing the amino acid sequence of SEQ ID NO:586, and heavy chains containing the amino acid sequence of SEQ ID NO:588; or

[0172] One or two light chains containing the amino acid sequence of SEQ ID NO:589, heavy chains containing the amino acid sequence of SEQ ID NO:590, and heavy chains containing the amino acid sequence of SEQ ID NO:592.

[0173] In any of the foregoing or related aspects, the IL-21 fusion protein comprises four polypeptide chains, wherein:

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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

[0191] 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:592, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO:589.

[0192] In any of the foregoing or related aspects, the IL-21 fusion protein is administered prior to the administration of the IL-2 fusion protein. In other aspects, the IL-2 fusion protein is administered prior to the administration of the IL-21 fusion protein. In some aspects, the IL-21 fusion protein and the IL-2 fusion protein are administered in parallel, sequentially, or simultaneously.

[0193] In any of the foregoing or related aspects, the IL-21 fusion protein is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some aspects, the IL-2 fusion protein is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some aspects, the IL-21 fusion protein and the IL-2 fusion protein are formulated as the same pharmaceutical composition. In other aspects, the IL-21 fusion protein and the IL-2 fusion protein are formulated as separate pharmaceutical compositions.

[0194] By incorporating via reference

[0195] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is expressly and individually indicated as being incorporated herein by reference in its entirety. Attached Figure Description

[0196] Figure 1 Schematic diagrams of the xmCD8-mIL21v1.1 and xmCD8.1-mIL2v fusion proteins are shown. Both fusions include anti-mouse CD8 IgG and a single copy of a cytokine (mIL21v1.1 or mIL2v) fused to the C-terminus of one heavy chain. In some cases, the Fc region of both schematic diagrams contains a club-and-mortar heterodimerization mutation. In some cases, the Fc region of both schematic diagrams does not contain a club-and-mortar heterodimerization mutation.

[0197] Figures 2A to 2D The efficacy study of MC38 compared single-drug administration with combinations of xmCD8-mIL21v1.1 and xmCD8.1-mIL2v administered simultaneously at different dose levels. The study showed efficacy at 0.3 mg / kg xmCD8.1-mIL2v and 0.1 mg / kg xmCD8-mIL21v1.1... Figure 2A ), 0.3 mg / kg xmCD8.1-mIL2v and 0.03 mg / kg xmCD8-mIL21v1.1 ( Figure 2B ), 0.1 mg / kg xmCD8.1-mIL2v and 0.1 mg / kg xmCD8-mIL21v1.1 ( Figure 2C ), 0.1 mg / kg xmCD8.1-mIL2v and 0.03 mg / kg xmCD8-mIL21v1.1 ( Figure 2D (Single drugs and combinations thereof)

[0198] Figure 3 The efficacy study of MC38 is shown, comparing single-agent therapy with combinations of xmCD8-mIL21v1.1 and xmCD8.1-mIL2v administered at different dose levels at 1-hour intervals. Separate groups of combination therapy are shown, where xmCD8-mIL21v1.1 or xmCD8.1-mIL2v is administered first. Single-agent therapy and combinations of 0.1 mg / kg xmCD8.1-mIL2v and 0.1 mg / kg xmCD8-mIL21v1.1 are shown. Detailed Implementation

[0199] This disclosure is at least in part based on the discovery that the combination of IL-2 and IL-21 fusion proteins (each containing an antibody that specifically binds to human CD8) reduces or inhibits tumor growth in vivo more effectively than administration of either fusion protein alone. Not wishing to be bound by theory, IL-2 and IL-21 selectively activate CD8+ T cells by targeting CD8+ T cells with anti-CD8-specific antibodies to induce an anti-tumor immune response. Since IL-2 activates CD8+ T cells via STAT5 phosphorylation and thus enhances cytotoxicity while promoting proliferation, and IL-21 activates CD8+ T cells via STAT3 phosphorylation and maintains T cell function, prevents exhaustion, promotes memory phenotype, and enhances cytotoxicity, it is believed that targeting these two independent, complementary, non-redundant pathways provides enhanced CD8+ T cell activation and thus therapeutic efficacy.

[0200] This disclosure particularly describes methods for treating diseases (e.g., cancer or infection) in mammalian subjects. 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 protein comprises 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.

[0201] definition

[0202] Unless the context clearly indicates otherwise, as used in this specification and the appended claims, the singular forms “a,” “an,” and “described” include a plurality of referred to objects. Thus, for example, reference to “molecule” optionally includes a combination of two or more such molecules, and so on.

[0203] It should be understood that aspects and implementations of this disclosure include aspects and implementations that "comprise", "consist of" aspects and implementations and "consist of substantially aspects and implementations".

[0204] As used herein, the term "about" refers to a common range of error for an individual value that is readily apparent to those skilled in the art. If the use of the term is unclear to those skilled in the art in the given context in which it is used, "about" will mean at most a particular value plus or minus 10%. In this document, reference to "about" a value or parameter includes (and describes) an implementation for said value or parameter itself.

[0205] As used herein, “area under curve” or “AUC” is a pharmacokinetic parameter, which refers to the area under a curve representing the plasma concentration of a drug (e.g., the IL-21 peptide or fusion protein described herein) relative to time after administration. In some embodiments, AUC measures a patient’s drug exposure and depends on dose, bioavailability, and clearance. Methods for determining AUC are known to those skilled in the art and include, but are not limited to, the rectangular method, the trapezoidal rule, and Simpson's rule.

[0206] As used herein, "immune cells" are the cells of the immune system that respond to organisms or other entities considered foreign to the host's immune system. They protect the host from foreign pathogens, organisms, and diseases. Immune cells, also known as white blood cells, participate in innate and adaptive immune responses against pathogens. The innate immune response occurs immediately after exposure to a pathogen and requires no additional initiation or learning process. The adaptive immune process requires initial initiation and subsequently develops memory, leading to enhanced reactivity upon subsequent encounters with the same pathogen. Innate immune cells include, but are not limited to, monocytes, macrophages, dendritic cells, innate lymphoid cells (ILCs), including natural killer (NK) cells, neutrophils, megakaryocytes, eosinophils, and basophils. Adaptive immune cells include B and T lymphocytes / cells. The T cell subset includes, but is not limited to, αβCD4+ T cells (native CD4+, memory CD4+, effector memory CD4+, effector CD4+, and regulatory CD4+) and αβCD8+ T cells (native CD8+, memory CD8+, effector memory CD8+, and effector CD8+). The B cell subset includes, but is not limited to, native B cells, memory B cells, and plasma cells. NK T cells and Tγδ (Tγδ) cells exhibit characteristics of both intrinsic and adaptive lymphocytes.

[0207] "T cells" or "T lymphocytes" are immune cells that play a crucial role in orchestrating immune responses in both health and disease. There are two main subsets of T cells with distinct functions and characteristics: T cells expressing the CD8 antigen (CD8+). +T cells are cytotoxic or killer T cells that can use cytotoxic proteins (such as granzymes and perforin) to lyse target cells; and T cells expressing the CD4 antigen (CD4+). + T cells are capable of regulating CD8+ + Helper T cells are a function of many other immune cell types, including T cells, B cells, and macrophages. Additionally, CD4+... + T cells are further subdivided into several subsets, such as T regulatory (Treg) cells, which suppress immune responses; and T helper 1 (Th1), T helper 2 (Th2), and T helper 17 (Th17) cells, which regulate different types of immune responses by secreting immunomodulatory proteins, such as cytokines. T cells recognize their targets via αβ T cell receptors that bind to unique antigen-specific motifs, and this recognition mechanism is generally required to trigger their cytotoxic and cytokine secretion functions. "Intrinsic lymphocytes" can also exhibit CD8+ expression. + and CD4 + T cells possess characteristics such as cytotoxic activity or the secretion of Th1, Th2, and Th17 cytokines. Some of these intrinsic lymphocyte subsets include NK cells as well as ILC1, ILC2, and ILC3 cells; and intrinsic-like T cells, such as Tγδ cells; and NK T cells. Typically, these cells respond rapidly to inflammatory stimuli from infected or injured tissues, such as immunomodulatory cytokines, but unlike αβ T cells, they can respond without needing to recognize antigen-specific patterns.

[0208] "Cytokines" are immunomodulatory polypeptide forms that mediate crosstalk between initiating / primary cells and target / effective cells. They can act as soluble forms or on the cell surface associated with binding to "cytokine receptors" on target immune cells to activate signal transduction. As used herein, "cytokine receptors" are polypeptides on the cell surface that activate intracellular signal transduction after binding to cytokines on the extracellular cell surface. Cytokines include, but are not limited to, chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor. Cytokines are produced by a wide range of cells, including immune cells, endothelial cells, fibroblasts, and stromal cells. A given cytokine can be produced by more than one cell type. Cytokines are pleiotropic; because receptors are expressed on multiple subsets of immune cells, a single cytokine can activate signal transduction pathways in multiple cells. However, depending on the cell type, cytokine signaling events can lead to different downstream cellular events, such as activation, proliferation, survival, apoptosis, effector function, and secretion of other immunomodulatory proteins.

[0209] The term "amino acid" as used here refers to naturally occurring carboxyl α-amino acids, including alanine (three-letter code: ala, single-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).

[0210] As used herein, "polypeptide" or "protein" refers to a molecule in which monomers (amino acids) are linearly linked together by peptide bonds (also known as amide bonds). The term "polypeptide" refers to any chain having two or more amino acids and does not refer to a specific length of the product. Therefore, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain having two or more amino acids, and the term "polypeptide" may be used in place of any of these terms, or the term "polypeptide" may be used interchangeably with any of these terms. The term "polypeptide" is also intended to indicate that the product of a polypeptide may be derived from a natural biological source or produced through recombinant technology and is not necessarily translated from a specified nucleic acid sequence. It can be produced in any way, including through chemical synthesis. Polypeptides typically have a defined three-dimensional structure, but they do not necessarily have such a structure. The size of the polypeptides disclosed herein 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. Polypeptides having a defined three-dimensional structure are called folded, and polypeptides that do not have a defined three-dimensional structure but can adopt many different conformations are called unfolded. Polypeptides can further form polymers, such as dimers, trimers, and higher-order oligomers, i.e., composed of more than one polypeptide molecule. The polypeptide molecules forming such dimers, trimers, etc., can be the same or different. The corresponding higher-order structures of such polymers are therefore called homodimers or heterodimers, homotrimers or heterotrimers, etc. The terms “peptide” and “protein” also refer to modified peptides / proteins, wherein post-expression modifications are achieved by, but are not limited to, the following: glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / capping groups, protein cleavage, or modification by non-naturally occurring amino acids.

[0211] As used herein, “identity” refers to the percentage (%) of sequence identity relative to a reference polypeptide sequence. This percentage is the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after sequence alignment and, where necessary, the introduction of vacancies to achieve the maximum sequence identity percentage, and without considering any conserved substitutions as part of sequence identity. Alignments for determining the amino acid sequence identity percentage can be performed in various known ways; for example, 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 full length of the compared sequences. However, for the purposes of this document, the sequence alignment computer program ALIGN-2 is used to generate the amino acid sequence identity % value. The ALIGN-2 sequence alignment computer program was designed by Genentech, Inc., and its source code is registered with the U.S. Copyright Office, Washington DC, 20559, under U.S. Copyright Registry No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from source code. The ALIGN-2 program can be compiled for use with UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0212] When using ALIGN-2 for amino acid sequence comparison, the amino acid sequence identity % of a given amino acid sequence A with respect to, and with, or against a given amino acid sequence B (which can be alternatively stated as the given amino acid sequence A having or containing a specific amino acid sequence identity % with respect to, and with, or against the given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X / Y, where X is the number of amino acid residues with the same matching score in the sequence alignment program ALIGN-2 in the A and B alignments of the procedure, and where Y is the total number of amino acid residues in B. It should be understood that if the lengths of amino acid sequences A and B are not equal, the amino acid sequence identity % of A with respect to B will not be equal to the amino acid sequence identity % of B with respect to A. Unless otherwise specifically stated, all amino acid sequence identity % values ​​used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0213] As used in this article, "residue" refers to a position in a protein and its identity with its associated amino acid. For example, Leu234 (also known as Leu234 or L234) is the residue at position 234 in the human antibody IgG1.

[0214] In this article, "wild-type" refers to the amino acid or nucleotide sequence found in nature, including allelic variations. Wild-type proteins have an unmodified amino acid or nucleotide sequence.

[0215] "Substitution" refers to a change in a polypeptide or nucleic acid encoding the polypeptide in which a naturally occurring amino acid or nucleotide in the wild-type sequence of the polypeptide or nucleic acid is replaced at the same position in the sequence with another non-naturally occurring amino acid or nucleotide. In some embodiments, one or more substitutions are introduced to alter the affinity of the polypeptide for its receptor, thereby altering its activity so that its affinity and activity differ from those of the wild-type homologous polypeptide. In some embodiments, substitutions improve the physiological properties of the polypeptide. Amino acid substitutions can be produced using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods for altering amino acid side chain groups by means other than genetic engineering, such as chemical modification, are also considered applicable.

[0216] "Mutation" refers to a change in a polypeptide or nucleic acid encoding the polypeptide in the following ways: i) an amino acid and / or nucleotide in the wild-type sequence has been substituted for another amino acid or nucleotide, ii) an amino acid and / or nucleotide in the wild-type sequence has been deleted, and / or iii) an amino acid and / or nucleotide has been inserted.

[0217] As used herein, “CD8” refers to any native human CD8. Unless otherwise explicitly indicated or contextually indicated, reference to “CD8” refers to CD8aa and / or CD8ab. The amino acid sequence of an exemplary human CD8b (the β chain of human CD8) is described under UniProt P10966 (CD8B_HUMAN). “CD8a” refers to the α chain of human CD8 (e.g., as described under 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 both unprocessed forms and mature forms produced by cellular processing. “CD8,” “CD8a,” “CD8b,” “CD8aa,” and “CD8ab” also include, but are not limited to, naturally occurring variants, such as one or more alleles or splice variants.

[0218] As used herein, unless otherwise indicated, “interleukin-2” or “IL-2” refers to any native human IL-2 or a variant thereof. In some cases, “IL-2” encompasses both unprocessed IL-2 and “mature IL-2” as a form of IL-2 produced by processing within the cell. An exemplary form of unprocessed native human IL-2 consists of an additional N-terminal amino acid signal peptide linked to mature IL-2. “IL-2” also includes, but is not limited to, naturally occurring variants of IL-2, such as one or more alleles or splice variants. An exemplary amino acid sequence of native human IL-2 is described under UniProt P60568(IL2_HUMAN).

[0219] “Affinity” or “binding affinity” refers to the sum strength of the 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 indicated, as used herein, “binding affinity” refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). Affinity can generally be determined by the dissociation constant (K0). D The ratio denoted by is the ratio of the dissociation rate constant to the binding rate constant (koff and kon, respectively). Therefore, equivalent affinity can contain different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by commonly known methods in the art, such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) techniques (e.g., BIAcore), biolayer interferometry (BLI) techniques (e.g., Octet), and other conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0220] As used herein, "binding" or "specific binding" refers to the ability of a polypeptide or antigen-binding molecule to selectively interact with a receptor for the polypeptide or target antigen, and this specific interaction may be distinct from non-targeted, undesirable, or non-specific interactions. Examples of specific binding include, but are not limited to, the binding of IL-2 cytokines to their specific receptors (e.g., IL-2Rα, IL-2Rβ, and IL-2Rγ), and the binding of antigen-binding molecules to specific antigens (e.g., CD8 or PD-1).

[0221] A "mutant IL-2 peptide" refers to an IL-2 peptide with reduced affinity for its receptor, whereby this reduced affinity results in decreased biological activity of the mutant. Reduced affinity and the resulting reduced activity can be achieved by introducing small amounts of amino acid mutations or substitutions. The peptide backbone of a mutant IL-2 peptide may also have other modifications, including but not limited to amino acid deletions, alterations, cyclizations, disulfide bonds, or post-translational modifications (e.g., glycosylation or altered carbohydrates); chemical or enzymatic modifications (e.g., linking PEG to the peptide backbone); addition of peptide tags or labels; or fusion with proteins or protein domains to produce a final construct with the desired characteristics (e.g., reduced affinity for IL-2Rβγ). The desired activity may also include improved physiological properties compared to the wild-type IL-2 peptide. Multiple modifications can be combined to achieve the desired activity alteration, such as reduced affinity or improved physiological properties. As a non-limiting example, a common N-linked glycosylated amino acid sequence can be incorporated into the peptide to allow glycosylation. Another non-limiting example is the incorporation of lysine residues into the peptide to enable polyethylene glycol (PEG) ligation. Preferably, one or more mutations are introduced into the peptide to alter its activity.

[0222] In some cases, a “mutant IL-21 peptide” refers to an IL-21 peptide with reduced affinity for its receptor, where such reduced affinity would result in decreased biological activity of the mutant. Reduced affinity and the resulting reduced activity can be achieved by introducing small amounts of amino acid mutations or substitutions. The peptide backbone of a mutant IL-21 peptide may also have other modifications, including but not limited to amino acid deletions, alterations, cyclizations, disulfide bonds, or post-translational modifications (e.g., glycosylation or altered carbohydrates); chemical or enzymatic modifications (e.g., linking PEG to the peptide backbone); addition of peptide tags or labels; or fusion with proteins or protein domains to produce a final construct with the desired characteristics (e.g., reduced affinity for IL-21R). The desired activity may also include improved physiological properties compared to the wild-type IL-21 peptide. Multiple modifications can be combined to achieve the desired activity alteration, such as reduced affinity or improved physiological properties. As a non-limiting example, a common N-linked glycosylated amino acid sequence may be incorporated into the peptide to allow glycosylation. Another non-limiting example is the incorporation of lysine into a polypeptide to enable polyethylene glycol esterification. Preferably, one or more mutations are introduced into the polypeptide to alter its activity.

[0223] The terms “antibody” and “immunoglobulin” are used interchangeably and in the broadest sense herein, and encompass a wide range of antibody structures, including but not limited to monoclonal antibodies (e.g., full-length or complete monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and single-domain antibodies (as described in more detail herein), as long as they exhibit the desired antigen-binding activity.

[0224] Antibodies (immunoglobulins) are proteins with structures substantially similar to those of native antibodies. "Native antibodies" are naturally occurring immunoglobulin molecules with different structures. For example, IgG-type native immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy chain domain or heavy chain variable domain; followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called the variable light chain domain or light chain variable domain; followed by the light chain constant domain (CL), also called the light chain constant region. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known and broadly described, for example, in Abbas et al., 2000, Cellular and Mol, and Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). Antibodies (immunoglobulins) are classified into different classes based on the amino acid sequence of their heavy chain constant domain. There are five main classes of antibodies: α (IgA), δ (IgD), γ (IgE), γ (IgG), or μ (IgM), some of which can be further subdivided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be classified into one of two types based on the amino acid sequence of their constant domain, referred to as kappa (κ) and lambda (λ). Immunoglobulins are basically composed of two Fab molecules and an Fc domain connected by an immunoglobulin hinge region.

[0225] 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 a constant region. The terminology includes native sequence Fc regions and variant Fc regions. Fc may refer to the last two constant immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD, and IgG; the last three constant immunoglobulin domains of IgE and IgM; and optionally, all or part of the flexible hinge at the N-terminus of these domains. For IgA and IgM, Fc may include a J-chain. The IgG Fc region contains the IgG CH2 and IgG CH3 domains and, in some cases, includes the hinge. Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known 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 an amino acid residue at approximately position 216 to an amino acid residue at approximately position 230. The hinge region as used herein may be a native hinge domain or a variant hinge domain. The "CH2 domain" of the human IgG Fc region typically extends from an amino acid residue at approximately position 231 to an amino acid residue at approximately position 340. The CH2 domain as used herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises an extension from the C-terminus of a residue in the Fc region to the CH2 domain, from an amino acid residue at approximately position 341 to an amino acid residue at approximately position 447 in IgG. The CH3 region as used herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "pump" ("pestle") in one chain and a correspondingly introduced "cavity" ("mortar") in the other chain; see U.S. Patent No. 5,821,333, which is expressly incorporated herein by reference). Therefore, the definition of an "Fc domain" includes amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or fragments thereof. In this case, the "Fc fragment" may contain fewer amino acids from either the N-terminus or C-terminus, but may still form a dimer with another Fc domain or Fc fragment, which can be detected using standard methods, typically based on size (e.g., non-denaturing chromatography, size exclusion chromatography, etc.). In this disclosure, the human IgG Fc domain is specifically used and may be an Fc domain derived from human IgG1, IgG2, or IgG4.

[0226] A “variant Fc domain” or “Fc variant” or “mutant Fc” contains amino acid modifications (e.g., substitutions, additions, and deletions) compared to the parental Fc domain. The term also includes naturally occurring allelic variants of the immunoglobulin Fc region. Generally, the variant Fc domain has at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with the corresponding parental human IgG Fc domain (using the identity algorithm discussed below, one embodiment of which utilizes the BLAST algorithm with default parameters as known in the art). Alternatively, the 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 modifications compared to the parental Fc domain. For example, the N-terminus or C-terminus of the immunoglobulin Fc region may be missing one or more amino acids without substantial loss of biological function. In addition, as discussed in this paper, the variant Fc domains in this paper retain the ability to form dimers with another Fc domain, as measured using known techniques as described in this paper, such as non-denaturing gel electrophoresis.

[0227] As used in this article, “Fcγ receptor,” “FcγR,” or “Fc gamma R” refers to any member of the protein family that binds to the Fc region of IgG antibodies and is encoded by the FcγR gene. In humans, this family includes, but is not limited to: FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including alloforms H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including alloforms V158 and F158) and FcγRIIIb (including alloforms FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, the entire reference is incorporated by reference) and any undiscovered human FcγR or FcγR isoforms or alloforms. FcγR can be derived from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγR includes but is not limited to FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(CD16-2), as well as any undiscovered mouse FcγR or FcγR isoforms or allotypes.

[0228] As used herein, “effective function” refers to a biochemical event resulting from the interaction of the antibody’s Fc region with an Fc receptor or ligand, which varies with antibody isotype. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), cytokine secretion, antigen uptake by immune complex-mediated antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcR (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause target cell lysis. ADCC is associated with binding to FcγRIIIa; increased binding to FcγRIIIa leads to increased ADCC activity. To assess the ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed, such as the in vitro ADCC assay described in U.S. Patent Nos. 5,500,362 or 5,821,337. As used herein, “ADCP” or antibody-dependent cell-mediated phagocytosis refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize a bound antibody on a target cell and subsequently induce phagocytosis of the target cell.

[0229] The terms “Fc null” and “Fc null variant” are used interchangeably and herein to describe modified Fcs with reduced or eliminated effector function. Such Fc nulls or Fc null variants reduce or eliminate the effector function of FcγR and / or complement receptors. Preferably, the effector function of such Fc nulls or Fc null variants is eliminated. Exemplary modification methods include, but are not limited to, chemical alterations, amino acid residue substitutions, insertions, and deletions. The exemplary amino acid positions (numbered according to the EU numbering scheme) on the Fc molecule in which one or more modifications are introduced to reduce the effector function of the resulting variant are at the following 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.Exemplary Fc molecules with reduced effector function include those having 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, L234F / L235E / P3 31S, 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 / A327 G and E233P / L234V / L235A / G236del, L234A / L235A / G237 are missing; ii) IgG2: A330S / A331S, V234A / G237A, V2 34A / 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.

[0230] As used herein, an epitope is a determinant, called a complementary site, that specifically binds to the variable region of an antibody molecule. Epitopes are groups of molecules, such as amino acids or sugar side chains, and typically possess specific structural and charge characteristics. A single antigen may have more than one epitope. An epitope may contain amino acid residues that directly participate in binding and other amino acid residues that do not directly participate in binding, such as amino acid residues that are effectively blocked by the antigen-binding peptide (in other words, the amino acid residues are located within the coverage region of the antigen-binding peptide). Epitopes may be conformational or linear. Epitopes typically comprise at least three and more often at least five or eight to ten amino acids. Antibodies that recognize the same epitope can be verified in a simple immunoassay (e.g., "binning") that demonstrates the ability of one antibody to block the binding of another antibody to a target antigen.

[0231] As used herein, a "linker" refers to a molecule that links two polypeptide chains. A linker can be a polypeptide linker or a synthetic chemical linker (e.g., see Protein Engineering, 9(3), 299-305, 1996). The length and sequence of a polypeptide linker are not specifically limited and can be selected by those skilled in the art for their purposes. A polypeptide linker comprises one or more amino acids. In some embodiments, the polypeptide linker is a peptide of at least 5 amino acids in length, preferably 5 to 100 amino acids, more preferably 10 to 50 amino acids. In one embodiment, the polypeptide linker is G, S, GS, SG, SGG, GGS, and GSG (where G = glycine and S = serine). In another embodiment, the peptide linker is (GGGS)xGn (SEQ ID NO:74), (GGGGS)xGn (SEQ ID NO:75), (GGGGGS)xGn (SEQ ID NO:76), S(GGGS)xGn (SEQ ID NO:386), 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 connector comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:79) or SGGGGSGGGGSGGGGS (SEQ ID NO:389). Synthetic chemical linkers include crosslinking agents commonly used to crosslink peptides, such as N-hydroxysuccinimide (NHS), disuccinimide succinate (DSS), bis(succinimide) succinate (BS3), dithiobis(succinimide propionate) (DSP), dithiobis(succinimide propionate) (DTSSP), ethylene glycol bis(succinimide succinate) (EGS), ethylene glycol bis(succinimide sulfonyl succinate) (sulfo-EGS), disuccinimide tartrate (DST), disulfonyl tartrate (sulfo-DST), bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone (BSOCOES), and bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES).

[0232] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct encoding the polypeptide of this disclosure, such as messenger RNA (mRNA), viral RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in the polynucleotide. In some aspects, one or more vectors (particularly expression vectors) containing such nucleic acids are provided. In one aspect, a method for manufacturing the polypeptide of this disclosure is provided, wherein the method includes culturing a host cell containing nucleic acid encoding the polypeptide under conditions suitable for polypeptide expression and recovering the polypeptide from the host cell. "Recombinant" means the production of a protein in a foreign host cell using recombinant nucleic acid technology. For the purposes of this disclosure, recombinant proteins expressed in host cells are considered isolated, including isolated, partially isolated, or partially or substantially purified native or recombinant proteins using any suitable technique.

[0233] When used to describe the various peptides disclosed herein, "isolated" means a peptide that has been identified, isolated, and / or recovered from cells or cell cultures expressing the peptide. Typically, isolated peptides are purified through at least one purification step. No desired purity level is specified; "purified" or "refined" means that the concentration of the target protein in the composition is increased relative to the concentration of contaminants 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.

[0234] The term "cancer" refers to a physiological disorder in mammals characterized by unregulated and abnormal cell growth with the potential to invade or spread to other parts of the body. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include lung cancer, small cell lung cancer, non-small cell lung cancer (NSCL), bronchioloalveolar cell lung cancer, squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, head and neck cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eye, thyroid cancer, uterine cancer, gastrointestinal cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer, colon cancer, breast cancer, endometrial cancer, uterine cancer, fallopian tube cancer, cervical cancer, vaginal cancer, vulvar cancer, and Hodgkin's disease. Diseases, including esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, bladder cancer, liver cancer, liver tumor, hepatocellular carcinoma, cervical cancer, salivary gland cancer, bile duct cancer, central nervous system (CNS) lesions, spinal axis tumors, brainstem gliomas, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, neuroblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas, and Ewings sarcoma, including refractory forms of any of the above cancers, or combinations of one or more cancers.

[0235] In some contexts, the terms "combination therapy" or "combination treatment" refer to the administration of two or more active agents or therapies in a single regimen that will provide a combined beneficial effect. This includes co-administration of active agents or therapies in a substantially simultaneous manner (e.g., as a single formulation of active agents in a fixed ratio) or in a sequential manner (e.g., as a combination of individual formulations of each active agent or therapy). Combination therapy also includes combinations in which individual elements (e.g., active ingredients or therapies) may act in combination at different times and / or through different pathways to provide a beneficial effect through synergistic action or the pharmacokinetic and pharmacodynamic effects of the individual agents or treatments of the combination therapy.

[0236] Fusion protein

[0237] This document provides IL-2 and IL-21 fusion proteins comprising any of the anti-CD8 antibodies, antigen-binding domains, or antibody fragments disclosed herein. In some embodiments, the fusion proteins of this disclosure comprise: a first portion comprising a human or humanized antibody or antigen-binding fragment thereof that specifically binds to CD8 (e.g., any 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.

[0238] In some embodiments, the fusion protein described herein induces activation of cells expressing human CD8ab heterodimers with at least 2-fold, at least 5-fold, or at least 10-fold higher efficacy than the activation induced in cells expressing human CD8aa homodimers. In some embodiments, the fusion protein induces activation of CD8+ T cells with at least 2-fold, at least 5-fold, or at least 10-fold higher efficacy than the activation induced in NK cells. In some embodiments, activation efficacy is measured using EC50 as assessed by cell proliferation. Exemplary assays are further described herein.

[0239] Fusion protein type

[0240] In some embodiments, the first portion comprises an antibody (e.g., an anti-CD8 antibody of this disclosure). In some embodiments, the first portion comprises an antibody fragment (e.g., an anti-CD8 antibody fragment of this 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 (e.g., an anti-CD8 antibody of this disclosure) one or two antibody heavy chain polypeptides and one or two antibody light chain polypeptides. In some embodiments, the first portion comprises three heavy chain CDRs and / or three light chain CDRs of a single anti-CD8 antibody of this disclosure, as shown in Tables 1-3, for example. In some embodiments, the first portion comprises the VH and / or VL domains of a single anti-CD8 antibody of this disclosure, as shown in Table 3, for example. In some embodiments, the first portion further comprises one or two human IgG Fc domains. In some embodiments, the one or two human IgG Fc domains are IgG1, IgG2, IgG3, or IgG4 Fc domains. In some embodiments, the one or two human IgG Fc domains do not have a C-terminal lysine residue. In some embodiments, the one or both human IgG Fc domains contain amino acid modifications (e.g., substitution, deletion, addition, etc.). In some embodiments, the Fc domain modifications promote heterodimer formation (e.g., as shown in Table 4). In some embodiments, the one or both Fc domains contain an Fcγ null mutation.

[0241] In some embodiments, the first portion comprises: two antibody heavy chain polypeptides having a structure according to formula [I] from the N-terminus to the C-terminus:

[0242] VH-CH1-Hinge-CH2-CH3 [I]

[0243] And two antibody light chain polypeptides, which contain the structure according to formula [II] from the N-terminus to the C-terminus:

[0244] VL-CL [II]

[0245] Wherein VH is the VH domain, CH1 is the antibody CH1 domain, the hinge is the antibody hinge domain, CH2-CH3 are the antibody Fc domains, VL is the VL domain, and CL is the antibody light chain constant domain. In some embodiments, the N-terminus of the second portion is fused to the C-terminus of one of the two CH3 domains.

[0246] In some embodiments, the first portion comprises: a first antibody heavy chain polypeptide having a structure according to formula [I] from the N-terminus to the C-terminus:

[0247] VH-CH1-hinge-CH2-CH3 [I],

[0248] Antibody light chain polypeptide, which comprises the structure according to formula [II] from the N-terminus to the C-terminus:

[0249] VL-CL [II],

[0250] The second antibody heavy chain polypeptide, which comprises, from the N-terminus to the C-terminus, the structure according to formula [III]:

[0251] Hinge -CH2-CH3 [III],

[0252] Wherein VH is the VH domain, CH1 is the antibody CH1 domain, the hinge is the antibody hinge domain, CH2-CH3 are the antibody Fc domains, VL is the VL domain, and CL is the antibody light chain constant 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.

[0253] In some embodiments, the first portion comprises: a first antibody heavy chain polypeptide having a structure according to formula [I] from the N-terminus to the C-terminus:

[0254] VH-CH1-hinge-CH2-CH3 [I],

[0255] Antibody light chain polypeptide, which comprises the structure according to formula [II] from the N-terminus to the C-terminus:

[0256] VL-CL [II],

[0257] The second antibody heavy chain polypeptide, which comprises, from the N-terminus to the C-terminus, the structure according to formula [III]:

[0258] Hinge -CH2-CH3 [III],

[0259] Wherein VH is the VH domain, CH1 is the antibody CH1 domain, the hinge is the antibody hinge domain, CH2-CH3 are the antibody Fc domains, VL is the VL domain, and CL is the antibody light chain constant domain. In some embodiments, the C-terminus of the second portion is fused to the N-terminus of the hinge domain of the second antibody heavy chain polypeptide.

[0260] In some embodiments, the anti-CD8 antibody of this disclosure is a multispecific (e.g., bispecific) antibody or antibody fragment. For example, in some embodiments, the multispecific antibody (e.g., 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 the target of interest. In some embodiments, the bispecific antibody may be generated by fusing an additional binding site to the heavy or light chain of an immunoglobulin. Examples of additional binding sites include, but are not limited to, variable regions, scFv, Fab, VHH, and peptides.

[0261] In some implementations, the recombinant bispecific antibodies disclosed herein can be very roughly classified into two categories: i) types generated by combining only the variable region and ii) types combining the variable region and the Fc domain. Representatives of the first category include tandem scFv (taFv), bifunctional antibodies (Db), DART, single-chain bifunctional antibodies (scDb), Fab-Fc, tandem Fab, dual-variable-region Fab, and tandem dAb / VHH. The two variable regions can be linked together via covalent bonds or non-covalent interactions.

[0262] Non-covalent interactions may involve using heterodimerization modules, such as leucine zippers, dock-and-lock methods using regulatory subunits of cAMP-dependent protein kinases (PKA), and anchoring domains or mortar-and-groove CH3 domains of kinase A-anchored proteins (AKAP) (US Patent Nos. 5,731,168; 7,695,936; Ridgway et al., ProtEng 9,617-621 (1996) and Carter, J Immunol Meth 248,7-15 (2001)) to pair variable regions.

[0263] In some implementations, bispecific antibodies are generated on a natural immunoglobulin architecture containing two pairs of heavy and light chains, each pair having different binding specificities. Homodimerization of the two heavy chains in IgG is mediated by CH3 interactions. To promote heterodimerization, genetic modifications are introduced into the two corresponding CH3 regions. Heterodimerization mutations typically involve steric repulsion, charge-directed interactions, or interchain disulfide bond formation. Exemplary and non-restrictive Fc modifications that promote heterodimerization include the following:

[0264] Table 4. Exemplary Fc modifications that promote heterodimerization.

[0265]

[0266]

[0267]

[0268] In some implementations, bispecific antibodies can be generated by post-production assembly from haptens, thus addressing heavy and light chain mismatch issues. These antibodies typically contain modifications that facilitate heterodimerization of the hapten. Exemplary systems include, but are not limited to, the mortar-and-mortar, IgG1 (EEE-RRR), IgG2 (EEE-RRRR) (Strop et al., J Mol Biol (2012)), and dimeric (F405L-K409R) listed in Table 4. In this case, the hapten is generated and purified individually in separate cell lines. The purified antibody is then moderately reduced to obtain the hapten, which is then assembled into a bispecific antibody. The heterodimeric bispecific antibody is then purified from the mixture using conventional purification methods.

[0269] In some implementations, a bispecific antibody generation strategy independent of preferred chain pairing can also be employed. These strategies typically involve introducing genetic modifications to the antibody in a manner that imparts different biochemical or physiological properties to the heterodimer compared to the homodimer; thus, the assembled or expressed heterodimer can be selectively purified from the homodimer. One example is the introduction of H435R / Y436F into the CH3 domain of IgG1 to eliminate the binding of Fc to protein A resin, followed by co-expression of an H435R / Y436F variant with wild-type Fc. Compared to the strong interaction of the homodimer wild-type antibody, the resulting homodimer containing two copies of H435R / Y436F cannot bind to the protein A column, while the heterodimer containing one mutant copy of H435R / Y436F exhibits reduced affinity for protein A (Tustian et al., Mabs 2016). Other examples include κ / λ antibodies (Fischer et al., Nature Communications 2015) and the introduction of different charges on individual chains (E357Q, S267K, or N208D / Q295E / N384D / Q418E / N421D) (US2018 / 0142040 A1; Strop et al. J MolBiol (2012)).

[0270] In some implementations, bispecific antibodies may be generated by fusing an additional binding site to the heavy or light chain of an immunoglobulin. Examples of additional binding sites include, but are not limited to, variable regions, scFv, Fab, VHH, and peptides.

[0271] Fc region of fusion protein

[0272] In some embodiments, the antibody or fusion protein of this 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., the 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., the IgG2 Fc region) comprises substitutions at one or more of the following positions: V234, G237, D265, H268, N297, V309, A330, A331, and K322. In some embodiments, the Fc region (e.g., the IgG4 Fc region) includes substitutions at one or more of the following locations: L235, G237, D265, and E318. In some embodiments, the Fc region (e.g., the IgG1 Fc region)... The Fc region contains one or more of the following mutations or mutation groups: 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 / G237 A / A330S / A331S, L234A / L235A / P329G, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L 235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G and E233P / L234V / L235A / G236del, L234A / L235A / G237del. In some implementations, the Fc region (e.g., the IgG2 Fc region) contains one or more of the following mutations or mutation groups: 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., 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: L234A, L235A, G237A, and K322A, according to the EU index numbering. In some embodiments, the Fc region comprises one, two, or all of the following mutations: L234A, L235A, and G237A, according to the EU index numbering.

[0273] In some embodiments, according to the EU numbering, the first Fc domain and the second Fc domain of the fusion protein comprise one or more of the following Fc mutations that reduce effector function: L234A, L235A, G237A, and K322A. In some embodiments, according to the EU numbering, the first Fc domain and the second Fc domain of the fusion protein comprise the following Fc mutations that reduce effector function: L234A, L235A, and G237A. In some embodiments, according to the EU numbering, the first Fc domain and the second Fc domain of the fusion protein comprise the following Fc mutations that reduce effector function: L234A, L235A, G237A, and K322A. In some embodiments, the first Fc domain and the second Fc domain of the fusion protein comprise the following amino acid substitutions that promote heterodimer formation: Y349C / T366W (杵) and S354C, T366S, L368A, and Y407V (臼).

[0274] In some embodiments, the heterodimer mutations and / or mutations that alter Fcγ receptor binding cause a decrease in Fc stability. Accordingly, additional mutations are added to the Fc region to increase 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, are introduced into the Fc region. Another example is the introduction of S228P into an IgG4-based bispecific antibody to stabilize the hinge disulfide bond. Additional examples include the introduction of the K338I, A339K, and K340S mutations to enhance Fc stability and aggregation resistance (Gao et al., 2019 Mol Pharm. 2019;16:3647).

[0275] Antibodies and antigen-binding domains

[0276] In some embodiments, the fusion protein described herein comprises an antibody, antibody fragment, or antigen-binding domain that specifically binds to human CD8b and / or human CD8ab. Any of the anti-CD8 antibodies disclosed herein (e.g., those that specifically bind to human CD8b and / or human CD8ab) may be used in the fusion proteins, methods, and uses disclosed herein.

[0277] In some embodiments, the anti-CD8 antibody of this disclosure specifically binds to human CD8b and / or human CD8ab with an affinity 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 higher than its affinity for binding to, for example, human CD8a and / or human CD8aa expressed on natural killer (NK) cells. In some embodiments, the anti-CD8 antibody of this disclosure specifically binds to human CD8b and / or human CD8ab with an affinity at least 10-fold higher than its affinity for binding to, for example, human CD8a and / or human CD8aa expressed on natural killer (NK) cells. In some embodiments, human CD8b and / or human CD8ab are expressed on the surface of human cells, such as human T cells.

[0278] In some embodiments, the anti-CD8 antibody of this disclosure specifically binds to cells (e.g., human T cells) expressing human CD8ab heterodimers on their surface at an EC50 of less than 1000 nM. In some embodiments, the anti-CD8 antibody of this disclosure specifically binds to human CD8+ T cells.

[0279] In some embodiments, the anti-CD8 antibody of this disclosure is a human antibody or antibody fragment. In some embodiments, the human antibody or antibody fragment contains a human-derived CDR and frame sequence in the variable domain, for example, isolated from humans or generated using a library containing human antibody sequences (e.g., CDR sequences). In some embodiments, the anti-CD8 antibody of this disclosure is a humanized antibody or antibody fragment. In some embodiments, the humanized antibody or antibody fragment contains a non-human-derived CDR (e.g., from mice, rabbits, goats, etc.) and a human-derived frame sequence in the variable domain. In some embodiments, the human or humanized antibody also contains a human Fc region. In some embodiments, the human Fc region also contains one or more Fc mutations, such as those disclosed herein. There are five main classes of complete antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0280] Various definitions of the CDR sequence of an antibody variable domain are known in the art. Unless otherwise specified, the CDR sequence is described herein according to the Kabat definition (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Vols. 1-3). However, other definitions are known and contemplated for use. For example, in some embodiments, the CDR sequence may be described by the Chothia definition (see, for example, Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Depending on the specific CDR definition used, the exact frame sequence may vary, but as is known in the art, the first frame sequence (FW-1) refers to the sequence from the N end of the VH or VL domain to the start of CDR-H1 / CDR-L1, the second frame sequence (FW-2) refers to the sequence from the end of CDR-H1 / CDR-L1 to the start of CDR-H2 / CDR-L2, the third frame sequence (FW-3) refers to the sequence from the end of CDR-H2 / CDR-L2 to the start of CDR-H3 / CDR-L3, and the fourth frame sequence (FW-4) refers to the sequence from the end of CDR-H3 / CDR-L3 to the C-end boundary of the VH or VL domain.

[0281] Other numbering systems known in the art are suitable for numbering the CDRs of the VH and / or VL amino acid sequences described herein. For example, IMGT (INTERN ATIONAL IMMUNOGENETICS INFORMATIONSYSTEM) numbering of variable regions is the numbering of residues in the variable heavy or light chains of immunoglobulins 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), and is explicitly incorporated herein by reference in its entirety. As used herein, “IMGT sequence numbering” or “numbering according to IMGT” means numbering the sequence encoding the variable region according to IMGT. For heavy chain variable domains, when numbered according to IMGT, the hypervariable region of CDR1 is in the range of amino acid positions 27 to 38, the hypervariable region of CDR2 is in the range of amino acid positions 56 to 65, and the hypervariable region of CDR3 is in the range of amino acid positions 105 to 117. For the light chain variable domains, when IMGT numbered, the hypervariable region of CDR1 is located at amino acid positions 27 to 38, the hypervariable region of CDR2 is located at amino acid positions 56 to 65, and the hypervariable region of CDR3 is located at amino acid positions 105 to 117. When CDRs are numbered according to IMGT, the heavy chain framework (FR) residues are located approximately at residues 1-26 (HC-FR1), 39-55 (HC-FR2), 66-104 (HC-FR3), and 118-128 (HC-FR4) in the heavy chain, and the light chain FR residues are located approximately at residues 1-26 (LC-FR1), 39-55 (LC-FR2), 66-104 (LC-FR3), and 118-128 (LC-FR4).

[0282] In some embodiments, the anti-CD8 antibody of this 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, the anti-CD8 antibody of this disclosure comprises 1, 2, or 3 heavy chain CDRs of antibody xhCD8v1 (e.g., shown in Tables 1-3) and / or 1, 2, or 3 light chain CDRs of antibody xhCD8v1 (e.g., shown in Tables 1-3). In some embodiments, the antibody is a humanized antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2 and CDR-H3 of the sequence from SEQ ID NO:58; and a VL domain comprising CDR-L1, CDR-L2 and CDR-L3 of the sequence from SEQ ID NO:59.

[0283] In some embodiments, the anti-CD8 antibody of this 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, the anti-CD8 antibody of this 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 a humanized antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2 and CDR-H3 of the sequence from SEQ ID NO:185; and a VL domain comprising CDR-L1, CDR-L2 and CDR-L3 of the sequence from SEQ ID NO:186.

[0284] In some embodiments, the anti-CD8 antibody of this 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 that is 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:63. In some embodiments, the anti-CD8 antibody of this 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 a humanized antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO:62; and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO:63.

[0285] In some embodiments, the anti-CD8 antibody of this 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 that is 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:65. In some embodiments, the anti-CD8 antibody of this 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 a humanized antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO:64; and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO:65.

[0286] In some embodiments, the anti-CD8 antibody of this 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 that is 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:67. In some embodiments, the anti-CD8 antibody of this 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 a humanized antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO:66; and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO:67.

[0287] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 a VL domain comprising: 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. In some embodiments, the VH domain comprises an amino acid sequence that is 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:69. In some embodiments, the anti-CD8 antibody of this 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 a humanized antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO:68; and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO:69.

[0288] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 a VL domain comprising: 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. In some embodiments, the VH domain comprises an amino acid sequence that is 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:71. In some embodiments, the anti-CD8 antibody of this 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 a human antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO:70; and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO:71.

[0289] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 a VL domain comprising: 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. In some embodiments, the VH domain comprises an amino acid sequence that is 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:73. In some embodiments, the anti-CD8 antibody of this 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 a human antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO:72; and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO:73.

[0290] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: CDR-H1, which comprises the amino acid sequence X1X2A1S, wherein X1 is S, K, G, N, R, D, T, or G, and wherein X2 is Y, L, H, or F (SEQ ID NO: 259); and CDR-H2, which comprises the amino acid sequence X1X2X3PX4X5X6X7X8X9YX. 10 QKFX 11 G, where 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, X 10 For A or N, and X 11The amino acid sequence X1X2X3GX4X5LFX6X7 is Q or T (SEQ ID NO:260); and CDR-H3, which contains the amino acid sequence X1X2X3GX4X5LFX6X7, wherein 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 S (SEQ ID NO:261); and the VL domain, which contains: CDR-L1, which contains the amino acid sequence X1X2SX3X4IX5GX6LN, wherein 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 (SEQ ID NO:261). NO:262); CDR-L2, which contains the amino acid sequence GX1X2X3LX4X5, wherein 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 (SEQ ID NO:263); and CDR-L3, which contains the amino acid sequence QX1X2X3X4X5PWT, wherein 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 (SEQ ID NO:264). In some embodiments, the VH domain further comprises: FW-1, which contains the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO:274); FW-2, which contains the sequence WVRQAPGQGLEWMG (SEQ ID NO:275); FW-3, which contains the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:276); and / or FW-4, which contains the sequence WGQGTLVTVSS (SEQ ID NO:277). In some embodiments, the VL field further comprises: FW-1, which contains the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:289); FW-2, which contains the sequence WYQQKPGKAPKLLIY (SEQ ID NO:290); FW-3, which contains the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:291); and / or FW-4, which contains the sequence FGGGTKVEIK (SEQ ID NO:292).

[0291] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 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:228. In some embodiments, the VH domain comprises an amino acid sequence that is 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:246. In some embodiments, the anti-CD8 antibody of this 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 a humanized antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO:245; and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO:246. In some embodiments, the VH domain further comprises: FW-1, which contains the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO:274); FW-2, which contains the sequence WVRQAPGQGLEWMG (SEQ ID NO:275); FW-3, which contains the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:276); and / or FW-4, which contains the sequence WGQGTLVTVSS (SEQ ID NO:277). In some embodiments, the VL field further comprises: FW-1, which contains the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:289); FW-2, which contains the sequence WYQQKPGKAPKLLIY (SEQ ID NO:290); FW-3, which contains the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:291); and / or FW-4, which contains the sequence FGGGTKVEIK (SEQ ID NO:292).

[0292] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 a VL domain comprising: 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. In some embodiments, the VH domain comprises an amino acid sequence that is 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 that is 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, the anti-CD8 antibody of this 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 a humanized antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO:251; and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO:252. In some embodiments, the VH domain further comprises: FW-1, which contains the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO:274); FW-2, which contains the sequence WVRQAPGQGLEWMG (SEQ ID NO:275); FW-3, which contains the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:276); and / or FW-4, which contains the sequence WGQGTLVTVSS (SEQ ID NO:277).In some embodiments, the VL field further comprises: FW-1, which contains the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:289); FW-2, which contains the sequence WYQQKPGKAPKLLIY (SEQ ID NO:290); FW-3, which contains the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:291); and / or FW-4, which contains the sequence FGGGTKVEIK (SEQ ID NO:292).

[0293] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 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:228. In some embodiments, the VH domain comprises an amino acid sequence that is 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:254. In some embodiments, the anti-CD8 antibody of this 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 a humanized antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 of the sequence from SEQ ID NO:253; and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 of the sequence from SEQ ID NO:254. In some embodiments, the VH domain further comprises: FW-1, which contains the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO:274); FW-2, which contains the sequence WVRQAPGQGLEWMG (SEQ ID NO:275); FW-3, which contains the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:276); and / or FW-4, which contains the sequence WGQGTLVTVSS (SEQ ID NO:277). In some embodiments, the VL field further comprises: FW-1, which contains the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:289); FW-2, which contains the sequence WYQQKPGKAPKLLIY (SEQ ID NO:290); FW-3, which contains the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:291); and / or FW-4, which contains the sequence FGGGTKVEIK (SEQ ID NO:292).

[0294] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: CDR-H1, which comprises the amino acid sequence X1YX2MS, wherein X1 is S, D, E, A, or Q, and X2 is A, G, or T (SEQ ID NO: 268); and CDR-H2, which comprises the amino acid sequence DIX1X2X3GX4X5TX6YADSVKG, wherein 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 (SEQ ID NO: 268). NO:269); and CDR-H3, which contains the amino acid sequence X1X2X3YX4WX5X6AX7DX8, wherein X1 is S or A, 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 (SEQ ID NO:270); and the VL domain, which contains: CDR-L1, which contains the amino acid sequence RASQSVSSNLA (SEQ ID NO:40); CDR-L2, which contains the amino acid sequence GASSRAT (SEQ ID NO:41); and CDR-L3, which contains the amino acid sequence QQYGSSPPVT (SEQ ID NO:42). In some embodiments, the VH domain further comprises: FW-1, which contains the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:281); FW-2, which contains the sequence WVRQAPGKGLEWVS (SEQ ID NO:282); FW-3, which contains the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:283); and / or FW-4, which contains the sequence WGQGTMVTVSS (SEQ ID NO:284) or WGQGTLVTVSS (SEQ ID NO:285). In some embodiments, the VL field further comprises: FW-1, which contains the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO:293); FW-2, which contains the sequence WYQQKPGQAPRLLIY (SEQ ID NO:294); FW-3, which contains the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:295); and / or FW-4, which contains the sequence FGQGTKVEIK (SEQ ID NO:296).

[0295] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 a VL domain comprising: 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. In some embodiments, the VH domain comprises an amino acid sequence that is 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:248. In some embodiments, the anti-CD8 antibody of this 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 a humanized antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO:247; and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO:248. In some embodiments, the VH domain further comprises: FW-1, which contains the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:281); FW-2, which contains the sequence WVRQAPGKGLEWVS (SEQ ID NO:282); FW-3, which contains the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:283); and / or FW-4, which contains the sequence WGQGTMVTVSS (SEQ ID NO:284) or WGQGTLVTVSS (SEQ ID NO:285).In some embodiments, the VL field further comprises: FW-1, which contains the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO:293); FW-2, which contains the sequence WYQQKPGQAPRLLIY (SEQ ID NO:294); FW-3, which contains the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:295); and / or FW-4, which contains the sequence FGQGTKVEIK (SEQ ID NO:296).

[0296] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 a VL domain comprising: 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. In some embodiments, the VH domain comprises an amino acid sequence that is 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 that is 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, the anti-CD8 antibody of this disclosure comprises 1, 2, or 3 heavy chain CDRs of antibody xhCD8v11 (e.g., as shown in Tables 1-3) and / or 1, 2, or 3 light chain CDRs of antibody xhCD8v11 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is a humanized antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 of the sequence from SEQ ID NO:249; and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 of the sequence from SEQ ID NO:250. In some embodiments, the VH domain further comprises: FW-1, which contains the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:281); FW-2, which contains the sequence WVRQAPGKGLEWVS (SEQ ID NO:282); FW-3, which contains the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:283); and / or FW-4, which contains the sequence WGQGTMVTVSS (SEQ ID NO:284) or WGQGTLVTVSS (SEQ ID NO:285).In some embodiments, the VL field further comprises: FW-1, which contains the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO:293); FW-2, which contains the sequence WYQQKPGQAPRLLIY (SEQ ID NO:294); FW-3, which contains the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:295); and / or FW-4, which contains the sequence FGQGTKVEIK (SEQ ID NO:296).

[0297] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 a VL domain comprising: 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. In some embodiments, the VH domain comprises an amino acid sequence that is 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:256. In some embodiments, the anti-CD8 antibody of this 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 a humanized antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO:255; and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO:256. In some embodiments, the VH domain further comprises: FW-1, which contains the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:281); FW-2, which contains the sequence WVRQAPGKGLEWVS (SEQ ID NO:282); FW-3, which contains the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:283); and / or FW-4, which contains the sequence WGQGTMVTVSS (SEQ ID NO:284) or WGQGTLVTVSS (SEQ ID NO:285).In some embodiments, the VL field further comprises: FW-1, which contains the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO:293); FW-2, which contains the sequence WYQQKPGQAPRLLIY (SEQ ID NO:294); FW-3, which contains the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:295); and / or FW-4, which contains the sequence FGQGTKVEIK (SEQ ID NO:296).

[0298] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 a VL domain comprising: 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. In some embodiments, the VH domain comprises an amino acid sequence that is 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:258. In some embodiments, the anti-CD8 antibody of this 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 a humanized antibody. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO:257; and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO:258. In some embodiments, the VH domain further comprises: FW-1, which contains the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:281); FW-2, which contains the sequence WVRQAPGKGLEWVS (SEQ ID NO:282); FW-3, which contains the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:283); and / or FW-4, which contains the sequence WGQGTMVTVSS (SEQ ID NO:284) or WGQGTLVTVSS (SEQ ID NO:285).In some embodiments, the VL field further comprises: FW-1, which contains the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO:293); FW-2, which contains the sequence WYQQKPGQAPRLLIY (SEQ ID NO:294); FW-3, which contains the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:295); and / or FW-4, which contains the sequence FGQGTKVEIK (SEQ ID NO:296).

[0299] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 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, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 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 anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 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 anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 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 anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 a VL domain comprising: 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. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 a VL domain comprising: 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. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 a VL domain comprising: 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. In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 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.

[0300] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: CDR-H1, which comprises the amino acid sequence 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 (SEQ ID NO: 265); and CDR-H2, which comprises the amino acid sequence X1PX2X3X4X5, wherein X1 is I, N, or M; X2 is G, N, H, S, R, I, or A; X3 is A, N, H, S, T, F, or Y; X4 is A, D, or G; and X5 is T, E, K, V, Q, or A (SEQ ID NO: 265). NO:266); and CDR-H3, which contains the amino acid sequence X1X2X3GX4X5LFX6X7, wherein 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 S (SEQ ID NO:267); and VL domain, which contains: CD R-L1, which contains the amino acid sequence X1X2SX3X4IX5GX6LN, wherein 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 (SEQ ID NO:267). SEQ ID NO:262); CDR-L2, which contains the amino acid sequence GX1X2X3LX4X5, wherein 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 (SEQ ID NO:263); and CDR-L3, which contains the amino acid sequence QX1X2X3X4X5PWT, wherein 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 (SEQ ID NO:264). In some embodiments, the VH domain further comprises: FW-1, which contains the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO:278); FW-2, which contains the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO:279); FW-3, which contains the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:280); and / or FW-4, which contains the sequence WGQGTLVTVSS (SEQ ID NO:277).In some embodiments, the VL field further comprises: FW-1, which contains the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:289); FW-2, which contains the sequence WYQQKPGKAPKLLIY (SEQ ID NO:290); FW-3, which contains the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:291); and / or FW-4, which contains the sequence FGGGTKVEIK (SEQ ID NO:292).

[0301] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 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:228. In some embodiments, the VH domain further comprises: FW-1, which contains the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO:278); FW-2, which contains the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO:279); FW-3, which contains the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:280); and / or FW-4, which contains the sequence WGQGTLVTVSS (SEQ ID NO:277). In some embodiments, the VL field further comprises: FW-1, which contains the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:289); FW-2, which contains the sequence WYQQKPGKAPKLLIY (SEQ ID NO:290); FW-3, which contains the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:291); and / or FW-4, which contains the sequence FGGGTKVEIK (SEQ ID NO:292).

[0302] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 a VL domain comprising: 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. In some embodiments, the VH domain further comprises: FW-1, which contains the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO:278); FW-2, which contains the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO:279); FW-3, which contains the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:280); and / or FW-4, which contains the sequence WGQGTLVTVSS (SEQ ID NO:277). In some embodiments, the VL field further comprises: FW-1, which contains the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:289); FW-2, which contains the sequence WYQQKPGKAPKLLIY (SEQ ID NO:290); FW-3, which contains the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:291); and / or FW-4, which contains the sequence FGGGTKVEIK (SEQ ID NO:292).

[0303] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 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:228. In some embodiments, the VH domain further comprises: FW-1, which contains the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO:278); FW-2, which contains the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO:279); FW-3, which contains the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:280); and / or FW-4, which contains the sequence WGQGTLVTVSS (SEQ ID NO:277). In some embodiments, the VL field further comprises: FW-1, which contains the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:289); FW-2, which contains the sequence WYQQKPGKAPKLLIY (SEQ ID NO:290); FW-3, which contains the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:291); and / or FW-4, which contains the sequence FGGGTKVEIK (SEQ ID NO:292).

[0304] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: CDR-H1, which comprises the amino acid sequence GFTFX1X2Y, wherein X1 is S, D, E, Q, S or A, and X2 is S, D, E, A or Q (SEQ ID NO: 271); and CDR-H2, which comprises the amino acid sequence X1X2X3GX4X5, wherein 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 (SEQ ID NO: 271). NO:272); and CDR-H3, which contains the amino acid sequence X1X2X3YX4WX5X6AX7DX8, wherein X1 is S or A, 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 (SEQ ID NO:273); and the VL domain, which contains: CDR-L1, which contains the amino acid sequence RASQSVSSNLA (SEQ ID NO:40); CDR-L2, which contains the amino acid sequence GASSRAT (SEQ ID NO:41); and CDR-L3, which contains the amino acid sequence QQYGSSPPVT (SEQ ID NO:42). In some embodiments, the VH domain further comprises: FW-1, which contains the sequence EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:286); FW-2, which contains the sequence AMSWVRQAPGKGLEWVSDI (SEQ ID NO:287); FW-3, which contains the sequence TAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:288); and / or FW-4, which contains the sequence WGQGTMVTVSS (SEQ ID NO:284) or WGQGTLVTVSS (SEQ ID NO:285). In some embodiments, the VL field further comprises: FW-1, which contains the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO:293); FW-2, which contains the sequence WYQQKPGQAPRLLIY (SEQ ID NO:294); FW-3, which contains the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:295); and / or FW-4, which contains the sequence FGQGTKVEIK (SEQ ID NO:296).

[0305] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 a VL domain comprising: 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. In some embodiments, the VH domain further comprises: FW-1, which contains the sequence EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:286); FW-2, which contains the sequence AMSWVRQAPGKGLEWVSDI (SEQ ID NO:287); FW-3, which contains the sequence TAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:288); and / or FW-4, which contains the sequence WGQGTMVTVSS (SEQ ID NO:284) or WGQGTLVTVSS (SEQ ID NO:285). In some embodiments, the VL field further comprises: FW-1, which contains the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO:293); FW-2, which contains the sequence WYQQKPGQAPRLLIY (SEQ ID NO:294); FW-3, which contains the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:295); and / or FW-4, which contains the sequence FGQGTKVEIK (SEQ ID NO:296).

[0306] In some embodiments, the anti-CD8 antibody of this disclosure comprises: a VH domain comprising: 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 a VL domain comprising: 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. In some embodiments, the VH domain further comprises: FW-1, which contains the sequence EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:286); FW-2, which contains the sequence AMSWVRQAPGKGLEWVSDI (SEQ ID NO:287); FW-3, which contains the sequence TAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:288); and / or FW-4, which contains the sequence WGQGTMVTVSS (SEQ ID NO:284) or WGQGTLVTVSS (SEQ ID NO:285). In some embodiments, the VL domain further comprises: FW-1, which comprises the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO:293); FW-2, which comprises the sequence WYQQKPGQAPRLLIY (SEQ ID NO:294); FW-3, which comprises the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:295); and / or FW-4, which comprises the sequence FGQGTKVEIK (SEQ ID NO:296). In some embodiments, this disclosure provides an anti-CD8 antibody comprising a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of the single antibodies listed in Table 1 and a VL domain comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of the single antibodies listed in Table 1. For example, the anti-CD8 antibody includes the antibodies 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, this disclosure provides an anti-CD8 antibody comprising a VH domain containing the CDR-H1, CDR-H2, and CDR-H3 sequences of the single antibodies listed in Table 2, and a VL domain containing the CDR-L1, CDR-L2, and CDR-L3 sequences of the single antibodies listed in Table 2. For example, the anti-CD8 antibody comprises the six CDRs of the antibody xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​xhCD8v15, V9 family, or V11 family shown in Table 2. In some embodiments, this disclosure provides a fusion protein comprising an anti-CD8 antibody, said anti-CD8 antibody comprising: a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of the single antibodies listed in Table 1; and a VL domain comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of the single antibodies listed in Table 1. For example, the anti-CD8 antibody of said fusion protein comprises the antibodies 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, this disclosure provides a fusion protein comprising an anti-CD8 antibody, said anti-CD8 antibody comprising: a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of the single antibodies listed in Table 2; and a VL domain comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of the single antibodies listed in Table 2. For example, the anti-CD8 antibody of said fusion protein comprises the antibodies 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 2.In some embodiments, this 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 domain and VL domain are from the same single antibody listed in Table 3). For example, the anti-CD8 antibody comprises the VH and VL of antibodies xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​or xhCD8v15 as shown in Table 3. In some embodiments, this disclosure provides a fusion protein 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 domain and VL domain are from the same single antibody listed in Table 3). In some embodiments, this disclosure provides an anti-CD8 antibody comprising the VH domain and VL domain sequences of a single antibody as listed in Table 3. In some embodiments, this disclosure provides a fusion protein comprising an anti-CD8 antibody comprising the VH domain and VL domain sequences of a single antibody as listed in Table 3. For example, the anti-CD8 antibody of the fusion protein comprises the VH and VL of antibodies xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14 or xhCD8v15 shown in Table 3.

[0307] Table 1. Anti-CD8 antibody CDR (Kabat)

[0308]

[0309]

[0310]

[0311] Table 2. Anti-CD8 antibody CDR (Chothia)

[0312]

[0313]

[0314] Table 3. Variable domain sequences of anti-CD8 antibody

[0315]

[0316]

[0317]

[0318]

[0319] Fusion protein cytokines

[0320] In some embodiments, the second part induces the activation of CD8+ T cells. In some embodiments, the second part comprises a polypeptide that induces signaling via IL-2Rβγ. In some embodiments, the second part comprises a polypeptide that induces signaling via IL-21R. In some embodiments, the second part comprises an IL-21 polypeptide (e.g., a human IL-21 polypeptide or a derivative thereof). In some embodiments, the second part comprises an IL-2 polypeptide (e.g., a human IL-2 polypeptide or a derivative thereof).

[0321] IL-2 peptide

[0322] In some implementations, the second part comprises an IL-2 polypeptide (e.g., a human IL-2 polypeptide or a derivative thereof).

[0323] In some embodiments, the binding affinity of the IL-2 polypeptide for IL-2Rα is reduced by 50% or more compared to the binding affinity of the wild-type IL-2 polypeptide containing the sequence of SEQ ID NO:81 for IL-2Rα. 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 binding affinity of the IL-2 polypeptide for IL-2Rβ is reduced by 50% or more compared to the binding affinity of the wild-type IL-2 polypeptide containing the sequence of SEQ ID NO:81 for IL-2Rβ. In some embodiments, the binding affinity of the IL-2 polypeptide for IL-2Rγ is reduced by 50% or more compared to the binding affinity of the wild-type IL-2 polypeptide containing the sequence of SEQ ID NO:81 for IL-2Rγ. The difference in binding affinity between the wild-type peptide and the disclosed peptide for IL-2Rα and IL-2Rβ can be measured, for example, in a standard surface plasmon resonance (SPR) assay familiar to those skilled in the art for measuring the affinity of protein-protein interactions. The difference in binding affinity between the wild-type peptide and the disclosed peptide for IL-2Rγ cannot be reliably measured by SPR assay because the wild-type IL-2 peptide has extremely low affinity for IL-2Rγ. In fact, its reduced IL-2Rγ affinity can be inferred by performing an in vitro assay measuring pSTAT5 and comparing the activity of the IL-2 peptide with and without the reduced IL-2Rγ affinity substitution on IL-2R-expressing cells. Exemplary sequences of IL-2Rα, IL-2Rβ, and IL-2Rγ are provided below.

[0324] IL-2Rα:

[0325] MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATER IYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI(SEQ ID NO:82)

[0326] IL-2Rβ:

[0327] (SEQ ID NO:83)

[0328] IL-2Rγ:

[0329] MLKPSLPFTSLLFLQLPLLGVGLNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET(SEQID NO:84)

[0330] In some embodiments, the IL-2 polypeptide is an IL-2 polypeptide containing one or more mutations, relative to a human IL-2 polypeptide containing the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:81). In some embodiments, the IL-2 polypeptide of this disclosure has one or more, two or more, or three or more affinity-reducing amino acid substitutions, relative to, for example, a wild-type mature IL-2 polypeptide having the amino acid sequence of SEQ ID NO:81. In some embodiments, 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, I92, T123, Q126, S127, I129, 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. A decreased affinity for IL-2Rβ can be achieved by substituting one or more of the following residues: E15, H16, L19, D20, D84, S87, N88, V91, and I92. A decreased affinity for IL-2Rγ can be achieved 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, I129, and S130.

[0331] In some embodiments, the IL-2 polypeptide contains an F42A or F42K amino acid substitution relative to the wild-type mature IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide contains an F42A or F42K amino acid substitution and an R38A, R38D, R38E, E62Q, E68A, E68Q, E68K, or E68R amino acid substitution relative to the wild-type mature IL-2 amino acid sequence, for example, as shown in SEQ ID NO:81. For example, in some embodiments, the IL-2 polypeptide contains 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 substitutions relative to the wild-type mature IL-2 amino acid sequence, for example. In some embodiments, the IL-2 polypeptide contains R38E and F42A amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide contains R38D and F42A amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide contains F42A and E62Q amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide contains R38A and F42K amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide contains R38D and F42A amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide contains R38A and F42K amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide contains F42A and E62Q amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide comprises, relative 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, T123E, T123K, T123Q, Q126A, Q126S, Q126T, Q126E, S127A, S127E,S127K or S127Q amino acid substitutions. In some embodiments, the IL-2 polypeptide comprises the following amino acid substitutions relative to the wild-type mature IL-2 amino acid sequence: 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; and relative to, for example, SEQ ID The wild-type mature IL-2 amino acid sequence shown in NO:81 has the following amino acid substitutions: 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, T123E, T123K, T123Q, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K, or S127Q. For example, in some embodiments, the IL-2 polypeptide contains amino acid substitutions of R38E, F42A, and H16E relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide contains amino acid substitutions of R38E, F42A, and H16D relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide contains amino acid substitutions of R38E, F42A, and D84K relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide contains amino acid substitutions of R38E, F42A, and D84R relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide contains amino acid substitutions of R38E, F42A, and N88S relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide contains amino acid substitutions of R38E, F42A, and N88A 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. In some embodiments, 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 N88T amino acid substitutions relative to the wild-type IL-2 amino acid sequence.F42A and N88D amino acid substitutions. 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 (relative to the sequence of SEQ ID NO:81) comprises SEQ ID NO:81 having one of the following amino acid substitution groups. The amino acid sequence of NO:81 is: 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, F42A and N88D; R38D, F42A and V91E; R38D, F42A and D84H; R38D, F42A and D84K; R38D, F42A and D84R; H16D, R3 8D 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 F42 K; 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; 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; R38E, F42A, N88T and C125A; R38E, F42A, N88D and C125A; R38E, F42A, V91E and C125A; R38E, F42A, D84 H 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, N 88S 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, N88A and C125A; R38A, F42K, N88G and C125A; R38A, F42K, N88R and C125A; R38A, F42K, N88T and C125A; R38A, F4 2K, N88D, 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, R 38A, 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; F42A, E62Q, N88D, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, D84H, and C125A; F42A, E62Q, D84K, and C125A; F42A, E62Q, D84R, and C125A; H16D, F42A, E62Q, and C125A; H16E, F42A, E62Q, and C125A; F42A, E62Q, C125A, and Q126S; F42A, N88S, and C1 25A; 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, relative to SEQ ID NO:81, the sequence of SEQ ID NO:81 having one, two, three, four, or five amino acid substitutions, and wherein said one, two, three, four, or five substitutions comprise substitutions at positions in SEQ ID NO:81 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, I92, T123, Q126, S127, I129, and S130. In some embodiments, the IL-2 polypeptide (relative to the sequence of SEQ ID NO:81) comprises SEQ ID NO:81 having one of the following amino acid substitution groups. Sequences of 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; R 38E, 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, 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; R38 A, 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 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38D, F42A, and N88S. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38D, F42A, and N88A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38D, F42A, and N88R. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38D, F42A, and N88T. In some embodiments, the IL-2 peptide comprises, relative to the sequence of SEQ ID NO:81, a sequence of SEQ ID NO:81 having the following amino acid substitutions: R38D,F42A and N88D. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38D, F42A, and V91E. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38D, F42A, and D84H. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38D, F42A, and D84R. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: H16D, R38D, and F42A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: H16E, R38D, and F42A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38A, F42K, and N88S. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38A, F42K, and N88A. In some embodiments, the IL-2 peptide comprises, relative to the sequence of SEQ ID NO:81, the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38A, F42K, and N88G. In some embodiments, the IL-2 peptide comprises, relative to the sequence of SEQ ID NO:81, the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38A, F42K, and N88R. In some embodiments, the IL-2 peptide comprises, relative to the sequence of SEQ ID NO:81, the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38A, F42K, and N88R.F42K and N88T. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38A, F42K, and N88D. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38A, F42K, and V91E. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38A, F42K, and D84K. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38A, F42K, and D84K. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38A, F42K, and D84R. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: H16D, R38A, and F42K. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: H16E, R38A, and F42K. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: R38A, F42K, and Q126S. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: F42A, E62Q, and N88S. In some embodiments, the IL-2 peptide comprises, relative to the sequence of SEQ ID NO:81, having the following amino acid substitutions: F42A, E62Q, and N88A. In some embodiments, the IL-2 peptide comprises, relative to the sequence of SEQ ID NO:81, having the following amino acid substitutions: F42A, E62Q, and N88G. In some embodiments, the IL-2 peptide comprises, relative to the sequence of SEQ ID NO:81, having the following amino acid substitutions: F42A, E62Q, and N88G.E62Q and N88R. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: F42A, E62Q, and N88T. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: F42A, E62Q, and N88D. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: F42A, E62Q, and V91E. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 with the following amino acid substitutions: F42A, E62Q, and D84H. In some embodiments, the IL-2 polypeptide comprises, relative to the sequence of SEQ ID NO:81, the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, E62Q, and D84K. In some embodiments, the IL-2 polypeptide comprises, relative to the sequence of SEQ ID NO:81, the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, E62Q, and D84R.

[0332] In some embodiments, the IL-2 polypeptide comprises, relative to SEQ ID NO:81, the sequence of SEQ ID NO:81 having another amino acid substitution at position C125. In some embodiments, the IL-2 polypeptide (relative to the sequence of SEQ ID NO:81) comprises SEQ ID NO:81 having one of the following amino acid substitutions. Sequence of 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, N88T, and C125A; R38E, F42A, V91E, and C 125A; 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, 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, N8 8T 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; 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 peptide has the following amino acid substitutions relative to the sequence contained in SEQ ID NO:81: R38E, F42A, and C125A. In some embodiments, the IL-2 peptide has the following amino acid substitutions relative to the sequence in SEQ ID NO:81: R38D, F42A, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, E62Q, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38A, F42K, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38E, F42A, N88S, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38E, F42A, and C125A.N88A and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38E, F42A, N88G, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38E, F42A, N88R, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38E, F42A, N88T, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38E, F42A, V91E, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38E, F42A, D84H, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38E, F42A, D84R, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: H16D, R38E, F42A, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: H16E, R38E, F42A, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38E, F42A, and C125A.C125A and Q126S. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38D, F42A, N88S, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38D, F42A, N88G, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38D, F42A, N88R, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38D, F42A, N88T, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38D, F42A, N88D, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38D, F42A, D84H, and C125A. In some embodiments, the IL-2 peptide comprises, relative to the sequence of SEQ ID NO:81, the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38D, F42A, D84K, and C125A. In some embodiments, the IL-2 peptide comprises, relative to the sequence of SEQ ID NO:81, the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38D, F42A, D84K, and C125A. In some embodiments, the IL-2 peptide comprises, relative to the sequence of SEQ ID NO:81, the sequence of SEQ ID NO:81 having the following amino acid substitutions: H16D, R38D,F42A and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: H16E, R38D, F42A, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38D, F42A, C125A, and Q126S. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38A, F42K, N88G, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38A, F42K, N88G, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38A, F42K, N88R, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38A, F42K, N88T, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38A, F42K, N88A, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38A, F42K, N88A, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38A, F42K, V91E, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38A, F42K, D84H, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38A, F42K, D84H, and C125A.D84K and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38A, F42K, D84R, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: H16D, R38A, F42K, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: H16E, R38A, F42K, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: R38A, F42K, C125A, and Q126S. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, E62Q, N88S, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, E62Q, N88G, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, E62Q, N88R, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, E62Q, N88T, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, E62Q, N88T, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, E62Q, N88T, and C125A.V91E and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, E62Q, and D84H and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, E62Q, and D84K and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, E62Q, and D84R and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: H16D, F42A, and E62Q and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: H16E, F42A, E62Q, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, E62Q, C125A, and Q126S. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, N88A, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, N88A, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, N88G, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, N88G, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, N88G, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, N88G, and C125A.N88D and C125A; F42A, V91E and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, D84H and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, D84K and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, D84R and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: H16D, F42A and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: H16E, F42A, and C125A. In some embodiments, the IL-2 peptide relative to the sequence of SEQ ID NO:81 comprises the sequence of SEQ ID NO:81 having the following amino acid substitutions: F42A, C125A, and Q126S.

[0333] In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 80). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 85). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 86). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 87). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:88).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 89). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 90). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 91). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 92). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 93).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 94). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 95). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 96). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 97). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 98).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 99). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 100). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 101). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 102). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:103).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 104). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 105). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 106). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 107). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:108).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 109). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 110). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 111). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 112). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:113).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 114). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 115). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 116). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 117). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:118).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 119). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 120). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 121). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 122). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:123).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 124). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 125). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 126). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 127). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:128).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 129). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 130). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 131). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 132). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:133).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 134). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 135). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 136). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 137). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:138).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 139). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 140). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 141). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 142). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:143).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 144). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 145). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 146). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 147). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO:148).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 149). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 150). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 151). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 152). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:153).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 154). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 155). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 190). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 191). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:192).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 193). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 194). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 195). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 196). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:197).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 198). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 199). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 200). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 201). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 202).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 203). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 204). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 205). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 206). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:207).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 208). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 209). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 210). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 211). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:212).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 213). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 214). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 215). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 216). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:297).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 354). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 355). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHL QCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:356). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:357). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:358).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 359). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 360). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:361). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:362). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:363).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:364). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:365). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:366). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:367). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:368).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 369). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 370). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:371). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:372). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:373).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:374). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:375). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:376). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:377). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:378).In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:379). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:380). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 381). In some embodiments, the IL-2 peptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 382). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 383). In some embodiments, the IL-2 polypeptide comprises the amino acid sequence of the IL-2 polypeptide listed in Table 6.

[0334] 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 with any of the amino acid sequences set forth in SEQ ID NO: 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 with any of the amino acid sequences set forth in SEQ ID NO: 80, 85-155, 190-216, 297, or 354-383. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence having at least 85% identity with any of the amino acid sequences set forth below: SEQ ID NO: 80, 85-155, 190-216, 297, or 354-383. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence having at least 90% identity with any of the amino acid sequences set forth below: SEQ ID NO: 80, 85-155, 190-216, 297, or 354-383. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence having at least 95% identity with any of the amino acid sequences set forth below: SEQ ID NO: 80, 85-155, 190-216, 297, or 354-383. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence having at least 96% identity with any of the amino acid sequences set forth below: SEQ ID NO: 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 with any of the amino acid sequences set forth below: SEQ ID NO: 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 with any of the amino acid sequences set forth below: SEQ ID NO: 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 with any of the amino acid sequences set forth below: SEQ ID NO: 80, 85-155, 190-216, 297, or 354-383. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence having 100% identity with any of the amino acid sequences set forth below: SEQ ID NO: 80, 85-155, 190-216, 297, or 354-383.

[0335] Table 6. Exemplary IL-2 peptide sequences

[0336]

[0337]

[0338] In some embodiments, the IL-2 peptide of this disclosure also contains other modifications, including but not limited to mutations and deletions, that provide additional advantages such as improved physiological properties. Improved physiological properties include, but are not limited to, improved thermal stability, 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; and the N-terminal A1 residue may be deleted, both of which are described in U.S. Patent No. 4,518,584. The IL-2 peptide may also include a mutation in residue M104, such as M104A, as described in U.S. Patent No. 5,206,344. Thus, in some embodiments, the IL-2 peptide of this disclosure comprises an amino acid substitution for C125A. In other embodiments, one, two, or three N-terminal residues are deleted.

[0339] Exemplary IL-2 fusion protein

[0340] In some embodiments, the fusion protein of this disclosure comprises one, two, or all three polypeptides shown in Table 5 for a single fusion protein. In some embodiments, the fusion protein of this disclosure comprises two light chains of a single fusion protein in Table 5, a heavy chain containing an IL-2 fusion compound, and a heavy chain not containing an IL-2 fusion compound. As is known in the art, the C-terminal lysine residue of some antibody heavy chain species can be cleaved in a portion of the molecule. Therefore, in some embodiments, the fusion protein of this disclosure comprises two light chains of a single fusion protein in Table 5, a heavy chain containing an IL-2 fusion compound, and a heavy chain not containing an IL-2 fusion compound, wherein the heavy chain not containing an IL-2 fusion compound comprises the sequence of the corresponding fusion protein: 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, the fusion protein of this disclosure comprises two light chains of a single fusion protein as shown in Table 5, a heavy chain containing an IL-2 fusion compound, and a heavy chain not containing an IL-2 fusion compound, wherein the heavy chain not containing an IL-2 fusion compound comprises the sequence of the corresponding fusion protein SEQ ID No: 217-224, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, or 353.In some embodiments, this disclosure provides a variety of fusion proteins of this disclosure (e.g., in mixture form), wherein each of the various fusion proteins comprises two light chains of a single fusion protein from Table 5, a heavy chain containing an IL-2 fusion compound, and a heavy chain not containing an IL-2 fusion compound, wherein the heavy chain not containing an IL-2 fusion compound comprises the sequence of the corresponding fusion protein 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, or the corresponding fusion protein SEQ ID No. Sequences No: 217-224, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, or 353, or the plurality of sequences comprising cleaved (e.g., sequences comprising the corresponding fusion protein, 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) species and uncleaved (e.g., sequences comprising the corresponding fusion protein, SEQ ID No: 217-224, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, or 353), or sequences comprising the corresponding fusion protein, SEQ ID No: 217-224, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, or 352) species and uncleaved (e.g., sequences comprising the corresponding fusion protein, SEQ ID No: 217-224, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 3 A mixture of species (sequences No:217-224, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349 or 353).

[0341] For example, in some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:156, a heavy chain containing the amino acid sequence of SEQ ID NO:157, and a heavy chain containing the amino acid sequence of SEQ ID NO:158. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:159, a heavy chain containing the amino acid sequence of SEQ ID NO:160, and a heavy chain containing the amino acid sequence of SEQ ID NO:161. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:162, a heavy chain containing the amino acid sequence of SEQ ID NO:163, and a heavy chain containing the amino acid sequence of SEQ ID NO:164. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:165, a heavy chain containing the amino acid sequence of SEQ ID NO:166, and a heavy chain containing the amino acid sequence of SEQ ID NO:167. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:168, a heavy chain containing the amino acid sequence of SEQ ID NO:169, and a heavy chain containing the amino acid sequence of SEQ ID NO:170. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:171, a heavy chain containing the amino acid sequence of SEQ ID NO:172, and a heavy chain containing the amino acid sequence of SEQ ID NO:173. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:174, a heavy chain containing the amino acid sequence of SEQ ID NO:175, and a heavy chain containing the amino acid sequence of SEQ ID NO:176. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:187, a heavy chain containing the amino acid sequence of SEQ ID NO:188, and a heavy chain containing the amino acid sequence of SEQ ID NO:189. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:298, a heavy chain containing the amino acid sequence of SEQ ID NO:299, and a heavy chain containing the amino acid sequence of SEQ ID NO:300. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:302, a heavy chain containing the amino acid sequence of SEQ ID NO:303, and a heavy chain containing the amino acid sequence of SEQ ID NO:304.In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:306, a heavy chain containing the amino acid sequence of SEQ ID NO:307, and a heavy chain containing the amino acid sequence of SEQ ID NO:308. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:310, a heavy chain containing the amino acid sequence of SEQ ID NO:311, and a heavy chain containing the amino acid sequence of SEQ ID NO:312. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:314, a heavy chain containing the amino acid sequence of SEQ ID NO:315, and a heavy chain containing the amino acid sequence of SEQ ID NO:316. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:318, a heavy chain containing the amino acid sequence of SEQ ID NO:319, and a heavy chain containing the amino acid sequence of SEQ ID NO:320. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:322, a heavy chain containing the amino acid sequence of SEQ ID NO:323, and a heavy chain containing the amino acid sequence of SEQ ID NO:324. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:326, a heavy chain containing the amino acid sequence of SEQ ID NO:327, and a heavy chain containing the amino acid sequence of SEQ ID NO:328. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:334, a heavy chain containing the amino acid sequence of SEQ ID NO:335, and a heavy chain containing the amino acid sequence of SEQ ID NO:336. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:338, a heavy chain containing the amino acid sequence of SEQ ID NO:339, and a heavy chain containing the amino acid sequence of SEQ ID NO:340. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:342, a heavy chain containing the amino acid sequence of SEQ ID NO:343, and a heavy chain containing the amino acid sequence of SEQ ID NO:344. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:346, a heavy chain containing the amino acid sequence of SEQ ID NO:347, and a heavy chain containing the amino acid sequence of SEQ ID NO:348.In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:350, heavy chains containing the amino acid sequence of SEQ ID NO:351, and heavy chains containing the amino acid sequence of SEQ ID NO:352.

[0342] In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:156, a heavy chain containing the amino acid sequence of SEQ ID NO:157, and a heavy chain containing the amino acid sequence of SEQ ID NO:217. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:159, a heavy chain containing the amino acid sequence of SEQ ID NO:160, and a heavy chain containing the amino acid sequence of SEQ ID NO:218. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:162, a heavy chain containing the amino acid sequence of SEQ ID NO:163, and a heavy chain containing the amino acid sequence of SEQ ID NO:219. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:165, a heavy chain containing the amino acid sequence of SEQ ID NO:166, and a heavy chain containing the amino acid sequence of SEQ ID NO:220. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:168, a heavy chain containing the amino acid sequence of SEQ ID NO:169, and a heavy chain containing the amino acid sequence of SEQ ID NO:221. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:171, a heavy chain containing the amino acid sequence of SEQ ID NO:172, and a heavy chain containing the amino acid sequence of SEQ ID NO:222. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:174, a heavy chain containing the amino acid sequence of SEQ ID NO:175, and a heavy chain containing the amino acid sequence of SEQ ID NO:223. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:187, a heavy chain containing the amino acid sequence of SEQ ID NO:188, and a heavy chain containing the amino acid sequence of SEQ ID NO:224. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:298, a heavy chain containing the amino acid sequence of SEQ ID NO:299, and a heavy chain containing the amino acid sequence of SEQ ID NO:301. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:302, a heavy chain containing the amino acid sequence of SEQ ID NO:303, and a heavy chain containing the amino acid sequence of SEQ ID NO:305.In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:306, a heavy chain containing the amino acid sequence of SEQ ID NO:307, and a heavy chain containing the amino acid sequence of SEQ ID NO:309. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:310, a heavy chain containing the amino acid sequence of SEQ ID NO:311, and a heavy chain containing the amino acid sequence of SEQ ID NO:313. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:314, a heavy chain containing the amino acid sequence of SEQ ID NO:315, and a heavy chain containing the amino acid sequence of SEQ ID NO:317. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:318, a heavy chain containing the amino acid sequence of SEQ ID NO:319, and a heavy chain containing the amino acid sequence of SEQ ID NO:321. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:322, a heavy chain containing the amino acid sequence of SEQ ID NO:323, and a heavy chain containing the amino acid sequence of SEQ ID NO:325. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:326, a heavy chain containing the amino acid sequence of SEQ ID NO:327, and a heavy chain containing the amino acid sequence of SEQ ID NO:329. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:330, a heavy chain containing the amino acid sequence of SEQ ID NO:331, and a heavy chain containing the amino acid sequence of SEQ ID NO:333. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:334, a heavy chain containing the amino acid sequence of SEQ ID NO:335, and a heavy chain containing the amino acid sequence of SEQ ID NO:337. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:338, a heavy chain containing the amino acid sequence of SEQ ID NO:339, and a heavy chain containing the amino acid sequence of SEQ ID NO:341. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:342, a heavy chain containing the amino acid sequence of SEQ ID NO:343, and a heavy chain containing the amino acid sequence of SEQ ID NO:345.In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:346, a heavy chain containing the amino acid sequence of SEQ ID NO:347, and a heavy chain containing the amino acid sequence of SEQ ID NO:349. In some embodiments, the fusion protein of this disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO:350, a heavy chain containing the amino acid sequence of SEQ ID NO:351, and a heavy chain containing the amino acid sequence of SEQ ID NO:353.

[0343] In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 156, a heavy chain containing the amino acid sequence of SEQ ID NO: 157, and a heavy chain containing the amino acid sequence of SEQ ID NO: 158 or 217. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 159, a heavy chain containing the amino acid sequence of SEQ ID NO: 160, and a heavy chain containing the amino acid sequence of SEQ ID NO: 161 or 218. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 162, a heavy chain containing the amino acid sequence of SEQ ID NO: 163, and a heavy chain containing the amino acid sequence of SEQ ID NO: 164 or 219. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 165, a heavy chain containing the amino acid sequence of SEQ ID NO: 166, and a heavy chain containing the amino acid sequence of SEQ ID NO: 167 or 220. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 168, a heavy chain containing the amino acid sequence of SEQ ID NO: 169, and a heavy chain containing the amino acid sequence of SEQ ID NO: 170 or 221. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 171, a heavy chain containing the amino acid sequence of SEQ ID NO: 172, and a heavy chain containing the amino acid sequence of SEQ ID NO: 173 or 222. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 174, a heavy chain containing the amino acid sequence of SEQ ID NO: 175, and a heavy chain containing the amino acid sequence of SEQ ID NO: 176 or 223. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 187, a heavy chain containing the amino acid sequence of SEQ ID NO: 188, and a heavy chain containing the amino acid sequence of SEQ ID NO: 189 or 224.In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species 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 or 301. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species 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 or 305. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species 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 or 309. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species 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 or 313. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species 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 or 317. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species 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 or 321. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species 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 or 325. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species 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 or 329.In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 330, a heavy chain containing the amino acid sequence of SEQ ID NO: 331, and a heavy chain containing the amino acid sequence of SEQ ID NO: 332 or 333. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 334, a heavy chain containing the amino acid sequence of SEQ ID NO: 335, and a heavy chain containing the amino acid sequence of SEQ ID NO: 336 or 337. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 338, a heavy chain containing the amino acid sequence of SEQ ID NO: 339, and a heavy chain containing the amino acid sequence of SEQ ID NO: 340 or 341. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 342, a heavy chain containing the amino acid sequence of SEQ ID NO: 343, and a heavy chain containing the amino acid sequence of SEQ ID NO: 344 or 345. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 346, a heavy chain containing the amino acid sequence of SEQ ID NO: 347, and a heavy chain containing the amino acid sequence of SEQ ID NO: 348 or 349. In some embodiments, this disclosure provides mixtures of fusion protein species, wherein each species comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 350, a heavy chain containing the amino acid sequence of SEQ ID NO: 351, and a heavy chain containing the amino acid sequence of SEQ ID NO: 352 or 353.

[0344] In some embodiments, this disclosure provides a fusion protein comprising two heavy chain sequences and two light chain sequences of a single fusion protein listed in Table 5, wherein one heavy chain sequence has an IL2 fusion compound and the other heavy chain sequence does not have an IL2 fusion compound, and wherein the two light chain sequences are identical. In some embodiments, the heavy chain sequence without the IL2 fusion compound contains a lysine residue at its C-terminus. In some embodiments, the fusion protein has type A as shown in Figure 7. For example, in some embodiments, the fusion protein comprises four polypeptide chains, wherein (1) 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; (2) 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; (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:339, the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:338, the fifth polypeptide chain comprises the amino acid sequence of SEQ ID NO:339, the sixth polypeptide chain comprises the amino acid sequence of SEQ ID NO:334, the seventh polypeptide chain comprises the amino acid sequence of SEQ ID NO:335, the eighth polypeptide chain comprises the amino acid sequence of SEQ ID NO:336, and the ninth polypeptide chain comprises the amino acid sequence of SEQ ID NO:334. The amino acid sequence of SEQ ID NO:341, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO:338.

[0345] Table 5. Sequence of anti-CD8:IL2 fusion protein

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367] Characteristics of IL-2 fusion proteins

[0368] In assays containing cells expressing antigens that also express IL-2Rβγ or IL-2Rαβγ and cells that do not express antigens, the preferential activity of a targeting IL-2 fusion protein containing an IL-2 peptide against antigen-expressing cells has been demonstrated. One such assay is an in vitro assay that measures STAT5 (pSTAT5) phosphorylation and / or the expression of the proliferation marker Ki-67 in human immune cells (e.g., human peripheral blood and / or tumor-infiltrating immune cells) after exposure to an IL-2 peptide. In one type of assay, the activity of the targeting IL-2 fusion protein against antigen-expressing and antigen-non-expressing cells was measured to demonstrate selectivity for antigen-expressing cells. In another type of assay, the activity of a targeting IL-2 fusion protein containing an IL-2 peptide against antigen-expressing cells was compared to the activity of a non-targeting IL-2 fusion protein containing the same IL-2 peptide and a control antibody against any antigen not recognizing the antigen-expressing cell to demonstrate the magnitude of IL-2 peptide signaling restoration upon fusion with an antigen-binding molecule.

[0369] In some embodiments, the fusion protein of this disclosure containing a CD8b antigen-binding molecule activates CD8b+IL-2Rβ+ cells at least 10-fold, at least 50-fold, or at least 100-fold more effectively than CD8b-IL-2Rβ+ cells. In some embodiments, the fusion protein activates CD8b+IL-2Rβ+ cells at 50-fold, 100-fold, or 200-fold more effectively than a fusion molecule containing the IL-2 mutant polypeptide and a control antibody that does not bind to any antigen expressed on the cells. The cell activation 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.

[0370] In some embodiments, the fusion protein of this disclosure exhibits one or more of the following: binding to human CD8 and not blocking CD8-class MHC interaction; and activating CD8+ T cells with at least 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, or 1000-fold higher potency than NK cell activation. In some embodiments, whether the anti-CD8 antibody or fusion protein of this disclosure blocks CD8-class MHC interaction can be determined, for example, by measuring the activation status of CD8+ T cells (e.g., after antigen stimulation) in the presence or absence of the anti-CD8 antibody or fusion protein. In some embodiments, activation of CD8+ T cells and / or NK cells can be measured, for example, by measuring one or more markers of proliferation (e.g., Ki67) (e.g., the proportion of treated cells expressing one or more markers), IL-2Rβ / γ downstream signaling, and / or STAT5 downstream signaling.

[0371] IL-21 peptide

[0372] Interleukin-21 (IL-21) is a cytokine expressed by T cells, B cells, NK cells, and bone marrow cells that regulates the activity of both innate and adaptive immune cells and improves T cell survival and effector function. Several Phase I and 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.

[0373] IL-21 has a four-helix bundle structure and exists in monomeric form. 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), with the first 29 forming the signal peptide; and the second IL-21 isoform contains 153 amino acids, with the first 29 forming the signal peptide, just like the first isoform.

[0374] IL-21 binds to a heterodimeric IL-21 receptor complex containing the IL-21 receptor (IL-21R) and a common γ chain (γc). The IL-21 receptor complex is expressed on the surface of T cells, B cells, and NK cells. The structure of the IL-21 receptor complex is similar to that of the IL-2 receptor complex because each of these cytokine receptor complexes contains γc.

[0375] When IL-21 binds to the IL-21 receptor complex, it activates the JAK / STAT signaling pathway to activate target genes. Although IL-21-induced signaling can be therapeutically desirable, the timing and location of this signaling pathway require careful consideration given IL-21's broad expression profile and its ability to enhance CD8+ T cell responses while inhibiting antigen presentation and T cell initiation.

[0376] In some embodiments, this disclosure provides an IL-21 polypeptide that contains at least one amino acid substitution relative to the human IL-21 amino acid sequence provided 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 containing at least one amino acid substitution relative to SEQ ID NO:390 are also referred herein as IL-21 mutant proteins. In some embodiments, the IL-21 polypeptide as described herein contains 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 greater. In some embodiments, the IL-21 polypeptide described herein comprises at least 35 amino acid substitutions compared to SEQ ID NO:390. In some embodiments, the IL-21 polypeptide described herein comprises an amino acid sequence differing from the amino acid sequence of human IL-21 (SEQ ID NO:390) by 10, 15, 20, or 25 amino acids. In some embodiments, the IL-21 polypeptide described herein comprises an amino acid sequence differing from the amino acid sequence of human IL-21 (SEQ ID NO:390) by no more than 16 amino acids. In some embodiments, the IL-21 polypeptide described herein comprises an amino acid sequence differing from the amino acid sequence of human IL-21 (SEQ ID NO:390) by 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids. In some embodiments, the IL-21 polypeptide described herein comprises an amino acid sequence differing from the amino acid sequence of human IL-21 (SEQ ID NO:390) by no more than 7 or no more than 5 amino acids. In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof, as described herein, comprises an amino acid sequence differing from the amino acid sequence of human IL-21 (SEQ ID NO: 390) by 10, 11, 12, 13, 14, 15, or 16 amino acids. In some embodiments, the IL-21 polypeptide, as described herein, comprises an amino acid sequence differing from the amino acid sequence of human IL-21 (SEQ ID NO: 390) by 3, 4, 5, or 6 amino acids. In some embodiments, the IL-21 polypeptide, as described herein, comprises an amino acid sequence differing from the amino acid sequence of human IL-21 (SEQ ID NO: 390) by 5 to 16 amino acids.In some embodiments, the IL-21 peptide described herein comprises an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO:390) by 3 to 6 amino acids or by 1 to 5 amino acids. In some embodiments, the IL-21 peptide described herein comprises an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO:390) by one or two amino acids.

[0377] Changed isoelectric point or charge distribution

[0378] In some embodiments, the IL-21 peptide contains at least one mutation that alters the isoelectric point and affects or does not affect its binding to IL-21R. In some embodiments, the IL-21 peptide contains at least one mutation that alters the isoelectric point without affecting its binding to IL-21R. An altered isoelectric point of a protein can change the surface charge distribution, thereby affecting the protein yield, nonspecific binding, and blood or circulating half-life of a given protein. In some embodiments, mutations in the IL-21 peptide lower the theoretical isoelectric point by a given unit, as measured by the publicly available database ProtParam (SwissProt) (which is hereby incorporated by reference).

[0379] In some embodiments, the IL-21 peptide 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 isoelectric point of the IL-21 peptide is about 6.0 to about 9.0. In some embodiments, the theoretical isoelectric point of the IL-21 peptide is about 6 to about 9. In some embodiments, the theoretical isoelectric point of the IL-21 peptide is at least about 6. In some embodiments, the theoretical isoelectric point of the IL-21 peptide is at most about 9.In some embodiments, the theoretical isoelectric point of the IL-21 peptide is about 9 to 8.9, about 9 to 8.8, about 9 to 8.7, about 9 to 8.5, about 9 to 8.3, about 9 to 8.1, about 9 to 7.9, about 9 to 7.7, about 9 to 7.5, about 9 to 7.3, about 9 to 7, about 9 to 6.5, about 9 to 6.0, about 8.9 to 8.8, about 8.9 to 8.7, about 8.9 to 8.5, about 8.9 to 8.3, about 8.9 to 8.1, about 8.9 to 7.9, about 8.9 to 7.7, about 8.9 to 7.5, about 8.9 to 7.3, and 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 about 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 8.1 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 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, the theoretical isoelectric point of the IL-21 polypeptide or its functional fragments or variants is 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, the theoretical isoelectric point of the IL-21 peptide is less than 9.0. In some embodiments, the theoretical isoelectric point of the IL-21 peptide is 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.

[0380] In some cases, altering the isoelectric point can increase the protein yield of the IL-21 peptide relative to human IL-21 during purification. In some embodiments, the increased protein yield is at least 5% higher than that of 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% higher than that of 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% higher than that of human IL-21. In some implementations, the increased protein yield is 5% to 10%, 5% to 15%, 5% to 20%, 5% to 25%, 5% to 50%, 5% to 75%, 5% to 100%, 5% to 125%, 5% to 150%, 5% to 175%, 5% to 200%, 5% to 225%, 5% to 250%, 5% to 275%, 5% to 300%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 75%, and 10% to 100%. 10% to 125%, 10% to 150%, 10% to 175%, 10% to 200%, 10% to 225%, 10% to 250%, 10% to 275%, 10% to 300%, 15% to 20%, 15% to 25%, 15% to 50%, 15% to 75%, 15% to 100%, 15% to 125%, 15% to 150%, 15% to 175%, 15% to 200%, 15% to 225%, 15% to 250%, 15% to 275%, 15% to 300%, 20% to 25%, 20% to 50%, 20% to 75%, 20% to 100%, 20% to 125%, 20% to 150%, 20% to 175%, 20% to 200%, 20% to 225%, 20% to 250%, 20% to 275%, 20% to 300%, 25% to 50%, 25% to 75%, 25% to 100%, 25% to 125%, 25% to 150%, 25% to 175%, 25% to 200%, 25% to 225%. 25% to 250%, 25% to 275%, 25% to 300%, 50% to 75%, 50% to 100%, 50% to 125%, 50% to 150%, 50% to 175%, 50% to 200%, 75% to 100%, 75% to 125%, 75% to 150%, 75% to 175%, 75% to 200%, 75% to 225%, 75% to 250%, 75% to 275%, 75% to 300%, 100% to 125%, 100% to 150%.100% to 175%, 100% to 200%, 100% to 225%, 100% to 250%, 100% to 275%, 100% to 300%, 125% to 150%, 125% to 175%, 125% to 200%, 125% to 225%, 125% to 250%, 125% 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 approximately 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% higher than that of human IL-21.

[0381] In some embodiments, human IL-21 comprises a region having 2-20 positively charged amino acid residues. In some embodiments, said region is 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, 4-1 6, 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 The IL-21 contains 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, 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 with 12 positively charged amino acid residues.In some implementations, human IL-21 includes a region with 2-20 amino acid residues, at least one of which is positively charged.In some implementations, 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, 11-17, 11-18, 11-19, 11-20, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 1 2-20, 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, 18-19, 18-20 or 19-20 amino acid residues, of which at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 are positively charged amino acid residues.In some embodiments, human IL-21 comprises a region having 12 amino acid residues, wherein 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 having 12 amino acid residues, wherein 2, 3, 4, 5, or 6 amino acid residues are positively charged.

[0382] In some embodiments, the amino acids in the region do not bind to the human IL-21 receptor. In some embodiments, the region comprises amino acid residues S80-T92 of SEQ ID NO:390. In some embodiments, the region comprises positively charged amino acid residues R85, R86, K88, H89, and R90. In some embodiments, the IL-21 polypeptide contains at least one amino acid substitution in the region of human IL-21. In some embodiments, the IL-21 polypeptide contains at least one amino acid substitution of S80-T92. In some embodiments, the IL-21 polypeptide contains at least four amino acid substitutions of S80-T92. In some embodiments, the IL-21 polypeptide contains at least four amino acid substitutions of S80-T92, provided that G84 is not substituted. In some embodiments, the IL-21 polypeptide contains at least one amino acid substitution at R85, R86, K88, H89, or R90. In some embodiments, the IL-21 polypeptide comprises amino acid substitutions at one or more of the following locations: S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, and T92.

[0383] In some embodiments, the IL-21 polypeptide of this disclosure has one or more, two or more, or three or more charge-reducing amino acid substitutions relative to, for example, a wild-type mature IL-21 polypeptide having the amino acid sequence of SEQ ID NO:390. In some embodiments, one or more, two or more, or three or more substituted residues are selected from the group consisting of: K56, S80, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92.

[0384] In some embodiments, the IL-21 peptide containing an altered isoelectric point relative to human IL-21 (SEQ ID NO:390) contains mutations at positions 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, the IL-21 peptide containing an altered isoelectric point relative to human IL-21 (SEQ ID NO:390) contains mutations at positions 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 peptide containing an altered isoelectric point relative to human IL-21 (SEQ ID NO:390) contains mutations at positions selected from the group consisting of: S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91 and T92 of SEQ ID NO:390.

[0385] In some embodiments, the IL-21 peptide contains a mutation at position K56 of SEQ ID NO:390. In some embodiments, the mutation includes K56G, K56S, K56E, K56D, or K56A. In some embodiments, the IL-21 peptide contains a mutation at position S80. In some embodiments, the mutation includes S80G, S80A, S80D, or S80E. In some embodiments, the IL-21 peptide contains a mutation at position T81 of SEQ ID NO:390. In some embodiments, the mutation includes T81G, T81S, T81E, T81D, or T81A. In some embodiments, the IL-21 peptide contains a mutation at position N82 of SEQ ID NO:390. In some embodiments, the mutation includes N82G, N82S, N82E, N82D, or N82A. In some embodiments, the IL-21 peptide contains 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 peptide contains 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 peptide contains 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 peptide contains 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 peptide contains 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 peptide contains a mutation at position K88 of SEQ ID NO:390. In some cases, the mutation comprises K88G, K88S, K88E, K88D, or K88A. In some embodiments, the IL-21 peptide contains 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 peptide contains 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 peptide contains a mutation at position L91 of SEQ ID NO:390. In some embodiments, the mutation includes L91G, L91S, L91E, L91D, or L91A. In some embodiments, the IL-21 peptide contains a mutation at position T92 of SEQ ID NO:390. In some embodiments, the mutation includes T92G, T92S, T92E, T92D, or T92A.

[0386] In some embodiments, the IL-21 polypeptide (relative to SEQ ID NO:390) comprises SEQ ID NO:390 having one of the following amino acid substitutions. 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, G81, 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, E85, G86, 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.

[0387] In some embodiments, the IL-21 peptide has at least 80% sequence identity with SEQ ID NO:390 and contains amino acids G85, G86, G88, and A90. In some embodiments, the IL-21 peptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:390 and contains amino acids G85, G86, G88, and A90. In some embodiments, the IL-21 peptide has at least 80% sequence identity with SEQ ID NO:390 and contains amino acids G85, G86, G88, and E90. In some embodiments, the IL-21 peptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:390 and contains amino acids G85, G86, G88, and E90. In some embodiments, the IL-21 peptide has at least 80% sequence identity with SEQ ID NO:390 and comprises amino acids A56, A75, G85, G86, G88, and E90. In some embodiments, the IL-21 peptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:390 and comprises amino acids A56, A75, G85, G86, G88, and E90. In some embodiments, the IL-21 peptide has at least 80% sequence identity with SEQ ID NO:390 and comprises amino acids A56, E75, G85, G86, G88, and A90. In some embodiments, the IL-21 peptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:390 and comprises amino acids A56, E75, G85, G86, G88, and A90. In some embodiments, the IL-21 peptide has at least 80% sequence identity with SEQ ID NO:390 and comprises amino acids A56, A75, G85, G86, G88, and A90. In some embodiments, the IL-21 peptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with 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 with SEQ ID NO:390 and contains the 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 with 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 with 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 with 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 with SEQ ID NO:390, and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 peptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:390, and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 peptide has at least 80% sequence identity with SEQ ID NO:390, and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 peptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:390 and contains amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 peptide has at least 80% sequence identity with SEQ ID NO:390 and contains amino acids G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 peptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:390 and contains amino acids G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO:390 and contains 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 with 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 with 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 with 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 with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and comprises the amino acids G85, G86, G87, G88, G89, and G90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and contains amino acids G85, G86, E87, G88, G89, and G90.

[0388] In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and contains the 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 with 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 with 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 with 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 with 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 with 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 with SEQ ID NO:390, and comprises amino acids G80, G81, G82, E83, G85, G86, E87, 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and comprises the 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 with SEQ ID NO:390 and contains 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 with SEQ ID NO:390 and contains 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 with 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 with 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 with 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 with 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 with SEQ ID NO:390, and contains amino acids G80, G81, E82, G83, G85, G86, G87, G88, G89, E90, G91, and G92.

[0389] In some embodiments, a charge-variant IL-21 polypeptide or a functional fragment or variant thereof is provided, comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences to those selected from the group consisting of SEQ ID NO. 391-421.

[0390] In some embodiments, the IL-21 peptide has about 75% to about 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO. 391-421. In some embodiments, the IL-21 peptide has at least about 75% sequence identity with a sequence selected from the group consisting of SEQ ID NO. 391-421. In some embodiments, the IL-21 peptide has at most about 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO. 391-421. In some embodiments, the IL-21 peptide has about 75% to about 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO. 391-421. The sequences in the group consisting of NO.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 have approximately 75% to approximately 80% sequence identity, approximately 75% to approximately 85% sequence identity, approximately 75% to approximately 90% sequence identity, approximately 75% to approximately 95% sequence identity, and approximately 75% to approximately 96% sequence identity. Sex, approximately 75% sequence identity to approximately 97% sequence identity, approximately 75% sequence identity to approximately 98% sequence identity, approximately 75% sequence identity to approximately 99% sequence identity, approximately 75% sequence identity to approximately 100% sequence identity, approximately 80% sequence identity to approximately 85% sequence identity, approximately 80% sequence identity to approximately 90% sequence identity, approximately 80% sequence identity to approximately 95% sequence identity, approximately 80% sequence identity to approximately 96% sequence identity, approximately 80% sequence identity to approximately 97% sequence identity, approximately 80% sequence identity to approximately 9 8% sequence identity, approximately 80% sequence identity to approximately 99% sequence identity, approximately 80% sequence identity to approximately 100% sequence identity, approximately 85% sequence identity to approximately 90% sequence identity, approximately 85% sequence identity to approximately 95% sequence identity, approximately 85% sequence identity to approximately 96% sequence identity, approximately 85% sequence identity to approximately 97% sequence identity, approximately 85% sequence identity to approximately 98% sequence identity, approximately 85% sequence identity to approximately 99% sequence identity, approximately 85% sequence identity to approximately 100% sequence identity, approximately 90% sequence identity. The sequence identity is approximately 95% to 96%; approximately 90% to 97%; approximately 90% to 98%; approximately 90% to 99%; approximately 90% to 100%; approximately 95% to 96%; approximately 95% to 97%; approximately 95% to 98%; approximately 95% to 99%; approximately 95% to 99%Approximately 95% sequence identity to approximately 100% sequence identity, approximately 96% sequence identity to approximately 97% sequence identity, approximately 96% sequence identity to approximately 98% sequence identity, approximately 96% sequence identity to approximately 99% sequence identity, approximately 96% sequence identity to approximately 100% sequence identity, approximately 97% sequence identity to approximately 98% sequence identity, approximately 97% sequence identity to approximately 99% sequence identity, approximately 97% sequence identity to approximately 100% sequence identity, approximately 98% sequence identity to approximately 99% sequence identity, approximately 98% sequence identity to approximately 100% sequence identity, or approximately 99% sequence identity to approximately 100% sequence identity. In some embodiments, the IL-21 peptide has approximately 75% sequence identity, approximately 80% sequence identity, approximately 85% sequence identity, approximately 90% sequence identity, approximately 95% sequence identity, approximately 96% sequence identity, approximately 97% sequence identity, approximately 98% sequence identity, approximately 99% sequence identity, or approximately 100% sequence identity with sequences selected from the group consisting of SEQ ID NO. 391-421.

[0391] In some embodiments, the IL-21 polypeptide or a functional fragment or variant thereof has at least 80% sequence identity with SEQ ID NO:392 and / or contains amino acids G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide or a functional fragment or variant thereof has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:392 and / or contains amino acids G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide or a functional fragment or variant thereof has at least 80% sequence identity with SEQ ID NO:391 and / or contains amino acids G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide or a functional fragment or variant thereof has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:391, and / or contains amino acids G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide or a functional fragment or variant thereof has at least 80% sequence identity with SEQ ID NO:393, and / or contains amino acids A56, A75, G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide or a functional fragment or variant thereof has at least 85%, 90%, 9...

Claims

1. A method of treating a subject suffering from a disease, the method comprising administering to the subject: (a) an effective amount of an IL-21 fusion protein comprising (i) a first antibody or an antigen-binding fragment thereof that specifically binds to human CD8, and (ii) an IL-21 polypeptide; and (b) an effective amount of an IL-2 fusion protein comprising (i) a second antibody or an antigen-binding fragment thereof that specifically binds to human CD8, and (ii) an IL-2 polypeptide.

2. A method for treating a subject having a disease, wherein the subject has received or is receiving treatment with an IL-21 fusion protein, the method comprising: An effective amount of an IL-2 fusion protein is administered to the subject, wherein the IL-21 fusion protein comprises (i) a first antibody or an antigen-binding fragment thereof that specifically binds to human CD8, and (ii) an IL-21 polypeptide, and wherein the IL-2 fusion protein comprises (i) a second antibody or an antigen-binding fragment thereof that specifically binds to human CD8, and (ii) an IL-2 polypeptide.

3. A method of treating a subject having a disease, wherein the subject has received or is receiving treatment with an IL-2 fusion protein, the method comprising: An effective amount of an IL-21 fusion protein is administered to the subject, wherein the IL-21 fusion protein comprises (i) a first antibody or an antigen-binding fragment thereof that specifically binds to human CD8, and (ii) an IL-21 polypeptide, and wherein the IL-2 fusion protein comprises (i) a second antibody or an antigen-binding fragment thereof that specifically binds to human CD8, and (ii) an IL-2 polypeptide.

4. The method of any one of claims 1-3, wherein the disease comprises cancer.

5. The method of any one of claims 1-3, wherein the disease comprises an infectious disease.

6. The method of any one of claims 1-3, wherein the treatment induces an immune response in the subject.

7. The method of any one of claims 1-3, wherein the treatment induces a T cell response in the subject.

8. The method of any one of claims 1-3, wherein the treatment delays cancer progression, reduces tumor volume, and / or reduces or inhibits tumor growth in the subject.

9. The method of any one of claims 1-3, wherein the treatment delays cancer progression, reduces tumor volume, and / or reduces or inhibits tumor growth in the subject more than treatment with the IL-2 fusion protein or the IL-21 fusion protein alone in the subject.

10. The method of any one of claims 1-3, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are the same.

11. The method of any one of claims 1-3, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are different.

12. The method of any one of claims 1-3, wherein the first antibody or antigen-binding fragment thereof 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 wherein in the first antibody or antigen-binding fragment thereof: 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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, which comprises the amino acid sequence of SEQ ID NO:229; CDR-H2, which comprises the amino acid sequence of SEQ ID NO:237; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:231; and the VL domain comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:40; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:41; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:

42.

13. The method of any one of claims 1-3, wherein the first antibody or antigen-binding fragment thereof 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 wherein in the first antibody or antigen-binding fragment thereof: 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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, which comprises the amino acid sequence of SEQ ID NO:240; CDR-H2, which comprises the amino acid sequence of SEQ ID NO:244; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:242; and the VL domain comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:40; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:41; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:

42.

14. The method of any one of claims 1-3, wherein the first antibody or antigen-binding fragment thereof 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 wherein the VH domain and the VL domain of the first antibody or antigen-binding fragment thereof comprise the following amino acid sequence: (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; (1) SEQ ID NO: 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.

15. The method of any one of claims 1-3, wherein the second antibody or antigen-binding fragment thereof 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 wherein in the second antibody or antigen-binding fragment thereof: 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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, which comprises the amino acid sequence of SEQ ID NO:229; CDR-H2, which comprises the amino acid sequence of SEQ ID NO:237; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:231; and the VL domain comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:40; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:41; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:

42.

16. The method of any one of claims 1-3, wherein the second antibody or antigen-binding fragment thereof 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 wherein in the second antibody or antigen-binding fragment thereof: 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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, which comprises the amino acid sequence of SEQ ID NO:240; CDR-H2, which comprises the amino acid sequence of SEQ ID NO:244; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:242; and the VL domain comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:40; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:41; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:

42.

17. The method of any one of claims 1-3, wherein the second antibody or antigen-binding fragment thereof 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 wherein the VH domain and the VL domain of the second antibody or antigen-binding fragment thereof comprise the following amino acid sequence: (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; (1) SEQ ID NO: 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.

18. The method of any one of claims 1-3, wherein the binding affinity of the IL-2 polypeptide to IL-2Rα is reduced by 50% or more compared to the binding affinity of a human IL-2 polypeptide comprising SEQ ID NO:

81.

19. The method of claim 18, wherein the IL-2 polypeptide comprises one or more amino acid mutations that reduce the binding affinity of the IL-2 polypeptide relative to the human IL-2 polypeptide comprising SEQ ID NO:

81.

20. The method of any one of claims 1-3, wherein the IL-2 polypeptide comprises an amino acid sequence of SEQ ID NO:81 having one, two, three, four or five amino acid substitutions relative to SEQ ID NO:81, and wherein the one, two, three, four or five substitutions comprise substitutions at positions of SEQ ID NO:81 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.

21. The method of claim 20, wherein the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with a set of amino acid substitutions relative to the sequence of SEQ ID NO: 81 selected from the group consisting of: 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, 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.

22. The method of claim 20, wherein the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 with another amino acid substitution at position C125 relative to SEQ ID NO:

81.

23. The method of claim 22, wherein the IL-2 polypeptide comprises SEQ ID NO: 81 with a set of amino acid substitutions. NO:81, wherein the set of amino acid substitutions is selected from the group consisting of: 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, N88T and C125A; R38E, F42A, V91E and C 125A; 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 5A; 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 C125 A; 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 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; H16D, F42A, 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. ; 24. The method of any one of claims 1-3, wherein the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 80 or SEQ ID NO:

297.

25. The method of any one of claims 1-3, wherein the IL-2 polypeptide comprises a sequence selected from the group consisting of: SEQ ID NO: 80, 85-155, 190-216, 297, and 354-383.

26. The method of any one of claims 1-3, wherein the IL-21 polypeptide comprises an amino acid sequence that is at least 80% identical to a human IL-21 polypeptide comprising SEQ ID NO:

390.

27. The method of claim 26, wherein the isoelectric point of the IL-21 polypeptide is at least about 0.6 units to about 5 units lower than the isoelectric point of the human IL-21 polypeptide.

28. The method of claim 27, wherein the IL-21 polypeptide comprises at least one amino acid substitution that lowers the isoelectric point of the IL-21 polypeptide by about 0.6 units to about 5 units relative to the human IL-21 polypeptide without the amino acid substitution.

29. The method of claim 27, wherein the IL-21 polypeptide comprises at least four amino acid substitutions that lower the isoelectric point of the IL-21 polypeptide by about 0.6 units to about 5 units relative to the human IL-21 polypeptide without the amino acid substitutions.

30. The method of claim 28 or 29, wherein the IL-21 polypeptide comprises at most 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions that lower the isoelectric point.

31. The method of claim 26, wherein the human IL-21 polypeptide has an isoelectric point of about 9.

42.

32. The method of claim 26, wherein the IL-21 polypeptide has an isoelectric point of about 7.12 to about 8.

72.

33. The method of claim 26, wherein the IL-21 polypeptide results in improved exposure relative to the human IL-21 polypeptide following administration to the subject as measured by at least about 1.5-fold higher area under the curve (AUC).

34. The method of claim 26, wherein the IL-21 polypeptide comprises a modified region, wherein relative to a region of the human IL-21 polypeptide comprising about 2 to 20 positively charged amino acid residues, the modified region comprises one or more amino acid substitutions having the about 2 to 20 positively charged amino acid residues.

35. The method of claim 34, wherein the modified region excludes amino acid residues that bind to human IL-21 receptor.

36. The method of claim 34 or 35, wherein the modified region comprises amino acid residues located at S80 to T92 of the human IL-21 polypeptide comprising SEQ ID NO:

390.

37. The method of claim 34, wherein 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 and does not bind to an IL-21 receptor, and wherein the IL-21 polypeptide comprises at least one amino acid substitution with the at least one positively charged amino acid residue.

38. The method of claim 34, wherein the IL-21 polypeptide does not comprise an amino acid substitution at G84 of the human IL-21 polypeptide comprising SEQ ID NO:

390.

39. The method of claim 26, wherein the IL-21 polypeptide comprises 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions relative to SEQ ID NO: 81 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.

40. The method of claim 39, 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.

41. The method of claim 39, wherein 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.

42. The method of any one of claims 1-3, wherein the IL-21 polypeptide comprises the amino acid sequence: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLP APEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPX1X2X3X4GX5X6X7X8X9X 10 X 11 X 12 CPSCDSYEKKPPKEFLERFKSLLQ KMIHQHLSSRTHGSEDS (SEQ ID NO: 554), where X1=G, S; X2=G, T; X3=G, E, N; 10 =A, E, S, G, R; X 11 = S, G, L; and X 12 =G, S, T, condition is X5, X6, X8, X9 and X 10 At least one of is not the amino acid residue at the same position as set forth in SEQ ID NO: 390, optionally wherein: (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,X 11 =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; or (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。 43. The method of any one of claims 1-3, wherein the IL-12 polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 391-421.

44. The method of claim 43, wherein the IL-21 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-421.

45. The fusion protein of claim 44, wherein the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO:

421.

46. ​​The method of any one of claims 1-3, wherein the IL-21 polypeptide comprises at least one amino acid substitution that reduces binding to an IL-21 receptor compared to binding by the human IL-21 polypeptide.

47. The method of claim 46, wherein the at least one amino acid substitution is at 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.

48. The method of claim 47, wherein the at least one amino acid substitution is selected from the group consisting of: R5F, R5A, R5E, R5S, R5T, R5N, R5Q, R5V, R5I, R5L, R5Y, I8E, 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.

49. The method of any one of claims 1-3, 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: 422-491.

50. The method of any one of claims 1-3, wherein the IL-21 polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 422-491.

51. The method of claim 50, wherein the IL-21 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 422-491.

52. The method of any one of claims 1-3, wherein 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 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.

53. The method of any one of claims 1-3, wherein 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; 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; 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.

54. The method of any one of claims 1-3, wherein 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 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.

55. The method of any one of claims 1-3, wherein 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; 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; 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; 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; 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; 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; 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; 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; 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:575, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:573; 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; 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; 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; 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; 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; 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; 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; 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.

56. The method of claim 1, wherein the IL-21 fusion protein is administered prior to administering the IL-2 fusion protein.

57. The method of claim 1, wherein the IL-2 fusion protein is administered prior to administering the IL-21 fusion protein.

58. The method of claim 1, wherein the IL-21 fusion protein and the IL-2 fusion protein are administered concurrently, sequentially, or simultaneously.

59. The method of claim 1, wherein the IL-21 fusion protein and the IL-2 fusion protein are administered sequentially.

60. The method of any one of claims 1-3, wherein the IL-21 fusion protein and the IL-2 fusion protein are formulated as a single pharmaceutical composition.

61. The method of any one of claims 1-3, wherein the IL-21 fusion protein and the IL-2 fusion protein are formulated as separate pharmaceutical compositions.

62. The method of any one of claims 1-3, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof each independently targets CD8ab or CD8b.

63. The method of claim 62, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof each target a different epitope on CD8ab or CD8b.

64. The method of claim 62, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof do not cross-compete for binding to CD8ab or CD8b.

65. A drug kit comprising one or more containers comprising (a) an IL-21 fusion protein comprising: (i) a first antibody or an antigen-binding fragment thereof that specifically binds to human CD8, and (ii) an IL-21 polypeptide; and (b) an IL-2 fusion protein comprising: (i) a second antibody or an antigen-binding fragment thereof that specifically binds to human CD8, and (ii) an IL-2 polypeptide; optionally a pharmaceutically acceptable carrier; optionally instructions for administering the IL-21 fusion protein and the IL-2 fusion protein to a subject in need thereof concurrently, sequentially or simultaneously.

66. A kit comprising a container comprising an IL-21 fusion protein, and instructions for administering the IL-21 fusion protein, optionally a pharmaceutically acceptable carrier, to a subject in need thereof who has received or is receiving treatment with an IL-2 fusion protein, wherein the IL-21 fusion protein comprises a first antibody or antigen-binding fragment thereof that specifically binds to human CD8 and an IL-21 polypeptide, and wherein the IL-2 fusion protein comprises a second antibody or antigen-binding fragment thereof that specifically binds to human CD8 and an IL-2 polypeptide.

67. A drug kit comprising a container comprising an IL-2 fusion protein, and instructions for administering the IL-2 fusion protein, optionally a pharmaceutically acceptable carrier, to a subject in need thereof who has received or is receiving treatment with an IL-21 fusion protein, wherein the IL-21 fusion protein comprises a first antibody or antigen-binding fragment thereof that specifically binds to human CD8 and an IL-21 polypeptide, and wherein the IL-2 fusion protein comprises a second antibody or antigen-binding fragment thereof that specifically binds to human CD8 and an IL-2 polypeptide.

68. A composition comprising (a) an IL-21 fusion protein comprising: (i) a first antibody or an antigen-binding fragment thereof that specifically binds to human CD8, and (ii) an IL-21 polypeptide; and (b) an IL-2 fusion protein comprising (i) a second antibody or an antigen-binding fragment thereof that specifically binds to human CD8, and (ii) an IL-2 polypeptide.

69. A pharmaceutical composition comprising the composition of claim 68 and a pharmaceutically acceptable carrier.

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