IL-21 Polypeptides and Targeting Constructs

Abstract

The present disclosure provides methods and compositions comprising IL-21 polypeptides, targeted cytokine constructs that selectively activate targeted immune cells (e.g., CD8+ T cells) over other immune cell types. The cytokines of the present disclosure may further comprise mutations that alter their charge distribution to improve their half-life in the blood.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 190,669, filed May 19, 2021; U.S. Provisional Application No. 63 / 223,684, filed July 20, 2021; PCT / US2021 / 056312, filed October 22, 2021; PCT / US2021 / 062458, filed December 8, 2021; and U.S. Provisional Application No. 63 / 297,631, filed January 7, 2022, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] Interleukin-21 (IL-21) is a T cell-derived pleiotropic cytokine that regulates the activity of both innate and adaptive immune cells. IL-21 can enhance T cell survival and effector function. Interleukin-21 is a type I cytokine and a member of the common cytokine receptor gamma chain (γc) family, which has emerged as a promising immunotherapeutic agent for the treatment of cancer. In some cases, IL-21 produced by activated CD4+ T cells and natural killer T (NKT) cells signals through a heterodimeric receptor complex containing the individual IL-21 receptor (IL-21R) subunit along with γc. In some cases, activation of the IL-21R complex leads to activation of the JAK / STAT signaling pathway. IL-21R is widely expressed in hematopoietic cells, including T and B lymphocytes, natural killer (NK) cells, and myeloid cells. IL-21 is a potent mitogen and survival factor for both NK cells and activated T cells. IL-21 can support the differentiation of CD4+ T helper 17 (Th17) and follicular helper T cells (Tfh) and block the differentiation of regulatory T cells (Treg). IL-21 can also enhance the survival of CD8+ T cells, resulting in a less activated but more persistent T cell phenotype, leading to improved tumor and viral control. A challenge with cytokine immunotherapy is that, in some cases, while activating immune cells to enhance immune responses, the same cytokines can also activate counterregulatory pathways, as exemplified by IL-2 and IFNγ. These counterregulatory pathways can activate regulatory T cell responses and inhibitory pathways. IL-21 has become an attractive target for several therapies because it plays a role in antitumor and antiviral responses in addition to exerting major effects on inflammatory reactions that lead to the development of autoimmune and inflammatory diseases.

[0003] There remains a need for treatment modalities that utilize IL-21, including sites that combine IL-21 modalities to target IL-21 to specific cell types. Summary of the Invention

[0004]

[0010] Provided herein in one embodiment of the present disclosure is an IL-21 polypeptide or functional fragment or variant thereof comprising a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 1, wherein the IL-21 polypeptide has an isoelectric point that is at least about 0.6 units to about 5 units lower than that of a wild-type IL-21 protein having the sequence of SEQ ID NO: 1. In some embodiments, the isoelectric point of SEQ ID NO: 1 is about 9.42. In some embodiments, the IL-21 polypeptide has an isoelectric point of about 7.12 to about 8.72. In some embodiments, the polypeptide provides improved exposure compared to wild-type IL-21, as measured by an AUC that is at least about 1.5-fold higher for the polypeptide compared to that of the wild-type IL-21 protein, when administered to a subject at an equivalent concentration. In some embodiments, the IL-21 polypeptide comprises at least one amino acid substitution that reduces the isoelectric point of the IL-21 polypeptide by about 0.6 units to about 5 units compared to a human IL-21 polypeptide without the amino acid substitution. In some embodiments, the IL-21 polypeptide comprises at least four amino acid substitutions that reduce the isoelectric point of the IL-21 polypeptide by about 0.6 units to about 5 units compared to a human IL-21 polypeptide without the amino acid substitutions, hi some embodiments, the IL-21 polypeptide comprises up to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions that reduce the isoelectric point.

[0005] In some embodiments, the present disclosure provides an IL-21 polypeptide, or a functional fragment or variant thereof, wherein the IL-21 polypeptide comprises at least one amino acid substitution of about 2-20 positively charged amino acid residues relative to a region of a human IL-21 polypeptide comprising about 2-20 positively charged amino acid residues. In some embodiments, this region of the human IL-21 polypeptide does not comprise amino acid residues that bind the human IL-21 receptor. In some embodiments, this region of the human IL-21 polypeptide comprises amino acid residues S80 to T92 of a human IL-21 polypeptide comprising SEQ ID NO:1. In some embodiments, the present disclosure provides an IL-21 polypeptide or functional fragment or variant thereof, 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, does not directly interact with or bind to a human IL-21 receptor, and the IL-21 polypeptide comprises at least one amino acid substitution of the at least one positively charged amino acid residue. In some embodiments, the present disclosure provides an IL-21 polypeptide that does not comprise an amino acid substitution at G84 of a human IL-21 polypeptide comprising SEQ ID NO:1.

[0006] In some embodiments, the IL-21 polypeptide comprises a mutation at one or more positions selected from the group consisting of K56, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92 of SEQ ID NO: 1. In some embodiments, the IL-21 polypeptide comprises a mutation (e.g., an amino acid substitution) at one or more 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: 1. In some embodiments, the IL-21 polypeptide comprises 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions at positions selected from S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, or T92 of SEQ ID NO:1. In some embodiments, the IL-21 polypeptide comprises 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions selected from S80G, T81G, N82G, N82E, A82G, A83E, A83S, R85G, R85E, R85S, R86G, R86E, Q87G, Q87E, Q87S, K88G, H89G, H89S, R90G, R90S, R90E, R90A, L91G, L91S, T92G, T92S. In some embodiments, an 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 embodiments, the disclosure provides an IL-21 polypeptide that does not comprise an amino acid substitution at G84 of a human IL-21 polypeptide comprising SEQ ID NO:1.

[0007] In some embodiments, the present disclosure provides a nucleic acid sequence comprising the amino acid sequence: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSCFQKAQLKSANTGNNERIINVSIKKLKRKPPX1X2X3X4GX5X6X7X8X9X 10 X 11 X 12CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 380) (wherein X1 = G,S; X2 = G,T; X3 = G,E,N; X4 = G,S,E,A; X5 = G,E,S,R; X6 = G,E,R; X7 = S,G,E,Q; X8 = G,K; X9 = G,S,H; X 10 =A, E, S, G, R;X 11 = S, G, L; and X 12 = G, S, T, but X 5、 X 6、 X 8、 X9 and X 10 is not the amino acid residue at the same position as shown in SEQ ID NO: 1, and optionally: (i) X5=G, X6=G, X8=G, X 10 =A; (ii) X5=G, X6=G, X8=G, X 10 =E; (iii)X1=G,X2=G,X3=G,X4=S,X5=E,X6=G,X7=S,X8=G,X9=G,X 10 =S; (iv)X1=G,X2=G,X3=G,X4=S,X5=G,X6=G,X7=S,X8=G,X9=G,X 10 =E; (v)X5=G, X6=G, X7=S, X8=G, X9=G, X 10 =E; (vi)X5=G, X6=G, X7=S, X8=G, X9=G, X 10 =S; (vii)X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E; (viii)X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =G; (ix)X5=G, X6=G, X7=E, X8=G, X9=G, X 10 =G; (x)X1=G, X2=G, X3=G, X4=S, X5=G, X6=G, X7=S, X8=G, X9=G, X 10 =G,X 11 =S, X 12 =G; (xi)X1=G、X2=G、X3=G、X4=G、X5=G、X6=G、X7=G、X8=G、X9=G、X 10 =G、X 11 =G、X 12 =G; (xii)X1=G、X2=G、X3=G、X4=S、X5=G、X6=G、X7=E、X8=G、X9=G、X 10 =G、X 11 =S、X 12 =G; (xiii)X1=G、X2=G、X3=G、X4=E、X5=G、X6=G、X7=E、X8=G、X9=G、X 10 =G、X 11 =S、X 12 =G; (xiv)X3=G、X4=G、X5=S、X6=G、X7=G、X8=G、X9=S、X 10 =G、X 11 =G、X 12 =S; (xv)X3=G、X4=G、X5=E、X6=G、X7=G、X8=G、X9=S、X 10 =G、X 11 =G、X 12 =S; (xvi)X1=G、X2=G、X3=G、X4=G、X5=E、X6=G、X7=G、X8=G、X9=G、X 10 =G、X 11 =G、X 12 =G; (xvii)X1=G、X2=G、X3=G、X4=S、X5=G、X6=G、X7=S、X8=G、X9=G、X 10 =E、X 11 =G、X 12 =G; (xviii)X1=G、X2=G、X3=G、X4=G、X5=G、X6=G、X7=G、X8=G、X9=G、X 10 =E、X 11 =G、X 12 =G; (xix)X3=G、X4=G、X5=S、X6=G、X7=G、X8=G、X9=S、X 10 =E、X 11 =G、X 12 =S; (xx)X3=G, X4=G, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E,X 11 =G,X 12 =G; (xxi)X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E,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) of a human IL-21 polypeptide comprising SEQ ID NO:1.

[0008] In some embodiments, the present disclosure provides an IL-21 polypeptide or functional fragment or variant thereof comprising an amino acid sequence at least 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to an amino acid sequence selected from SEQ ID NOs: 2, 4-15, and 23-40. In some embodiments, the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15, and 23-40. In some embodiments, the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO: 40.

[0009] In some embodiments, the present disclosure provides an IL-21 polypeptide or functional fragment or variant thereof comprising at least one amino acid substitution that reduces binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide, in some embodiments, the at least one amino acid substitution is in one or more amino acid residues at positions R5, 18, R9, R11, L13, 114, 116, V17, D18, K72, K73, L74, K75, R76, K77, or K117 of SEQ ID NO:1. In some embodiments, 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, In some embodiments, the at least one amino acid substitution is selected from I16R, V17I, V17A, D18A, K72A, K72E, K73A, K73E, K75A, K75E, L74I, L74F, L74M, L74V, R76E, R76F, R76A, R76N, R76D, R76S, R76T, R76Q, R76V, R76I, R76L, R76Y, R76M, K77A, K77E, and K117A. In some embodiments, the at least one amino acid substitution is R76E or R76Q. In some embodiments, the IL-21 polypeptide comprises a mutation at a position selected from the group consisting of R5, 18, R9, R11, Q12, 114, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117 of SEQ ID NO: 1. In some embodiments, the IL-21 polypeptide comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.In some embodiments, an IL-21 polypeptide comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. In some embodiments, an IL-21 polypeptide comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a sequence selected from SEQ ID NOs: 2, 4-15, and 23-40. In some embodiments, an IL-21 polypeptide comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a sequence selected from SEQ ID NOs: 16-21, 41-98, and 374-379. In some embodiments, an IL-21 polypeptide comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a sequence selected from SEQ ID NOs: 16-21, 41-93, and 374-379. In some embodiments, an IL-21 polypeptide comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a sequence selected from SEQ ID NOs: 94-98.

[0010]

[0010] In one embodiment of the present disclosure, there is provided herein a targeted cytokine construct comprising an IL-21 polypeptide or functional fragment or variant thereof according to the present disclosure and an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to CD8+ T cells. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to at least one of CD8α, CD8αα, or CD8αβ. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to CD8β.

[0011] Provided herein in one embodiment of the present disclosure is a targeted cytokine construct comprising: a) an IL-21 polypeptide or a functional fragment or variant thereof comprising an amino acid sequence at least 80% identical to SEQ ID NO: 1; and b) an antibody or antigen-binding fragment thereof that specifically binds to at least one of CD8α, CD8αα, or CD8αβ.

[0012] Provided herein, in one embodiment of the present disclosure, is a targeted cytokine construct comprising: a) an IL-21 polypeptide or a functional fragment or variant thereof comprising an amino acid sequence at least 80% identical to SEQ ID NO: 1; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8β.

[0013] In some embodiments, the IL-21 polypeptide comprises a mutation at one or more amino acid positions of SEQ ID NO: 1. In some embodiments, the IL-21 polypeptide comprises a mutation at one or more positions selected from the group consisting of K56, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92 of SEQ ID NO: 1. In some embodiments, the IL-21 polypeptide comprises a mutation at a position selected from the group consisting of R5, 18, R9, R11, Q12, 114, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117 of SEQ ID NO: 1. In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0014] In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. In some embodiments, the construct activates CD8+ T cells with at least about 10-fold or greater potency compared to activation of NK or CD4+ T cells. In some embodiments, the construct activates CD8+ T cells with at least about 10-fold to about 100,000-fold greater potency compared to activation of NK or CD4+ T cells. In some embodiments, the antibody or antigen-binding fragment thereof comprises: i) a first polypeptide comprising, arranged from N to C-terminus, a variable light chain (VL) amino acid sequence and a light chain constant region amino acid sequence (CL1); ii) a second polypeptide comprising, arranged from N to C-terminus, a variable heavy chain (VH) amino acid sequence, a heavy chain CH1 constant region amino acid sequence, a hinge region amino acid sequence, a heavy chain CH2 constant region amino acid sequence, and a heavy chain CH3 constant region amino acid sequence; and iii) a third polypeptide comprising, arranged from N to C-terminus, a hinge region amino acid sequence, a heavy chain CH2 constant region amino acid sequence, and a heavy chain CH3 constant region amino acid sequence, wherein the CH2 and CH3 domains of each of the second and third polypeptides form an Fc domain. In some embodiments, the third polypeptide comprises, arranged from N to C-terminus, a variable heavy chain (VH) amino acid sequence, a heavy chain CH1 constant region amino acid sequence, a hinge region amino acid sequence, a heavy chain CH2 constant region amino acid sequence, and a heavy chain CH3 constant region amino acid sequence, and the antibody or antigen-binding fragment thereof further comprises iv) a fourth polypeptide comprising, arranged from N to C-terminus, a variable light chain (VL) amino acid sequence, and a light chain constant amino acid sequence (CL1). In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof and the antibody or antigen-binding fragment thereof are operably linked to each other.In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, is linked to the N-terminus or C-terminus of the antibody, or antigen-binding fragment thereof. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, is conjugated to the C-terminus of a second or third polypeptide. In some embodiments, the targeted cytokine construct comprises at least one molecule of an IL-21 polypeptide. In some embodiments, the Fc domain is a human IgG Fc domain. In some embodiments, the Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the Fc domain comprises one or more modifications that promote heterodimerization. In some embodiments, the second polypeptide comprises a knob modification in the CH2 or CH3 domain and the third polypeptide comprises a hole modification in the CH2 or CH3 domain; or the third polypeptide comprises a knob modification in the CH2 or CH3 domain and the second polypeptide comprises a hole modification in the CH2 or CH3 domain. In some embodiments, at least one of the second and third polypeptides comprises the following mutations, numbered according to the EU index: L234A, L235A, and G237A.

[0015] Provided herein in one embodiment of the present disclosure is a targeted cytokine construct comprising: a) an IL-21 polypeptide or a functional fragment or variant thereof comprising an amino acid sequence at least 80% identical to SEQ ID NO: 1; and b) an antibody or antigen-binding fragment thereof comprising a first antigen-binding arm and a second antigen-binding arm, wherein the first and second antigen-binding arms bind to two different antigens, and at least one of the first and second antigen-binding arms specifically binds to CD8α, CD8αα, or CD8αβ.

[0016] Provided herein in one embodiment of the present disclosure is a targeted cytokine construct comprising: a) an IL-21 polypeptide or a functional fragment or variant thereof comprising an amino acid sequence at least 80% identical to SEQ ID NO: 1; and b) an antibody or antigen-binding fragment thereof comprising a first antigen-binding arm and a second antigen-binding arm, wherein the first and second antigen-binding arms bind to two different antigens, and at least one of the first and second antigen-binding arms specifically binds to CD8β. In some embodiments, the targeted cytokine construct activates CD8+ T cells with at least about 10-fold or greater potency compared to activation of NK cells or CD4+ T cells. In some embodiments, the targeted cytokine construct activates CD8+ T cells with at least about 10-fold to about 100,000-fold greater potency compared to activation of NK cells or CD4+ T cells. In some embodiments, the IL-21 polypeptide, or functional fragment thereof, or variant thereof, comprises a mutation at a position selected from the group consisting of K56, T81, N82, A83, R85, G84, R86, Q87, K88, H89, R90, L91, and T92 of SEQ ID NO: 1. In some embodiments, the IL-21 polypeptide, or functional fragment thereof, or variant thereof, comprises a mutation at a position selected from the group consisting of R5, 18, R9, R11, Q12, 114, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117 of SEQ ID NO: 1. In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the sequence of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

[0017] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising: (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO:1, wherein the one or more amino acid substitutions are within S80 to T92 of SEQ ID NO:1, and at least one amino acid substitution is at residue R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by the human IL-21 polypeptide; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β In some embodiments, the IL-21 polypeptide does not include a G84 substitution. In some embodiments, the at least one amino acid substitution that provides reduced binding is R76E or R76Q.

[0018] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising: (i) four or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO:1, wherein the four or more amino acid substitutions are within S80 to T92 of SEQ ID NO:1, and at least four amino acid substitutions are at residues R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by the human IL-21 polypeptide; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β The present invention provides a fusion protein comprising:

[0019] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising: (i) four or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO:1, wherein the four or more amino acid substitutions are within S80 to T92 of SEQ ID NO:1, and wherein at least four amino acid substitutions are at residues R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide, wherein at least one amino acid substitution is at a residue selected from R5, 18, R9, R11, L13, 114, 116, V17, D18, K72, K73, L74, K75, R76, K77, K117, and a combination thereof; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β The present invention provides a fusion protein comprising:

[0020] In some embodiments, the present disclosure provides a fusion protein comprising: a) (i) four or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO:1, wherein the four or more amino acid substitutions are within S80 to T92 of SEQ ID NO:1, and at least four amino acid substitutions are at residues R85, R86, K88, H89, R90, or a combination thereof, and (ii) (a) R11D; (b) R11E; (c) I14D, D18A, and K117A; (d) R76E; (e) R5F; (f) R76F ;(g)I8E;(h)R5A;(i)R5E;(j)R5S;(k)R5T;(l)R5N;(m)R5Q;(n)R5V;(o)R5I;(p)R5L;(q)R5Y;(r)R76A;(s)R76N;(t)R76D;(u)R76S;(v )R76T;(w)R76Q;(x)R76V;(y)R76I;(z)R76L;(aa)R76Y;(bb)K77A;(cc)K77E;(dd)K72A;(ee)K72E;(ff)K75A;(gg)K75E;(hh)K73A;(ii )K73E;(jj)R5F and K77A;(kk)R5F and K77E;(ll)R5F and K72A;(mm)R5F and K72E;(nn)R5F and K76A;(oo)R5F and K76E;(pp)K73A and K76F;(qq)K73E and K76F;(rr)R9A;(ss)R9D;(tt)R9E;(uu)R9H;(vv)R9S;(ww)R9T;(xx)R9N;(zz)R9G;(aaa)R9V;(bbb)R9I;(ccc)R9L;(ddd)R9Y;(eee) (hhh) K75E and R76F; (iii) V17I and L74I; (jjj) I16A and L74F; (kkk) I16S, V17I, and L74V; (lll) I16R, V17I, and L74I; (mmm) L13F, I16A, V17A, and L74M; and (nnn) an IL-21 polypeptide comprising at least one amino acid substitution selected from L13R, I16A, V17I, and L74I; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β The present invention provides a fusion protein comprising:

[0021] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising: (i) four or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the at least four amino acid substitutions are at residues S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, T92, or a combination thereof, and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide, wherein the at least one amino acid substitution is at a residue selected from R5, 18, R9, R11, L13, 114, 116, V17, D18, K72, K73, L74, K75, R76, K77, K117, and a combination thereof; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β The present invention provides a fusion protein comprising:

[0022] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising: (i) four or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO:1, wherein the at least four amino acid substitutions are at residues S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, T92, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by the human IL-21 polypeptide, wherein the at least one amino acid substitution is at residue R76; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β The present invention provides a fusion protein comprising:

[0023] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising (i) an amino acid sequence selected from SEQ ID NOs: 2, 4-15, and 23-40, and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β The present invention provides a fusion protein comprising:

[0024] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising: (i) an amino acid sequence selected from SEQ ID NOs: 2, 4-15, and 23-40; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide, wherein the at least one amino acid substitution is at a residue selected from R5, 18, R9, R11, L13, 114, 116, V17, D18, K72, K73, L74, K75, R76, K77, K117, and a combination thereof; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β The present invention provides a fusion protein comprising:

[0025] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising: (i) an amino acid sequence selected from SEQ ID NOs: 2, 4-15, and 23-40; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide, wherein the at least one amino acid substitution is at residue R76; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β The present invention provides a fusion protein comprising:

[0026] In some embodiments, the IL-21 polypeptide and the antibody or antigen-binding fragment thereof are linked to each other via a linker. In some embodiments, the IL-21 polypeptide is linked to the N-terminus or C-terminus of the antibody or antigen-binding fragment thereof. In some embodiments, the IL-21 polypeptide is linked to the C-terminus of the antibody or antigen-binding fragment thereof.

[0027] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a knob modification; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the VH chain and the VL chain comprise CDRs that specifically bind to CD8αβ or CD8β; The IL-21 polypeptide comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO:1, wherein the one or more amino acid substitutions are within S80 to T92 of SEQ ID NO:1, and at least one amino acid substitution is at residue R85, R86, K88, H89, R90, or a combination thereof, and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide.

[0028] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the VH chain and the VL chain comprise CDRs that specifically bind to CD8αβ or CD8β; The IL-21 polypeptide comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO:1, wherein the one or more amino acid substitutions are within S80 to T92 of SEQ ID NO:1, and at least one amino acid substitution is at residue R85, R86, K88, H89, R90, or a combination thereof, and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide.

[0029] In one embodiment of the present disclosure, a polynucleotide encoding the IL-21 polypeptide according to any one of claims 1 to 8 or a functional fragment or variant thereof is provided herein.

[0030] Provided herein in one embodiment of the present disclosure is a polynucleotide encoding a targeted cytokine construct comprising an IL-21 polypeptide or a functional fragment or variant thereof and an antibody or antigen-binding fragment thereof according to the present disclosure, wherein the polynucleotide comprises a coding sequence for the IL-21 polypeptide and a coding sequence for the antibody or antigen-binding fragment thereof.

[0031] Provided herein, in one embodiment of the present disclosure, is a vector comprising a polynucleotide of the present disclosure.

[0032] Provided herein, in one embodiment of the disclosure, is a host cell comprising a polynucleotide or vector of the disclosure.

[0033] Provided herein, in one embodiment of the present disclosure, is a pharmaceutical composition comprising an IL-21 polypeptide according to the present disclosure or a functional fragment or variant thereof and a pharmaceutically acceptable carrier.

[0034] Provided herein, in one embodiment of the present disclosure, is a pharmaceutical composition comprising a targeted cytokine construct according to the present disclosure and a pharmaceutically acceptable carrier.

[0035] Provided herein, in one embodiment of the present disclosure, is a method for selective activation of CD8+ T cells, the method comprising contacting a population of cells comprising CD8+ T cells, CD4+ T cells, and NK cells with a targeted cytokine construct according to the present disclosure. In some embodiments, the selective activation comprises activation of CD8+ T cells with at least about 10-fold or greater potency compared to activation of NK cells or CD4+ T cells in the population of cells. In some embodiments, the selective activation comprises activation of CD8+ T cells with at least about 10-fold to about 100,000-fold greater potency compared to activation of NK cells or CD4+ T cells in the population of cells. In some embodiments, the selective activation of CD8+ T cells results in increased STAT3 phosphorylation in CD8+ T cells compared to STAT3 phosphorylation in NK cells or CD4+ T cells in the population of cells.

[0036] Provided herein in one embodiment of the present disclosure is a method of treating a disease in a subject, the method comprising administering an IL-21 polypeptide or functional fragment or variant thereof according to the present disclosure, a targeted cytokine construct described herein, or a pharmaceutical composition according to the present disclosure. In some embodiments, the method further comprises an additional therapeutic agent. In some embodiments, the disease comprises cancer or a chronic infection. In some embodiments, the disease comprises cancer, including acute lymphoblastic leukemia (ALL) (including non-T cell ALL), acute myeloid leukemia, B cell prolymphocytic leukemia, B cell acute lymphoblastic leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), hairy cell leukemia, Hodgkin's disease, Malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma (NHL), plasma cell proliferative disorders (asymptomatic myeloma (including smoldering multiple myeloma or indolent myeloma), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytoma (including plasma cell dyscrasia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (Crow-Fukase syndrome; Takatsuki disease;and PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoblastic leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma, or Waldenstrom's macroglobulinemia, mantle cell lymphoma (MCL), transformed Follicular lymphoma (TFL), primary mediastinal B-cell lymphoma (PMBCL), multiple myeloma, hairy cell lymphoma / leukemia, lung cancer, small cell lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, squamous cell carcinoma, adenocarcinoma of the lung, squamous cell carcinoma of the lung, cancer of the peritoneum, head and neck cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, thyroid cancer, uterine cancer, gastrointestinal cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer cancer), gastric cancer, colon cancer, breast cancer, endometrial cancer, uterine cancer, cancer of the fallopian tubes, cancer of the cervix, cancer of the vagina, cancer of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, cancer of the renal pelvis, mesothelioma, bladder cancer, liver cancer, hepatocellular carcinoma, cervical cancer, salivary gland cancer, bile duct cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and Ewing's sarcoma (including refractory versions of any of the above cancers, or a combination of one or more of the above cancers);

[0037] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; (a) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 137, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 138, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 139; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 140, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 141, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; (b) a) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 149, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 150, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 151; a VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 152, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 153, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 154; (c) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 155, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 156, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 157; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 158, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 159, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 160; (d) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 161, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 162, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 163; and the VL domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 164. (e) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 167, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 168, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 169; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 170, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 171, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 172;(f) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 173, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; (g) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 179, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 180, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 181. (h) the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 182, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 183, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 184; (h) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 144, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148;(i) the VH domain comprises a CDR-H1 comprising the amino acid sequence of X1X2AIS, where X1 is S, K, G, N, R, D, T, or G, and X2 is Y, L, H, or F (SEQ ID NO: 185), X1X2X3PX4X5X6X7X8X9YX10QKFX11G, 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, and 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, X10 is A or N, and X11 is Q or T (SEQ ID NO: 186), and CDR-H2 comprising the amino acid sequence of 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 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: 188). 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: 190);(j) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 200, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 201; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 152, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 153, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 202; (k) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20 the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (l) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 152, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 153, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 206; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 202; (m) the VH domain comprises a CDR-H1 comprising the amino acid sequence of X1YX2MS (wherein X1 is S, D, E, A, or Q, and X2 is A, G, or T) (SEQ ID NO: 208), 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, and X5 is S or I; X6 is A or G) (SEQ ID NO: 209), and CDR-H3 comprising the amino acid sequence of 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: 210);(n) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; (o) the VH domain comprises CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 176), CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 177), and CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 178). the VL domain comprises CDR-H1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 260, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; the VL domain comprises CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 176), CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 177), and CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 178); or (p) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 252, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 253, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 254; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 255, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 256, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 257;

[0038] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; and (a) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 226, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 227, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 151; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 152, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 153, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 154; (b) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 228, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 227, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 157; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 158, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 159, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 160; (c) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 223, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 227 and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 163; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 164, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 165, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 166; (d) 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: 227, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 169; the VL domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 170. (e) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 230, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 231, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178;(f) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 230, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 232, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 181; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 182, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 183, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 184; (g) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 223, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 224, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 225. (h) the VL domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 140, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 141, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; (h) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 233, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 234, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148. X1PX2X3X4X5 (wherein Xi is I, N, or M, X2 is G, N, H, S, R, I, or A, and X3 is A, N, H, S, or P); X4 is S, K, G, N, R, D, T, or G, and X5 is Y, L, H, or F) (SEQ ID NO: 235); X1 is T, F, or Y, X4 is A, D, or G, and X5 is T, E, K, V, Q, or A (SEQ ID NO: 236), and CDR-H3 is an amino acid sequence of 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: 237);The VL domain comprises CDR-L1 comprising the amino acid sequence of 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: 188); 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: 190); (j) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241; and CDR-H2 comprising the amino acid sequence of SEQ ID NO: 204; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 152, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 153, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 202; (k) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and the VL domain comprises the amino acid sequence of SEQ ID NO: 205. (l) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 152, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 153, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 202;(m) the VH domain comprises a CDR-H1 having the amino acid sequence of GFTFX1X2Y (wherein X1 is S, D, E, Q, S, or A, and X2 is S, D, E, A, or Q) (SEQ ID NO: 244); a CDR-H2 having the amino acid sequence of 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: 245); X is S or A; X is N, H, A, D, L, Q, Y, or R; X is A, N, S, or G; X is A, V, R, E, or S; X is D or S; X is D, N, Q, E, S, T, or L; X is L, F, or M; and X is I, Y, or V) (SEQ ID NO: 246); and the VL domain comprises a CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 176), GASSRAT (SEQ ID NO: 176). (n) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; and the VL domain comprises a CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 176), a CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 177), and a CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 178). (o) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 261, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; the VL domain comprises a CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 176), a CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 177), and a CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 178);or (p) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 223, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 224, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 284; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 285, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 286, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 287;

[0039] In some embodiments, (a) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 109, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 110; (b) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 111, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 112. (c) 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: 113, and 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: 114; (d) 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: 115. (e) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 117, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 118; (f) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 119, (g) 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: 123; and 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: 124;(h) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 129, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 130; (i) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 131; and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 132. (j) 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: 125; 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: 126; (k) 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: 127. (l) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 128; (l) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 133; the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 134; (m) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 135. (n) 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: 107; and 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: 108;(o) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 121; the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 122; or (p) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 258; the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 259;

[0040] In some embodiments, (a) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (b) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (c) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; or (d) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178.

[0041] In some embodiments, (a) 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: 129, and 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: 130; or (b) 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: 127, and 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: 128.

[0042] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising: (i) four or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO:1, wherein the four or more amino acid substitutions are within S80 to T92 of SEQ ID NO:1, and at least four amino acid substitutions are at residues R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by the human IL-21 polypeptide; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β, wherein the antibody or antigen-binding fragment thereof is (i) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (ii) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (iii) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; or (iv) a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178. The present invention provides a fusion protein comprising:

[0043] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising: (i) four or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO:1, wherein the four or more amino acid substitutions are within S80 to T92 of SEQ ID NO:1, and wherein at least four amino acid substitutions are at residues R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide, wherein at least one amino acid substitution is at a residue selected from R5, 18, R9, R11, L13, 114, 116, V17, D18, K72, K73, L74, K75, R76, K77, K117, and a combination thereof; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β, wherein the antibody or antigen-binding fragment thereof is (i) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (ii) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (iii) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; or (iv) a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178. The present invention provides a fusion protein comprising:

[0044] In some embodiments, the present disclosure provides a fusion protein comprising: a) (i) four or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO:1, wherein the four or more amino acid substitutions are within S80 to T92 of SEQ ID NO:1, and at least four amino acid substitutions are at residues R85, R86, K88, H89, R90, or a combination thereof, and (ii) (a) R11D; (b) R11E; (c) I14D, D18A, and K117A; (d) R76E; (e) R5F; (f) R76F ;(g)I8E;(h)R5A;(i)R5E;(j)R5S;(k)R5T;(l)R5N;(m)R5Q;(n)R5V;(o)R5I;(p)R5L;(q)R5Y;(r)R76A;(s)R76N;(t)R76D;(u)R76S;(v )R76T;(w)R76Q;(x)R76V;(y)R76I;(z)R76L;(aa)R76Y;(bb)K77A;(cc)K77E;(dd)K72A;(ee)K72E;(ff)K75A;(gg)K75E;(hh)K73A;(ii )K73E;(jj)R5F and K77A;(kk)R5F and K77E;(ll)R5F and K72A;(mm)R5F and K72E;(nn)R5F and K76A;(oo)R5F and K76E;(pp)K73A and K76F;(qq)K73E and K76F;(rr)R9A;(ss)R9D;(tt)R9E;(uu)R9H;(vv)R9S;(ww)R9T;(xx)R9N;(zz)R9G;(aaa)R9V;(bbb)R9I;(ccc)R9L;(ddd)R9Y;(eee) (hhh) K75E and R76F; (iii) V17I and L74I; (jjj) I16A and L74F; (kkk) I16S, V17I, and L74V; (lll) I16R, V17I, and L74I; (mmm) L13F, I16A, V17A, and L74M; and (nnn) an IL-21 polypeptide comprising at least one amino acid substitution selected from L13R, I16A, V17I, and L74I; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β, wherein the antibody or antigen-binding fragment thereof is (i) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (ii) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (iii) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; or (iv) a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178. The present invention provides a fusion protein comprising:

[0045] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising: (i) four or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the at least four amino acid substitutions are at residues S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, T92, or a combination thereof, and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide, wherein the at least one amino acid substitution is at a residue selected from R5, 18, R9, R11, L13, 114, 116, V17, D18, K72, K73, L74, K75, R76, K77, K117, and a combination thereof; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β, wherein the antibody or antigen-binding fragment thereof is (i) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (ii) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (iii) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; or (iv) a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178. The present invention provides a fusion protein comprising:

[0046] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising: (i) four or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO:1, wherein the at least four amino acid substitutions are at residues S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, T92, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by the human IL-21 polypeptide, wherein the at least one amino acid substitution is at residue R76; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β The present invention provides a fusion protein comprising:

[0047] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising (i) an amino acid sequence selected from SEQ ID NOs: 2, 4-15, and 23-40, and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β, wherein the antibody or antigen-binding fragment thereof is (i) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (ii) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (iii) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; or (iv) a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178. The present invention provides a fusion protein comprising:

[0048] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising: (i) an amino acid sequence selected from SEQ ID NOs: 2, 4-15, and 23-40; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide, wherein the at least one amino acid substitution is at a residue selected from R5, 18, R9, R11, L13, 114, 116, V17, D18, K72, K73, L74, K75, R76, K77, K117, and a combination thereof; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β, wherein the antibody or antigen-binding fragment thereof is (i) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (ii) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (iii) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; or (iv) a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178. The present invention provides a fusion protein comprising:

[0049] In some embodiments, the present disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising: (i) an amino acid sequence selected from SEQ ID NOs: 2, 4-15, and 23-40; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide, wherein the at least one amino acid substitution is at residue R76; and b) an antibody or antigen-binding fragment thereof that specifically binds to CD8αβ or CD8β, wherein the antibody or antigen-binding fragment thereof is (i) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (ii) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (iii) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; or (iv) a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178. The present invention provides a fusion protein comprising:

[0050] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the one or more amino acid substitutions are within S80 to T92 of SEQ ID NO: 1, and at least one amino acid substitution is at residue R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide.

[0051] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the one or more amino acid substitutions are within S80 to T92 of SEQ ID NO: 1, and at least one amino acid substitution is at residue R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide.

[0052] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the one or more amino acid substitutions are within S80 to T92 of SEQ ID NO: 1, and at least one amino acid substitution is at residue R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide.

[0053] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the one or more amino acid substitutions are within S80 to T92 of SEQ ID NO: 1, and at least one amino acid substitution is at residue R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide.

[0054] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide.

[0055] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide.

[0056] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide.

[0057] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide.

[0058] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. and the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16 to 21, 41 to 98, and 374 to 379.

[0059] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 199, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 203, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. and the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16 to 21, 41 to 98, and 374 to 379.

[0060] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. and the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16 to 21, 41 to 98, and 374 to 379.

[0061] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. and the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16 to 21, 41 to 98, and 374 to 379.

[0062] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the one or more amino acid substitutions are within S80 to T92 of SEQ ID NO: 1, and at least one amino acid substitution is at residue R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide.

[0063] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the one or more amino acid substitutions are within S80 to T92 of SEQ ID NO: 1, and at least one amino acid substitution is at residue R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide.

[0064] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a knob modification, and the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the one or more amino acid substitutions are within S80 to T92 of SEQ ID NO: 1, and at least one amino acid substitution is at residue R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide.

[0065] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the one or more amino acid substitutions are within S80 to T92 of SEQ ID NO: 1, and at least one amino acid substitution is at residue R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to a human IL-21 receptor compared to binding by a human IL-21 polypeptide.

[0066] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide.

[0067] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide.

[0068] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a knob modification, and the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide.

[0069] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide.

[0070] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. and the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16 to 21, 41 to 98, and 374 to 379.

[0071] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. wherein the VH chain and the VL chain comprise CDRs that specifically bind to CD8αβ or CD8β; The IL-21 polypeptide provides a fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 16-21, 41-98, and 374-379.

[0072] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a knob modification, and the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. and the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16 to 21, 41 to 98, and 374 to 379.

[0073] In some embodiments, the present disclosure provides a fusion protein comprising: a) a first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; b) a second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and wherein the VH chain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; c) a third polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and the HC constant domain comprises a hole modification, and wherein the VH chains of the second polypeptide and the third polypeptide are the same; and d) a fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. and the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16 to 21, 41 to 98, and 374 to 379.

[0074] In some embodiments, the fusion protein comprises four polypeptides: the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:262, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:263, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:264, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:262; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:266, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:267, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:268, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:266; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:270, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:271, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:272, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:270; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:274, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:275, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:276, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:274; or The first polypeptide chain comprises the amino acid sequence of SEQ ID NO:278, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:279, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:280, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:278.

[0075] In some embodiments, the fusion protein comprises four polypeptides: the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:262, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:263, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:265, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:262; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:266, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:267, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:269, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:266; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:270, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:271, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:273, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:270; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:274, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:275, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:277, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:274; or The first polypeptide chain comprises the amino acid sequence of SEQ ID NO:278, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:279, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:281, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:278.

[0076] In some embodiments, the fusion protein comprises four polypeptides: (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:297, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:298, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:299, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:297; (b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 301, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 302, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 303, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 301; (c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 305, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 306, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 307, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 305; or (d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 309, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 310, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 311, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 309.

[0077] In some embodiments, the fusion protein comprises four polypeptides: (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:297, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:298, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:300, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:297; (b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 301, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 302, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 304, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 301; (c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 305, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 306, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 308, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 305; or (d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 309, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 310, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 312, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 309.

[0078] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0079] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0080] [Figure 1] AC show the amino acid sequences of the following polypeptides: mature IL-21 (SEQ ID NO: 1) (A), IL-21R (SEQ ID NO: 381) (B), and common gamma chain (SEQ ID NO: 382) (C). [Figure 2] 1 shows the amino acid sequence of the mature wild-type IL-21 polypeptide (SEQ ID NO: 1) and modifications to generate the charge variants. "X" indicates the amino acid position that was substituted in the sequence of the wild-type IL-21 polypeptide to generate the charge variant. [Figure 3A] Figure 1 shows ion exchange chromatography traces of fusion proteins of anti-CD8 antibody and either wild-type IL-21 or IL-21 charge variants after Protein A purification. Fusion proteins of xhCD8 and either wild-type IL-21 or exemplary IL-21 charge variants v1-v6 are shown, as measured by HPLC. In all cases, the left peak(s) are unwanted products, primarily homodimers, while the right peak is the desired heterodimeric fusion protein; the percentage of the total for each indicated region is shown. [Figure 3B]Figure 1 shows ion exchange chromatography traces of fusion proteins of anti-CD8 antibody and either wild-type IL-21 or IL-21 charge variants after Protein A purification. Fusion proteins of xhCD8.1 and exemplary IL-21 charge variant v2, or variants v7-v31, as measured by FPLC, are shown. In all cases, the peak(s) on the left are unwanted products, primarily homodimers, while the peaks on the right are the desired heterodimeric fusion proteins. The percentage of the total for each indicated region is shown. [Figure 3C] Figure 1 shows ion exchange chromatography traces of fusion proteins of anti-CD8 antibody and either wild-type IL-21 or IL-21 charge variants after Protein A purification. Fusion proteins of xhCD8.1 and exemplary IL-21 charge variant v2, or variants v7-v31, as measured by FPLC, are shown. In all cases, the peak(s) on the left are unwanted products, primarily homodimers, while the peaks on the right are the desired heterodimeric fusion proteins. The percentage of the total for each indicated region is shown. [Figure 3D] Figure 1 shows ion exchange chromatography traces of fusion proteins of anti-CD8 antibody and either wild-type IL-21 or IL-21 charge variants after Protein A purification. Fusion proteins of xhCD8.1 and exemplary IL-21 charge variant v2, or variants v7-v31, as measured by FPLC, are shown. In all cases, the peak(s) on the left are unwanted products, primarily homodimers, while the peaks on the right are the desired heterodimeric fusion proteins. The percentage of the total for each indicated region is shown. [Figure 3E]Figure 1 shows ion exchange chromatography traces of fusion proteins of anti-CD8 antibody and either wild-type IL-21 or IL-21 charge variants after Protein A purification. Fusion proteins of xhCD8.1 and exemplary IL-21 charge variant v2, or variants v7-v31, as measured by FPLC, are shown. In all cases, the peak(s) on the left are unwanted products, primarily homodimers, while the peaks on the right are the desired heterodimeric fusion proteins. The percentage of the total for each indicated region is shown. [Figure 3F] Figure 1 shows ion exchange chromatography traces of fusion proteins of anti-CD8 antibody and either wild-type IL-21 or IL-21 charge variants after Protein A purification. Fusion proteins of xhCD8.1 and wild-type IL-21 are shown, as measured by FPLC. In all cases, the left peak(s) are unwanted products, primarily homodimers, while the right peak is the desired heterodimeric fusion protein; the percentage of the total for each indicated region is shown. [Figure 4A] 1 shows mouse pharmacokinetic data for the fusion proteins. Serum concentrations are shown for 1 mg / kg of control antibody (xCtrl) with either wild-type IL-21 or the exemplary charge variant IL-21 polypeptide IL-21 v2 dosed intravenously. [Figure 4B] 1 shows mouse pharmacokinetic data for the fusion protein. Serum concentrations are shown for 1 mg / kg of control antibody (xCtrl) with either wild-type IL-21 or the exemplary charge variant IL-21 polypeptide IL-21v2 dosed subcutaneously. [Figure 4C] 1 shows mouse pharmacokinetic data for the fusion protein. In a separate experiment, serum concentrations are additionally shown for 1 mg / kg subcutaneously dosed wild-type IL-21, or exemplary charge variant IL-21 polypeptides, IL-21v2 or IL-21v31. [Figure 4D]1 shows mouse pharmacokinetic data for the fusion protein. In a separate experiment, serum concentrations are additionally shown for 1 mg / kg of wild-type IL-21 or exemplary charge variant IL-21 polypeptides, IL-21v2 or IL-21v31, dosed intravenously. [Figure 5] Figures A-B show activation of STAT3 by wild-type IL-21 in CD8+ T cells, CD4+ T cells, NK cells, and B cells from human peripheral blood mononuclear cells (PBMCs) (A) or whole blood (B). STAT3 activation was measured by flow cytometry, and all cell types were potently activated by wild-type IL-21. [Figure 6] FIG. 1 is a schematic diagram showing the general mechanism of how targeted fusions of mutant IL-21 polypeptides with CD8 antigen binding molecules and non-targeted fusions with mutant IL-21 polypeptides work to stimulate cells that do or do not express the CD8 antigen. [Figure 7] 1 shows activation of STAT3 in human CD8+ T cells by fusion proteins of anti-CD8 antibodies and either wild-type IL-21, exemplary IL-21 polypeptide charge variants IL-21v1, IL-21v2, or IL-21v6. [Figure 8A] Figure 1 shows activation of STAT3 in human PBMCs by fusion proteins of the anti-human CD8 antibody xhCD8 and various attenuated versions of IL-21v2. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8 and the exemplary IL-21 polypeptide IL-21v2.1 containing attenuating mutations all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximum STAT3 activation. [Figure 8B]Figure 1 shows activation of STAT3 in human PBMCs by fusion proteins of the anti-human CD8 antibody xhCD8 and various attenuated versions of IL-21v2. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8 and the exemplary IL-21 polypeptide IL-21v2.2 containing attenuating mutations all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximum STAT3 activation. [Figure 8C] Figure 1 shows activation of STAT3 in human PBMCs by fusion proteins of the anti-human CD8 antibody xhCD8 with various attenuated versions of IL-21v2. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8 with the exemplary IL-21 polypeptide IL-21v2.3 containing attenuating mutations all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximum STAT3 activation. [Figure 8D] Figure 1 shows activation of STAT3 in human PBMCs by fusion proteins of the anti-human CD8 antibody xhCD8 with various attenuated versions of IL-21v2. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8 with the exemplary IL-21 polypeptide IL-21v2.4 containing attenuating mutations all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximum STAT3 activation. [Figure 8E]Figure 1 shows activation of STAT3 in human PBMCs by fusion proteins of the anti-human CD8 antibody xhCD8 and various attenuated versions of IL-21v2. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8 and the exemplary IL-21 polypeptide IL-21v2.5 containing attenuating mutations all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximum STAT3 activation. [Figure 8F] Figure 1 shows activation of STAT3 in human PBMCs by fusion proteins of the anti-human CD8 antibody xhCD8 with various attenuated versions of IL-21v2. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8 with the exemplary IL-21 polypeptide IL-21v2.6 containing attenuating mutations all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximum STAT3 activation. [Figure 9A] Figure 1 shows activation of STAT3 in human PBMCs by a fusion protein of the anti-human CD8 antibody xhCD8.1 with an additional attenuated version of IL-21v2 or a non-targeting Fc fusion to IL-21v2 as a control. STAT3 activation in CD8+ T cells is shown. [Figure 9B] Figure 1 shows activation of STAT3 in human PBMCs by a fusion protein of the anti-human CD8 antibody xhCD8.1 with an additional attenuated version of IL-21v2 or a non-targeting Fc fusion to IL-21v2 as a control. STAT3 activation in CD4+ T cells is shown. [Figure 9C] Figure 1 shows activation of STAT3 in human PBMCs by a fusion protein of the anti-human CD8 antibody xhCD8.1 with an additional attenuated version of IL-21v2 or a non-targeting Fc fusion to IL-21v2 as a control. STAT3 activation in CD8+ T cells is shown. [Figure 9D]Figure 1 shows activation of STAT3 in human PBMCs by a fusion protein of the anti-human CD8 antibody xhCD8.1 with an additional attenuated version of IL-21v2 or a non-targeting Fc fusion to IL-21v2 as a control. STAT3 activation in CD4+ T cells is shown. [Figure 9E] Figure 1 shows activation of STAT3 in human PBMCs by a fusion protein of the anti-human CD8 antibody xhCD8.1 with an additional attenuated version of IL-21v2 or a non-targeting Fc fusion to IL-21v2 as a control. STAT3 activation in CD8+ T cells is shown. [Figure 9F] Figure 1 shows activation of STAT3 in human PBMCs by a fusion protein of the anti-human CD8 antibody xhCD8.1 with an additional attenuated version of IL-21v2 or a non-targeting Fc fusion to IL-21v2 as a control. STAT3 activation in CD4+ T cells is shown. [Figure 9G] Figure 1 shows activation of STAT3 in human PBMCs by a fusion protein of the anti-human CD8 antibody xhCD8.1 with an additional attenuated version of IL-21v2 or a non-targeting Fc fusion to IL-21v2 as a control. STAT3 activation in CD8+ T cells is shown. [Figure 9H] Figure 1 shows activation of STAT3 in human PBMCs by a fusion protein of the anti-human CD8 antibody xhCD8.1 with an additional attenuated version of IL-21v2 or a non-targeting Fc fusion to IL-21v2 as a control. STAT3 activation in CD4+ T cells is shown. [Figure 10A]Figure 1 shows activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, B cells, and myeloid cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), IL-21v31.48 (D), and IL-21v31.51 (E), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximum STAT3 activation. For xhCD8.1-hIL21v31.6, data from two separate experiments are shown (B). Non-targeted, non-attenuated Fc-hIL21v2 is shown in F, and activation was observed in all cell types shown. [Figure 10B] Figure 1 shows activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, B cells, and myeloid cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), IL-21v31.48 (D), and IL-21v31.51 (E), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximum STAT3 activation. For xhCD8.1-hIL21v31.6, data from two separate experiments are shown (B). Non-targeted, non-attenuated Fc-hIL21v2 is shown in F, and activation was observed in all cell types shown. [Figure 10C]Figure 1 shows activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, B cells, and myeloid cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), IL-21v31.48 (D), and IL-21v31.51 (E), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximum STAT3 activation. For xhCD8.1-hIL21v31.6, data from two separate experiments are shown (B). Non-targeted, non-attenuated Fc-hIL21v2 is shown in F, and activation was observed in all cell types shown. [Figure 10D] Figure 1 shows activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, B cells, and myeloid cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), IL-21v31.48 (D), and IL-21v31.51 (E), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximum STAT3 activation. For xhCD8.1-hIL21v31.6, data from two separate experiments are shown (B). Non-targeted, non-attenuated Fc-hIL21v2 is shown in F, and activation was observed in all cell types shown. [Figure 10E]Figure 1 shows activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, B cells, and myeloid cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), IL-21v31.48 (D), and IL-21v31.51 (E), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximum STAT3 activation. For xhCD8.1-hIL21v31.6, data from two separate experiments are shown (B). Non-targeted, non-attenuated Fc-hIL21v2 is shown in F, and activation was observed in all cell types shown. [Figure 10F] Figure 1 shows activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, B cells, and myeloid cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), IL-21v31.48 (D), and IL-21v31.51 (E), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximum STAT3 activation. For xhCD8.1-hIL21v31.6, data from two separate experiments are shown (B). Non-targeted, non-attenuated Fc-hIL21v2 is shown in F, and activation was observed in all cell types shown. [Figure 11A]Figure 1 shows STAT3 activation in mouse splenocytes. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Activation profiles are shown for recombinant wild-type mouse IL-21 (A); a fusion protein of the non-targeting control antibody xCtrl and the mouse IL-21 charge variant mIL-21v1 (B); and a fusion protein of the anti-mouse CD8 antibody xmCD8 and the attenuated mouse IL-21 charge variant mIL21v1.1 (C). As measured by EC50 and maximum STAT3 activation, all cell types are activated for both recombinant wild-type mouse IL-21 and xCtrl-mIL21v1, while for xmCD8-mIL21v1.1, there is preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells. [Figure 11B] Figure 1 shows STAT3 activation in mouse splenocytes. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Activation profiles are shown for recombinant wild-type mouse IL-21 (A); a fusion protein of the non-targeting control antibody xCtrl and the mouse IL-21 charge variant mIL-21v1 (B); and a fusion protein of the anti-mouse CD8 antibody xmCD8 and the attenuated mouse IL-21 charge variant mIL21v1.1 (C). As measured by EC50 and maximum STAT3 activation, all cell types are activated for both recombinant wild-type mouse IL-21 and xCtrl-mIL21v1, while for xmCD8-mIL21v1.1, there is preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells. [Figure 11C]Figure 1 shows STAT3 activation in mouse splenocytes. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Activation profiles are shown for recombinant wild-type mouse IL-21 (A); a fusion protein of the non-targeting control antibody xCtrl and the mouse IL-21 charge variant mIL-21v1 (B); and a fusion protein of the anti-mouse CD8 antibody xmCD8 and the attenuated mouse IL-21 charge variant mIL21v1.1 (C). As measured by EC50 and maximum STAT3 activation, all cell types are activated for both recombinant wild-type mouse IL-21 and xCtrl-mIL21v1, while for xmCD8-mIL21v1.1, there is preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells. [Figure 12] (a) Tumor growth curves in the MC38 syngeneic tumor model treated with a single dose of either PBS; 0.3 or 1 mg / kg (mpk) of the non-targeting control antibody xCtrl and the fusion protein xCtrl-mIL21v1 of the non-attenuated murine IL-21 charge variant IL-21v1; or 0.1, 0.3, or 1 mpk of the anti-mouse CD8 antibody xmCD8 and the fusion protein xmCD8-mIL21v1.1 of the attenuated murine IL-21 charge variant IL21v1.1. Each subpanel shows the tumor growth curve from each individual animal; the fraction indicates a complete response (CR), and the arrow indicates the time of treatment. [Figure 13] Figures A-D show the effect of FTY720 on the antitumor efficacy of xmCD8-mIL21v1.1 in the MC38 tumor model. Figure A shows the dosing scheme for FTY720 and xmCD8-mIL21v1.1, and tumor growth curves for 0.1 mpk (B), 0.3 mpk (C), or 1 mpk (D) of xmCD8-mIL21v1.1. Plotted values ​​are mean tumor volumes, and error bars indicate standard error of the mean. Each group contains 8 animals. [Figure 14A]Figure 1 shows activation of STAT3 in human whole blood from additional donors by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), IL-21v31.48 (D), and IL-21v31.51 (E), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50. For xhCD8.1-hIL21v31.4 and xhCD8.1-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (A and C). Non-targeting, non-attenuated Fc-hIL21v31 is shown in F, where activation was observed in all cell types shown. [Figure 14B] Figure 1 shows activation of STAT3 in human whole blood from additional donors by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), IL-21v31.48 (D), and IL-21v31.51 (E), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50. For xhCD8.1-hIL21v31.4 and xhCD8.1-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (A and C). Non-targeting, non-attenuated Fc-hIL21v31 is shown in F, where activation was observed in all cell types shown. [Figure 14C]Figure 1 shows activation of STAT3 in human whole blood from additional donors by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), IL-21v31.48 (D), and IL-21v31.51 (E), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50. For xhCD8.1-hIL21v31.4 and xhCD8.1-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (A and C). Non-targeting, non-attenuated Fc-hIL21v31 is shown in F, where activation was observed in all cell types shown. [Figure 14D] Figure 1 shows activation of STAT3 in human whole blood from additional donors by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), IL-21v31.48 (D), and IL-21v31.51 (E), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50. For xhCD8.1-hIL21v31.4 and xhCD8.1-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (A and C). Non-targeting, non-attenuated Fc-hIL21v31 is shown in F, where activation was observed in all cell types shown. [Figure 14E]Figure 1 shows activation of STAT3 in human whole blood from additional donors by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), IL-21v31.48 (D), and IL-21v31.51 (E), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50. For xhCD8.1-hIL21v31.4 and xhCD8.1-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (A and C). Non-targeting, non-attenuated Fc-hIL21v31 is shown in F, where activation was observed in all cell types shown. [Figure 14F] Figure 1 shows activation of STAT3 in human whole blood from additional donors by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), IL-21v31.48 (D), and IL-21v31.51 (E), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50. For xhCD8.1-hIL21v31.4 and xhCD8.1-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (A and C). Non-targeting, non-attenuated Fc-hIL21v31 is shown in F, where activation was observed in all cell types shown. [Figure 15A]Figure 1 shows activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v11 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), and IL-21v31.51 (D), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50. For xhCD8v11-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (C). [Figure 15B] Figure 1 shows activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v11 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), and IL-21v31.51 (D), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50. For xhCD8v11-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (C). [Figure 15C]Figure 1 shows activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v11 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), and IL-21v31.51 (D), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50. For xhCD8v11-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (C). [Figure 15D] Figure 1 shows activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v11 with exemplary IL-21 polypeptides containing attenuating mutations, IL-21v31.4 (A), IL-21v31.6 (B), IL-21v31.23 (C), and IL-21v31.51 (D), all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50. For xhCD8v11-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (C). [Figure 16A]Figure 1 shows activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various versions of IL-21v2 charge variants that also contain additional mutations. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v.1 with exemplary IL-21 polypeptides IL-21v2.63 (A), IL-21v2.64 (B), and IL-21v2.65 (C), which contain attenuating mutations, all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50. [Figure 16B] Figure 1 shows activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various versions of IL-21v2 charge variants that also contain additional mutations. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v.1 with exemplary IL-21 polypeptides IL-21v2.63 (A), IL-21v2.64 (B), and IL-21v2.65 (C), which contain attenuating mutations, all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50. [Figure 16C] Figure 1 shows activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various versions of IL-21v2 charge variants that also contain additional mutations. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v.1 with exemplary IL-21 polypeptides IL-21v2.63 (A), IL-21v2.64 (B), and IL-21v2.65 (C), which contain attenuating mutations, all show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50. [Figure 17](A-C) show ion-exchange chromatography traces of fusion proteins of anti-CD8 antibody and IL-21R-attenuated IL-21 charge variants after Protein A purification. Traces are shown for xhCD8v11-hIL21v31.23 (A), xhCD8.1-hIL21v31.23 (B), and xhCD8.1-hIL21v31.4 (C) as measured by FPLC. In all cases, the peak(s) to the left of the major peak are unwanted products, primarily homodimers, while the major peak in the center is the desired heterodimeric fusion protein; the percentage of the total desired peak is shown. [Figure 18] Pharmacokinetic data for fusion proteins in wild-type C57BL6 mice are shown. Serum concentrations are shown for intravenously administered fusion proteins of anti-human CD8 antibodies xhCD8.1 or xhCD8v11 with either hIL-21v31.4, hIL-21v31.23, or hIL-21v0.4. Anti-human CD8 antibodies xhCD8.1 and xhCD8v11 are not cross-reactive with mouse CD8. Exposure is shown for xhCD8.1-hIL21v31.4, xhCD8v11-hIL21v31.23, xhCD8.1-hIL21v31.23, and xhCD8.1-hIL21v0.4. [Figure 19A] Figure 1 shows characterization data for recombinant cytokine variants based on wild-type IL-21 or IL-21v31 charge variants. SDS-PAGE of material purified by IMAC followed by preparative SEC is shown. Analytical SEC of material purified by IMAC followed by preparative SEC is shown. Retention times and peak widths are also reported to assess column interactions. In summary, recombinant cytokine variants based on IL-21v31 charge variants have shorter retention times and narrower peak widths compared to variants based on wild-type IL-21, suggesting fewer column interactions and more desirable biophysical properties for IL-21v31-based constructs. [Figure 19B]Figure 1 shows characterization data for recombinant cytokine variants based on wild-type IL-21 or IL-21v31 charge variants. SDS-PAGE of material purified by IMAC followed by preparative SEC is shown. Analytical SEC of material purified by IMAC followed by preparative SEC is shown. Retention times and peak widths are also reported to assess column interactions. In summary, recombinant cytokine variants based on IL-21v31 charge variants have shorter retention times and narrower peak widths compared to variants based on wild-type IL-21, suggesting fewer column interactions and more desirable biophysical properties for IL-21v31-based constructs. [Figure 20] 1 shows the exposure of recombinant (non-fusion) wild-type human IL-21 and IL-21v31 charge variants in mice. IL-21v31 shows increased in vivo exposure compared to wild-type IL-21. [Figure 21A] 1A and 1B provide structural schematics showing the interactions between wild-type IL-21 (SEQ ID NO: 1) and IL-21R (SEQ ID NO: 381), respectively. Residues S80-T92 of SEQ ID NO: 1 form a largely unstructured flexible loop (circled). Residues P78 and P79 (Pro-Pro), and C93 and P94 (Cys-Pro) surrounding the flexible loop are shown. [Figure 21B] 1A-1D provide structural schematics showing the interactions between wild-type IL-21 (SEQ ID NO: 1) and IL-21R (SEQ ID NO: 381), respectively. Residues S80-T92 of SEQ ID NO: 1 form a largely unstructured flexible loop (circled). The charge of wild-type IL-21 is shown, indicating that the flexible loop is highly charged. [Figure 22]This figure shows the activity of IL-21 fusion molecules in cultures of alloreactive T cells and activated dendritic cells isolated from two different human PBMC donors. The molecules tested included two CD8+ T cell-targeting fusions, xhCD8.1-hIL21v31.4 and xhCD8.1-hIL21v31.23, as well as the non-targeting, non-attenuated Fc-hIL21v31. IL-2 release in the cultures was measured to determine the level of alloreactive T cell activation. In summary, Fc-hIL21v31 suppressed alloreactive T cell activation, whereas the CD8+ T cell-targeting fusion did not. [Figure 23A] Figure 1 shows in vitro activation of STAT3 in cynomolgus monkey whole blood by fusion proteins of anti-human CD8 antibodies xhCD8.1 or xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, and B cells. The CD8+ T cell-targeting fusion proteins xhCD8.1-IL-21v31.4 (A), xhCD8.1-IL-21v31.23 (B), and xhCD8v11-IL-21v31.23 (C) all show preferential STAT3 activation of CD8+ T cells over B and CD4+ T cells, as measured by EC50. Non-targeting, non-attenuated Fc-hIL21v31 is shown in D, and activation was observed in all cell types shown. [Figure 23B] Figure 1 shows in vitro activation of STAT3 in cynomolgus monkey whole blood by fusion proteins of anti-human CD8 antibodies xhCD8.1 or xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, and B cells. The CD8+ T cell-targeting fusion proteins xhCD8.1-IL-21v31.4 (A), xhCD8.1-IL-21v31.23 (B), and xhCD8v11-IL-21v31.23 (C) all show preferential STAT3 activation of CD8+ T cells over B and CD4+ T cells, as measured by EC50. Non-targeting, non-attenuated Fc-hIL21v31 is shown in D, and activation was observed in all cell types shown. [Figure 23C] Figure 1 shows in vitro activation of STAT3 in cynomolgus monkey whole blood by fusion proteins of anti-human CD8 antibodies xhCD8.1 or xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, and B cells. The CD8+ T cell-targeting fusion proteins xhCD8.1-IL-21v31.4 (A), xhCD8.1-IL-21v31.23 (B), and xhCD8v11-IL-21v31.23 (C) all show preferential STAT3 activation of CD8+ T cells over B and CD4+ T cells, as measured by EC50. Non-targeting, non-attenuated Fc-hIL21v31 is shown in D, and activation was observed in all cell types shown. [Figure 23D] Figure 1 shows in vitro activation of STAT3 in cynomolgus monkey whole blood by fusion proteins of anti-human CD8 antibodies xhCD8.1 or xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, and B cells. The CD8+ T cell-targeting fusion proteins xhCD8.1-IL-21v31.4 (A), xhCD8.1-IL-21v31.23 (B), and xhCD8v11-IL-21v31.23 (C) all show preferential STAT3 activation of CD8+ T cells over B and CD4+ T cells, as measured by EC50. Non-targeting, non-attenuated Fc-hIL21v31 is shown in D, and activation was observed in all cell types shown. [Figure 24] Figure 1 shows in vivo activation of STAT3 in cynomolgus monkeys by xhCD8.1-IL-21v31.4. STAT3 activation levels in CD8+ T cells, CD4+ T cells, NK cells, and B cells were assessed 20 minutes after dosing. Results show that xhCD8.1-IL-21v31.4 preferentially activates STAT3 in CD8+ T cells over CD4+ T cells, NK cells, and B cells, as measured by STAT3 MFI. DETAILED DESCRIPTION OF THE INVENTION

[0081] In some embodiments, the present disclosure provides an IL-21 polypeptide, or a functional fragment or variant thereof, that comprises improved properties relative to wild-type IL-21 (SEQ ID NO: 1). Examples of improved properties include, but are not limited to, (i) improved systemic exposure (e.g., as measured by an increased area under the curve (AUC) upon administration of the IL-21 polypeptide, or a functional fragment or variant thereof, to a subject compared to administration of the wild-type IL-21 protein); (ii) attenuated binding to the IL-21 receptor; (iii) reduced cytotoxicity, or any combination thereof.

[0082] As described herein, the present disclosure provides IL-21 polypeptides or functional fragments or variants thereof having a reduced charge (i.e., isoelectric point) and reduced binding affinity to the human IL-21 receptor compared to the human IL-21 polypeptide (SEQ ID NO: 1). In particular, the present disclosure provides IL-21 polypeptides having one or more amino acid substitutions in a positively charged region located within residues S80-T92 of human IL-21. As shown in Figure 21B, residues S80-T92 of human IL-21 form a flexible loop that does not directly interact with the human IL-21 receptor. Without wishing to be bound by theory, residues P78, P79, C93, and P94 of human IL-21 are believed to provide structural rigidity around the loop. The present disclosure provides IL-21 polypeptides having one or more amino acid substitutions in the flexible loop that have a reduced isoelectric point and improved bioavailability and biological activity. As described herein, IL-21 polypeptides of the present disclosure having a reduced isoelectric point relative to human IL-21 polypeptide demonstrate increased circulating half-life and reduced multireactivity (e.g., with positively charged proteins abundant in the circulation). As shown in Figures 4A-4D, fusion proteins having an IL-21 polypeptide having a reduced isoelectric point relative to human IL-21 have improved half-life. The present disclosure further provides fusion proteins of an IL-21 polypeptide to an anti-CD8 antibody that selectively activate CD8+ T cells over CD4+ T cells, NK cells, and B cells, including in vivo in non-human primates (see Figure 24). Without being bound by theory, fusion proteins of an IL-21 polypeptide and an antigen-binding domain that specifically binds to CD8ab or CD8b selectively activate CD8+ T cells over NK cells expressing CD8aa. IL-21 polypeptides with reduced isoelectric points also have lower binding affinities to proteins abundant in the circulation, such as heparin and hemoglobin (see Table 5).Furthermore, without being bound by theory, IL-21 polypeptides of the present disclosure having glycine and / or serine substitutions in the soft loops may have reduced immunogenicity compared to unmodified human IL-21 after administration to a subject.

[0083] The present disclosure also provides IL-21 polypeptides with reduced binding to the human IL-21 receptor. As shown herein, the reduced binding of the IL-21 polypeptides of the present disclosure to their receptor reduces the IL-21-mediated suppressive effect on alloreactive T cells and dendritic cells (see Figure 22). Without wishing to be bound by theory, the reduced binding of the IL-21 polypeptides of the present disclosure to their receptor is expected to avoid the reduced effect of binding on B cells and NK cells. The therapeutic efficacy of fusion proteins comprising the IL-21 polypeptides of the present disclosure and an anti-CD8 targeting antibody is also demonstrated herein. Without being bound by theory, linking an IL-21 polypeptide with reduced binding to an anti-CD8 targeting antibody provides selective restoration of the attenuated IL-21 polypeptide. Specifically, Figure 12 shows that such fusion proteins provide anti-tumor efficacy in vivo. This efficacy is at least in part due to peripheral activation of CD8+ T cells, as blocking lymphocyte trafficking reduces the therapeutic response (see Figures 13A-13D). Without being bound by theory, T cells in the tumor microenvironment may be dysfunctional and express inhibitory molecules, thus peripheral activation of CD8+ T cells by fusion proteins provides an anti-tumor immune response. Thus, the present disclosure provides novel IL-21 polypeptides and fusion proteins comprising the IL-21 polypeptides with improved biological properties, as well as methods of using such polypeptides to treat disease in subjects.

[0084] Various embodiments of the present disclosure provide methods and compositions that involve the use of cytokines (e.g., interleukin-21 (IL-21) or IL-21 polypeptides, or functional fragments or variants thereof, comprising one or more mutated amino acid residues relative to the wild-type IL-21 polypeptide sequence) that are induced or targeted to activate specific cell subsets (e.g., CD8+ T cells), herein referred to as targeted cytokine constructs. Without wishing to be bound by any particular theory, it is contemplated that the ability to selectively activate certain cell types may allow for a more robust response and the ability to limit the activation of other cell types that would be counterproductive to a successful immune response. In some cases, the methods and compositions disclosed herein allow for the targeted delivery of IL-21 polypeptides to desired cell types and the administration of IL-21 at relatively low concentrations to induce the selective activation of CD8+ T cells. In some cases, use of the targeted cytokine constructs disclosed herein provides more selective cell (e.g., CD8+ T cell) activation compared to activation of other cell types, e.g., natural killer (NK) cells, CD4+ T cells, or B cells.

[0085] definition Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. The meaning and scope of terms should be clear, but in case of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or exogenous definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. In this application, the use of "or" means "and / or" unless otherwise stated. Furthermore, the use of the term "including," as well as other variants such as "includes" and "included," is not limiting.

[0086] Generally, the nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0087] As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "chimeric transmembrane receptor polypeptide" includes a plurality of chimeric transmembrane receptor polypeptides.

[0088] The term "about" or "approximately" means within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, according to practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in this application and claims, unless otherwise stated, the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0089] As used herein, "area under the curve" or "AUC" is a pharmacokinetic parameter that refers to the area under a plot of plasma concentration of a drug (e.g., an IL-21 polypeptide or fusion protein described herein) versus time after dosing. In some embodiments, AUC measures a patient's exposure to a drug and is dependent 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 rule, the trapezoidal rule, and Simpson's rule.

[0090] A "cytokine" is a form of immunomodulatory polypeptide that can mediate crosstalk between progenitor / primary cells and target / effector cells. It can function as a soluble form or be associated with the cell surface, binding to a "cytokine receptor" on target immune cells and activating signal transduction. As used herein, a "cytokine receptor" is a polypeptide on the cell surface that activates intracellular signal transduction upon binding of the cytokine on the extracellular cell surface. Cytokines can include, but are not limited to, chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. 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 multiple cell types. Cytokines are pleiotropic because receptors are expressed on multiple immune cell subsets, and a single cytokine can activate signal transduction pathways in multiple cells. However, depending on the cell type, signal transduction events for cytokines can result in different downstream cellular events, such as activation, proliferation, survival, apoptosis, effector function, and secretion of other immunomodulatory proteins. The given cytokine, in some embodiments, is a wild-type cytokine polypeptide, a fragment thereof, or a variant thereof, e.g., a mutated cytokine polypeptide (also referred to herein as a mutant cytokine, e.g., a mutant IL-21 or an IL-21 mutant).

[0091] The term "antigen," as used herein, refers to a molecule or fragment thereof that can be bound by a selective binding agent. As an example, an antigen can be a ligand, such as a receptor, that can be bound by a selective binding agent. As another example, an antigen can be an antigenic molecule, such as an immunological protein (e.g., an antibody), that can be bound by a selective binding agent. An antigen can also refer to a molecule or fragment thereof that can be used in an animal to generate antibodies capable of binding to that antigen.

[0092] The term "antibody," as used herein, refers to any antigen-binding molecule containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. The term "antibody" includes immunoglobulin molecules containing four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of an antibody (or antigen-binding portion thereof) can be identical to human germline sequences or can be modified naturally or artificially. An amino acid consensus sequence can be defined based on a parallel analysis of two or more CDRs.

[0093] The term "intact antibody" refers to an antibody comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. In one embodiment, the antibody is an intact antibody. In one embodiment, the intact antibody is an intact human IgG1, IgG2, or IgG4 isotype. In certain embodiments, the antibody, or antigen-binding fragment thereof, is a human IgG1, IgG2, or IgG4 isotype.

[0094] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, or "antibody fragment," and the like, include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using, for example, any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, involving the manipulation and expression of DNA encoding antibody variable and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids, etc.

[0095] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs), e.g., CDR3 peptides), or constrained FR3-CDR3-FR4 peptides).

[0096] The term "variable region" or "variable domain" of an antibody, or a fragment thereof, as used herein refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of the complementarity-determining regions (CDRs; i.e., CDR-1, CDR-2, and CDR-3) and framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. According to the method used in this disclosure, the amino acid positions assigned to the CDRs and FRs may be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)). The amino acid numbering of an antibody or antigen-binding fragment also follows that of Kabat.

[0097] The term "complementarity-determining region" or "CDR," as used herein, refers to a complementarity-determining region within an antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions, designated CDR1, CDR2, and CDR3 for each variable region. The term "CDR set," as used herein, refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently in different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia et al., J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) have used the Kabat CDRs to It has been found that certain subportions within the CDRs adopt nearly identical peptide backbone conformations despite the high degree of diversity at the amino acid sequence level. These subportions are designated L1, L2, and L3 or H1, H2, and H3 ("L" and "H" refer to the light chain and heavy chain regions, respectively). These regions may be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)).Still other CDR boundary definitions may not strictly adhere to one of the above systems, but will nevertheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. While the methods used herein may utilize CDRs defined according to any of these systems, preferred embodiments use Kabat- or Chothia-defined CDRs.

[0098] The term "framework region" (hereinafter, FR), as used herein, refers to those variable domain residues other than the CDR residues. Each variable domain typically has four FRs, identified as FR1, FR2, FR3, and FR4. Common structural features among the variable regions of antibodies, or functional fragments thereof, are well known in the art. DNA sequences encoding particular antibodies can usually be found according to well-known methods, for example, those described in Kabat, et al., 1987, Sequence of Proteins of Immunological Interest, USDapartment of Health and Human Services, Bethesda, MD (incorporated herein by reference). A general method for cloning functional variable regions from antibodies can also be found in Chaudhary, VK, et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066 (incorporated herein by reference).

[0099] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one "amino acid modification," as defined herein. A variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acid substitutions in the native-sequence Fc region or the Fc region of a parent polypeptide. In one embodiment, a variant Fc region herein may have a sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, or at least about 99% identical to a native-sequence Fc region. According to another embodiment, a variant Fc region herein may have a sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, or at least about 99% identical to the Fc region of a parent polypeptide.

[0100] The term "Fc receptor" or "FcR," as used herein, generally refers to a receptor capable of binding to the Fc region of an antibody, or any derivative, variant, or fragment thereof. In certain embodiments, FcR is one that binds IgG antibodies (gamma receptors, FcgammaR), including receptors of the FcgammaRI (CD64), FcgammaRII (CD32), and FcgammaRIII (CD16) subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcgammaRII receptors include FcgammaRIIA (an "activating receptor") and FcgammaRIIB (an "inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The term "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus.

[0101] As used herein, "effector function" refers to a biochemical event resulting from the interaction of an antibody Fc region with an Fc receptor or ligand, which varies depending on the 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, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcRs (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on target cells and subsequently cause lysis of the target cells. ADCC correlates with binding to FcγRIIIa; increased binding to FcγRIIIa results in increased ADCC activity. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent Nos. 5,500,362 or 5,821,337, may be performed. "ADCP" or antibody-dependent cell-mediated phagocytosis, as used herein, refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibody on target cells and subsequently cause phagocytosis of the target cells.

[0102] "Fc null" and "Fc null variant" are used interchangeably herein to refer to a modified Fc with reduced or abolished effector function. Such Fc null or Fc null variants have reduced or abolished FcγR and / or complement receptors. Preferably, such Fc null or Fc null variants have abolished effector function. Exemplary methods for modification include, but are not limited to, chemical alterations, amino acid residue substitutions, insertions, and deletions. Exemplary amino acid positions on the Fc molecule at which one or more modifications may be introduced to reduce the effector function of the resulting variant are (numbered according to the EU numbering scheme): 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 with one or more of the following substitutions: i) IgG1: N297A, N297Q, D265A / N297A, D265A / N297Q, C220S / C226S / C229S / P238S, S267E / L328F, C226S / C229S / E233P / L234V / L235A, L234F / L235E / P331S, L234A / L235A, L234A / L235A / G237A, L234A / L235A / G237A / K322A, L234A / L235A / G237A / A330S / A331S, L234A / L235A / P329G, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G23 6del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A3 27G and E233P / L234V / L235A / G236del, L234A / L235A / G237 deletion; ii) IgG2:A330S / A331S, V234A / G237A, V 234A / 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.

[0103] As used herein, "epitope" refers to a determinant capable of specific binding to the variable region of an antibody molecule, known as the paratope. An epitope is a grouping of molecules, e.g., amino acids or sugar side chains, that typically possess specific structural and charge characteristics. A single antigen may have multiple epitopes. An epitope may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding, e.g., amino acid residues effectively blocked by the antigen-binding peptide (i.e., amino acid residues within the footprint of the antigen-binding peptide). Epitopes may be conformational or linear. An epitope typically contains at least three, more commonly 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," demonstrating the ability of one antibody to block the binding of another antibody to a target antigen.

[0104] As used herein, "linker" refers to a molecule that connects two polypeptide chains. The linker may be a polypeptide linker or a synthetic chemical linker (for example, as disclosed in Protein Engineering, 9(3), 299-305, 1996). The length and sequence of the polypeptide linker are not particularly limited and can be selected by those skilled in the art according to the purpose. The polypeptide linker contains one or more amino acids. Preferably, the polypeptide linker is a peptide having a length of at least 5 amino acids, preferably 5 to 100, more preferably 10 to 50 amino acids. In one embodiment, the peptide linker is G, S, GS, SG, SGG, GGS, or GSG (G = glycine and S = serine). In another embodiment, the peptide linker is (GGGS) x G n (SEQ ID NO: 294) or (GGGGS) x G n (SEQ ID NO: 295), (GGGGGS) x G n(SEQ ID NO: 296), S(GGGS)xGn (SEQ ID NO: 289), S(GGGGS)xGn (SEQ ID NO: 290), or S(GGGGGS)xGn (SEQ ID NO: 291) (x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 and n=0, 1, 2, or 3). In some embodiments, the linker is (GGGGS) x G n (x=2, 3, or 4 and n=0) (SEQ ID NO: 372); in some embodiments, the linker is (GGGGS) x G n (x=3 and n=0) (SEQ ID NO: 373). In some embodiments, the linker comprises the sequence SGGGGSGGGGSGGGGS (SEQ ID NO: 292), or SGGGGSGGGGSGGGG (SEQ ID NO: 293). Synthetic chemical linkers include cross-linking agents conventionally used to cross-link peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(succinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(succinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0105] The term "nucleotide," as used herein, generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term "nucleotide," as used herein, refers in some instances to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled by well-known techniques. Labeling may also be performed with quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides may include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif.; FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; Boehringer Fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2′-dATP available from Mannheim, Indianapolis, Ind.; and Molecular Chromosomal labeled nucleotides available from Probes, Eugene, Oreg., include BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP. Nucleotides can also be labeled or marked by chemical modification. The chemically modified single nucleotide can be a biotin-dNTP.Some non-limiting examples of biotinylated dNTPs can include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0106] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, in either single-stranded, double-stranded, or multi-stranded form. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or a fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. A polynucleotide can contain one or more analogs (e.g., modified backbones, sugars, or nucleobases). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acids, xenonucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent-based analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queusine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci (locuses) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides, including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.

[0107] The term "gene," as used herein, refers to a nucleic acid (e.g., DNA, e.g., genomic DNA and cDNA) and its corresponding nucleotide sequence involved in encoding an RNA transcript. When used herein with respect to genomic DNA, the term includes intervening non-coding and regulatory regions, which may include the 5' and 3' ends. In some uses, the term encompasses the transcribed sequence, including the 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region contains an "open reading frame" that encodes a polypeptide. In some uses of the term, a "gene" includes only the coding sequence (e.g., an "open reading frame" or "coding region") necessary to encode a polypeptide. In some cases, a gene does not encode a polypeptide, e.g., a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. In some cases, the term "gene" includes not only the transcribed sequence, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene, in some cases, refers to an "endogenous gene" or a native gene in its natural location in the genome of an organism. A gene, in some cases, refers to an "exogenous gene" or a non-native gene. A non-native gene, in some cases, refers to a gene that is not normally found in the host organism but is introduced into the host organism by gene transfer. A non-native gene, in some cases, refers to a gene that is not in its natural location in the genome of an organism. A non-native gene also, in some cases, refers to a naturally occurring nucleic acid or polypeptide sequence that contains mutations, insertions, and / or deletions (e.g., a non-native sequence).

[0108] The term "regulating," as used herein, with respect to expression or activity refers to altering the level of expression or activity. Regulation can occur at the transcriptional and / or translational level.

[0109] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by a peptide bond(s). The terms do not imply a particular length of the polymer, nor are they intended to indicate or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or naturally occurring. The terms apply to naturally occurring amino acid polymers and amino acid polymers containing at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The terms include amino acid chains of any length, including full-length proteins and proteins with or without secondary and / or tertiary structure (e.g., domains). The terms also encompass amino acid polymers modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation, such as conjugation with a labeling component. The terms "amino acid" and "amino acids," as used herein, generally refer to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids can include natural amino acids and unnatural amino acids, which are chemically modified to include groups or chemical moieties that do not naturally occur in amino acids. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D- and L-amino acids.

[0110] The terms "derivative," "variant," and "fragment," as used herein with respect to a polypeptide, refer to a polypeptide that is related to a wild-type polypeptide, for example, by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Derivatives, variants, and fragments of a polypeptide can include one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, as compared to the wild-type polypeptide.

[0111] The term "residue," as used herein, refers to a position in a protein and its associated amino acid identity. For example, Leu 234 (also referred to as Leu234 or L234) is the residue at position 234 in human antibody IgG1.

[0112] The term "wild-type," as used herein, refers to an amino acid or nucleotide sequence found in nature, including allelic variations. A wild-type protein has an amino acid or nucleotide sequence that has not been intentionally modified.

[0113] The term "substitution" or "mutation" refers to a change to the polypeptide backbone in which an amino acid naturally occurring in the wild-type sequence of the polypeptide is substituted with another amino acid that does not naturally occur at the same position in the polypeptide. In some cases, the mutation(s) are introduced to modify the affinity of the polypeptide for its receptor, thereby altering its activity so that it differs from the affinity and activity of the wild-type cognate polypeptide. Mutations may also improve the biophysical properties of the polypeptide. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is contemplated that methods other than genetic engineering, such as modifying the side chain group of an amino acid by chemical modification, may also be useful.

[0114] The term "affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). Affinity is usually measured by the dissociation constant (K), which is the ratio of the dissociation and association rate constants (koff and kon, respectively). D) Thus, equivalent affinities may involve different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) technology (e.g., BIAcore), biolayer interferometry (BLI) technology (e.g., Octet), and other conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In some instances, binding affinity is determined by Scatchard analysis, which involves generating a Scatchard plot, which is a plot of the ratio of the concentration of bound ligand to the concentration of unbound ligand against the concentration of bound ligand.

[0115] The terms "binding" or "specific binding," as used herein, may refer to the ability of a polypeptide or antigen-binding domain to selectively interact with a polypeptide or receptor for a target antigen, respectively, and this specific interaction may be distinguished from non-target or unwanted or non-specific interactions. Examples of specific binding may include, but are not limited to, IL-21 cytokine binding to its specific receptor (e.g., IL-21R and common gamma chain) and antigen-binding domain binding to a specific antigen (e.g., CD8 or PD-1).

[0116] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, e.g., a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Also encompassed are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0117] The terms "treatment" and "treating," as used herein, refer to an approach for obtaining beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. For example, treatment may include administering a system or cell population disclosed herein. Therapeutic benefit refers to any therapeutically relevant improvement or effect of one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, a composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom may not yet be manifest.

[0118] The terms "effective amount," or "therapeutically effective amount," or "effective dose," or "effective dosage" refer to the amount of a composition, e.g., a composition of immune cells, e.g., lymphocytes (e.g., T lymphocytes and / or NK cells), that can be combined with a targeted cytokine construct of the present disclosure, that is sufficient to result in a desired activity following administration to a subject in need thereof. Within the context of the present disclosure, the term "therapeutically effective" refers to the amount of a composition sufficient to delay the onset of, halt the progression of, or alleviate or relieve at least one symptom of a disorder treated by the methods of the present disclosure.

[0119] As used herein, the term "amino acid sequence identity (%)" in relation to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific sequence, after aligning the sequences and introducing gaps as necessary to achieve maximum sequence identity, without considering any conservative substitutions as part of the sequence identity.Alignment for determining amino acid sequence identity can be achieved by various methods within the skill of the art, for example, using publicly available computer software, such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment across the entire length of the sequences being compared.Alignment for determining amino acid sequence identity can be achieved, for example, using the publicly available sequence comparison computer program ALIGN-2. The source code for the ALIGN-2 sequence comparison computer program is available, along with user documentation, at the U.S. Copyright Office, Washington, DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program can be compiled for use on UNIX operating systems, such as Digital UNIX v4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0120] For all amino acid positions discussed in this disclosure, in the context of antibodies or antigen-binding fragments thereof, numbering is according to the EU index. "EU index" or "EU index as in Kabat et al." or "EU numbering scheme" refers to EU antibody numbering (see Edelman et al., 1969; Kabat et al., 1991).

[0121] Interleukin-21 Polypeptide Interleukin-21 (IL-21) is a cytokine expressed by T cells, B cells, NK cells, and myeloid cells that can regulate the activity of both innate and adaptive immune cells and improve T cell survival and effector function. Several Phase I and Phase II clinical trials include 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.

[0122] IL-21 has a four-helix bundle structure and exists as a monomer. In humans, two isoforms of IL-21 are known, each derived from a precursor molecule. The first IL-21 isoform contains 162 amino acids (aa), of which the first 29 constitute a signal peptide; the second IL-21 isoform contains 153 aa, of which the first 29 constitute a signal peptide, like the first isoform.

[0123] IL-21 binds to the heterodimeric IL-21 receptor complex, which contains the IL-21 receptor (IL-21R) and the common gamma chain (γc). The IL-21 receptor complex is expressed on the surface of T, B, and NK cells. The IL-21 receptor complex is structurally similar to the IL-2 receptor complex in that each of these cytokine receptor complexes contains γc.

[0124] When IL-21 binds to the IL-21 receptor complex, the JAK / STAT signaling pathway is activated, activating target genes. Although IL-21-induced signaling can be therapeutically desirable, careful consideration of the timing and location of signaling is required, given the broad expression profile of IL-21 and the fact that IL-21 has the ability to enhance CD8+ T cell responses and suppress antigen presentation and T cell priming.

[0125] The present disclosure provides, in some embodiments, an IL-21 polypeptide or functional fragment or variant thereof that comprises at least one amino acid substitution relative to the wild-type IL-21 amino acid sequence provided herein as SEQ ID NO: 1. Unless otherwise stated, the terms "wild-type IL-21," "wild-type IL-21 polypeptide," "human IL-21," and "human IL-21 polypeptide" are used interchangeably and may refer to the amino acid sequence of SEQ ID NO: 1. Such IL-21 polypeptides that comprise at least one amino acid substitution relative to SEQ ID NO: 1 are also referred to herein as IL-21 variants. In exemplary aspects, an IL-21 polypeptide or functional fragment or variant thereof described herein comprises at least one and no more than X amino acid substitutions, where X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof described herein comprises at least 35 amino acid substitutions compared to SEQ ID NO: 1. In exemplary embodiments, an IL-21 polypeptide or functional fragment or variant thereof described herein comprises an amino acid sequence that differs by 10 amino acids, 15 amino acids, 20 amino acids, or 25 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1). In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof described herein comprises an amino acid sequence that differs by 16 or fewer amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1). In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof described herein comprises an amino acid sequence that differs by 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1). In exemplary embodiments, an IL-21 polypeptide or functional fragment or variant thereof described herein comprises an amino acid sequence that differs by 7 or fewer amino acids or 5 or fewer amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1).In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof described herein comprises an amino acid sequence that differs by 10, 11, 12, 13, 14, 15, or 16 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1). In exemplary embodiments, an IL-21 polypeptide or functional fragment or variant thereof described herein comprises an amino acid sequence that differs by 3, 4, 5, or 6 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1). In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof described herein comprises an amino acid sequence that differs by 5 to 16 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1). In exemplary embodiments, an IL-21 polypeptide or functional fragment or variant thereof described herein comprises an amino acid sequence that differs by 3 to 6 amino acids or 1 to 5 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1). In exemplary embodiments, an IL-21 polypeptide or functional fragment or variant thereof described herein comprises an amino acid sequence that differs by 1 or 2 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1).

[0126] Altered isoelectric point or charge distribution In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least one mutation that alters its isoelectric point with or without affecting its binding to IL-21R. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least one mutation that alters its isoelectric point without affecting binding to IL-21R. An altered isoelectric point of a protein can change the surface charge distribution, thereby affecting protein yield, nonspecific binding, and blood or circulatory half-life for a given protein. In some embodiments, a mutation in an IL-21 polypeptide, or functional fragment or variant thereof, reduces the theoretical isoelectric point by a given unit as measured by the publicly available database ProtParam (SwissProt) (incorporated herein by reference).

[0127] In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, has an isoelectric point difference of about 0.6 to about 5.0 units compared to the isoelectric point of SEQ ID NO: 1, which has an isoelectric point of about 9.42. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, has an isoelectric point of about 6.0 to about 9.0. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises a theoretical isoelectric point of about 6 to about 9. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises a theoretical isoelectric point of at least about 6. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises a theoretical isoelectric point of at most about 9.In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof has a nucleotide sequence of about 9 to about 8.9, about 9 to about 8.8, about 9 to about 8.7, about 9 to about 8.5, about 9 to about 8.3, about 9 to about 8.1, about 9 to about 7.9, about 9 to about 7.7, about 9 to about 7.5, about 9 to about 7.3, about 9 to about 7, about 9 to about 6.5, about 9 to about 6.0, about 8.9 to about 8.8, about 8.9 to about 8.7, about 8.9 to about 8.5, about 8.9 to about 8.3, about 8.9 to about 8.1, about 8.9 to about 7.9, about 8.9 to about 7.7, about 8.9 to about 7.5, about 8.9 to about 8. 9 to about 7.3, 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 The theoretical isoelectric points include 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 7.3, about 7.5 to about 7, or about 7.3 to about 7, about 7.3 to about 6.5, about 7.3 to about 6.0, about 7.0 to about 6.5, about 7.0 to about 6.0, or about 6.5 to about 6.0.In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises a theoretical isoelectric point of about 9, about 8.9, about 8.8, about 8.79, about 8.7, about 8.5, about 8.39, about 8.3, about 8.1, about 7.9, about 7.7, about 7.5, about 7.3, about 7, about 6.5, or about 6.0. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises a theoretical isoelectric point of less than 9.0. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises a theoretical isoelectric point of less than 8.9, less than 8.8, less than 8.7, less than 8.5, less than 8.3, less than 8.1, less than 7.9, less than 7.7, less than 7.5, less than 7.3, less than 7.2, less than 7.0, less than 6.5, or less than 6.2.

[0128] In some examples, the altered isoelectric point may increase the protein yield of an IL-21 polypeptide or functional fragment or variant thereof during purification compared to wild-type IL-21. In some embodiments, the increased protein yield is at least 5% higher than wild-type IL-21. In some embodiments, the increased protein yield is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% higher than wild-type IL-21. In some embodiments, the increased protein yield is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% higher than wild-type IL-21. In some embodiments, the increased protein yield is between 5% and 10%, between 5% and 15%, between 5% and 20%, between 5% and 25%, between 5% and 50%, between 5% and 75%, between 5% and 100%, between 5% and 125%, between 5% and 150%, between 5% and 175%, between 5% and 200%, between 5% and 225%, between 5% and 250%, between 5% and 275%, between 5% and 300%, between 10% and 15%, between 10% and 20%, between 10% and 25%. 25%, 10%~50%, 10%~75%, 10%~100%, 10%~125%, 10%~150%, 10%~175%, 10%~200%, 10%~225%, 10%~250%, 10%~275%, 10%~300%, 15%~20%, 15%~25%, 15%~50%, 15%~75%, 15%~100%, 15%~125%, 15%~150% , 15%~175%, 15%~200%, 15%~225%, 15%~250%, 15%~275%, 15%~300%, 20%~25%, 20%~50%, 20%~75%, 20%~100%, 20%~125%, 20%~150%, 20%~175%, 20%~200%, 20%~225%, 20%~250%, 20%~275%, 20%~300% , 25%~50%, 25%~75%, 25%~100%, 25%~125%, 25%~150%, 25%~175%, 25%~200%, 25%~225%, 25%~250%, 25%~275%, 25%~300%, 50%~75%, 50%~100%, 50%~125%, 50%~150%, 50%~175%, 50%~200%, 75%~100%,75%~125%, 75%~150%, 75%~175%, 75%~200%, 75%~225%, 75%~250%, 75%~275%, 75%~300%, 100%~125%, 100%~150%, 100%~175%, 100%~200%, 100%~225%, 100%~250%, 100%~275%, 100%~300%, 125%~150%, 125%~175%, 125%~200%, 125%~225%, 125%~25 0%, 125%-275%, 125%-300%, 150%-175%, 150%-200%, 175%-200%, 175%-225%, 175%-250%, 175%-275%, 175%-300%, 200%-225%, 200%-250%, 200%-275%, 200%-300%, 225%-250%, 225%-275%, 225%-300%, 250%-275%, 250%-300%, or 275%-300%. In some embodiments, the increased protein yield is about 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300%.

[0129] In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprises at least one amino acid substitution of one or more positively charged amino acid residues relative to a region of a wild-type IL-21 polypeptide comprising one or more positively charged amino acid residues. In some cases, the region of wild-type IL-21 comprises between 2 and 20 positively charged amino acid residues.In some embodiments, the region in wild-type IL-21 is 2 to 3, 2 to 4, 2 to 5, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 11, 3 to 12, 3 to 13, 3 to 14, 3 to 15, 3 to 16, 3 to 17, 3 to 18, 3 to 19, 3 to 20, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 4 to 11, 4 to 12, 4 to 13, 4 to 14, 4 to 15, 4 to 16, 4 to 17, 4 to 18, 4 to 19, 3 to 20, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 4 to 11, 4 to 12, 4 to 13, 4 to 1 4, 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- and 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, this region of wild-type IL-21 comprises 12 positively charged amino acid residues.In some embodiments, the region of wild-type IL-21 comprises between 2 and 20 amino acid residues, at least one of which is positively charged.In some embodiments, the region of wild-type IL-21 has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 positively charged amino acid residues. , 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, 12-20, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 14-15, 14-16, 14-17, 3-18, 14-19, 14-20, 15-16, 15-17, 15-18, 15-19, 15-20, 16-17, 16-18, 16-19, 16-20, 17-18, 17-19, 17-20, 18-19, 18-20, or 19-20 amino acid residues.In some embodiments, this region of wild-type IL-21 comprises 12 amino acid residues, of which 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues are positively charged. In some embodiments, this region of wild-type IL-21 comprises 12 amino acid residues, of which 2, 3, 4, 5, or 6 amino acid residues are positively charged.

[0130] In some embodiments, amino acids within this region of wild-type IL-21 do not bind the human IL-21 receptor. In some embodiments, this region of wild-type IL-21 comprises amino acid residues S80 through T92 of SEQ ID NO: 1. In some embodiments, this region of wild-type IL-21 comprises positively charged amino acid residues R85, R86, K88, H89, and R90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least one amino acid substitution within this region of wild-type IL-21. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least one amino acid substitution within S80 through T92. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least four amino acid substitutions within S80 through T92. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least four amino acid substitutions within S80 through T92, with the exception that G84 is not substituted. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least one amino acid substitution at R85, R86, K88, H89, or R90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises an amino acid substitution at one or more of S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, and T92. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises an amino acid substitution at S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, and T92.

[0131] In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprising an altered isoelectric point compared to wild-type IL-21 (SEQ ID NO: 1) comprises a mutation at a position selected from the group consisting of K56, S80, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92 of SEQ ID NO: 1. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprising an altered isoelectric point compared to wild-type IL-21 (SEQ ID NO: 1) comprises a mutation at a position selected from the group consisting of K56, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92 of SEQ ID NO: 1. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprising an altered isoelectric point compared to wild-type IL-21 (SEQ ID NO: 1) comprises a mutation at a position selected from the group consisting of S80, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92 of SEQ ID NO: 1. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprising an altered isoelectric point compared to wild-type IL-21 (SEQ ID NO: 1) comprises a mutation at a position selected from the group consisting of S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, and T92 of SEQ ID NO: 1.

[0132] In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises a mutation at position K56 of SEQ ID NO: 1. In some examples, the mutation comprises K56G, K56S, K56E, K56D, or K56A. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises a mutation at position S80. In some embodiments, the mutation comprises S80G, S80A, S80D, or S80E. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises a mutation at position T81 of SEQ ID NO: 1. In some examples, the mutation comprises T81G, T81S, T81E, T81D, or T81A. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises a mutation at position N82 of SEQ ID NO: 1. In some examples, the mutation comprises N82G, N82S, N82E, N82D, or N82A. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises a mutation at position A83 of SEQ ID NO: 1. In some examples, the mutation comprises A83G, A83S, A83E, or A83D. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises a mutation at position G84 of SEQ ID NO: 1. In some examples, the mutation comprises G84A, G84S, G84E, or G84D. In some embodiments, G84 is not mutated. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises a mutation at position R85 ​​of SEQ ID NO: 1. In some examples, the mutation comprises R85G, R85S, R85E, R85D, or R85A. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises a mutation at position R86 of SEQ ID NO: 1. In some examples, the mutation comprises R86G, R86S, R86E, R86D, or R86A. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises a mutation at position Q87 of SEQ ID NO: 1. In some examples, the mutation comprises Q87G, Q87S, Q87E, Q87D, or Q87A.In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises a mutation at position K88 of SEQ ID NO: 1. In some examples, the mutation comprises K88G, K88S, K88E, K88D, or K88A. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises a mutation at position H89 of SEQ ID NO: 1. In some examples, the mutation comprises H89G, H89S, H89E, H89D, or H89A. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises a mutation at position R90 of SEQ ID NO: 1. In some examples, the mutation comprises R90G, R90S, R90E, R90D, or R90A. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises a mutation at position L91 of SEQ ID NO: 1. In some examples, the mutation comprises L91G, L91S, L91E, L91D, or L91A. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises a mutation at position T92 of SEQ ID NO: 1. In some examples, the mutation comprises T92G, T92S, T92E, T92D, or T92A.

[0133] In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof having an altered isoelectric point compared to IL-21 of SEQ ID NO: 1 comprises an amino acid sequence that is at least 75% identical to the sequence of SEQ ID NO: 1, and comprises a mutation at least one position selected from the group consisting of positions K56 and R90 of SEQ ID NO: 1. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof having an altered isoelectric point compared to IL-21 of SEQ ID NO: 1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 1, and comprises a mutation at least one position selected from the group consisting of positions K56 and R90 of SEQ ID NO: 1. The mutation at position K56 is, in some instances, K56G, K56S, K56E, K56D, or K56A; and the mutation at position R90 is, in some instances, R90G, R90S, R90E, R90D, or R90A. The altered isoelectric point is, in some instances, a reduced isoelectric point compared to that of IL-21 of SEQ ID NO: 1 (which is about 9.42), for example, a reduction of at least about 0.6 units.

[0134] In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and is selected from the group consisting of 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; G8 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; or G85, G86, E87, G88, G89, and G90 (position numbers correspond to SEQ ID NO: 1).

[0135] In some embodiments, an IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, G88, and A90. In some embodiments, an IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, G88, and A90. In some embodiments, an IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, G88, and E90. In some embodiments, an IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids A56, A75, G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids A56, A75, G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids A56, E75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids A56, E75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids A56, A75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids A56, A75, G85, G86, G88, and A90.In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids E56, A75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids E56, A75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, S83, E85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, S83, E85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, S87, G88, G89, and E90.In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, G87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, G87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, G87, G88, G89, and G90. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, G87, G88, G89, and G90. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, E87, G88, G89, and G90. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, E87, G88, G89, and G90.In some embodiments, a charge-variant IL-21 polypeptide or functional fragment or variant thereof is provided, comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.

[0136] In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, S83, G85, G86, E87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, S83, G85, G86, E87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, E83, G85, G86, E87, G88, G89, G90, S91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, E83, G85, G86, E87, G88, G89, G90, S91 and G92.In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G82, G83, S85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G82, G83, S85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G82, G83, E85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G82, G83, E85, G86, G87, G88, S89, G90, G91 and S92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, G83, E85, G86, G87, G88, G89, G90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, G83, E85, G86, G87, G88, G89, G90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, E90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, E90, G91 and G92.In some embodiments, an IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, an IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, an IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G82, G83, S85, G86, G87, G88, S89, E90, G91, and S92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G82, G83, S85, G86, G87, G88, S89, E90, G91 and S92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G82, G83, G85, G86, G87, G88, G89, E90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G82, G83, G85, G86, G87, G88, G89, E90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, G87, G88, G89, E90 and G91. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G85, G86, G87, G88, G89, E90 and G91. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G82, G83, G85, G86, G87, G88, G89, E90, and G91.In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G82, G83, G85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G82, G83, G85, E86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G82, G83, G85, E86, G87, G88, G89, E90, and G91. In some embodiments, an IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G82, G83, E85, G86, G87, G88, G89, E90, and G91. In some embodiments, an IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G82, G83, E85, G86, G87, G88, G89, E90, and G91. In some embodiments, an IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 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 comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 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 comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, E83, G85, G86, G87, G88, G89, E90, G91 and G92.In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, G82, E83, G85, G86, G87, G88, G89, E90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, E82, G83, G85, G86, G87, G88, G89, E90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 and comprises amino acids G80, G81, E82, G83, G85, G86, G87, G88, G89, E90, G91 and G92. In some embodiments, a charge-variant IL-21 polypeptide or functional fragment or variant thereof is provided that comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 23-40. In some embodiments, there is provided a charge-variant IL-21 polypeptide or functional fragment or variant thereof comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 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.

[0137] In some embodiments, the IL-21 polypeptide comprises about 75% sequence identity to about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. In some embodiments, the IL-21 polypeptide comprises at least about 75% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. In some embodiments, the IL-21 polypeptide comprises up to about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. In some embodiments, the IL-21 polypeptide has about 75% to about 80% sequence identity, about 75% to about 85% sequence identity, about 75% to about 90% sequence identity, about 75% to about 95% sequence identity, about 75% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. Sequence identity: about 96% to about 97% sequence identity, about 75% to about 98% sequence identity, about 75% to about 99% sequence identity, about 75% to about 100% sequence identity, about 80% to about 85% sequence identity, about 80% to about 90% sequence identity, about 80% to about 95 ... Sequence identity to about 96% sequence identity, about 80% sequence identity to about 97% sequence identity, about 80% sequence identity to about 98% sequence identity, about 80% sequence identity to about 99% sequence identity, about 80% sequence identity to about 100% sequence identity, about 85% sequence identity to about 90% sequence identity, about 85% sequence identity to about 95% sequence identity, about 85% sequence identity to about 96% sequence identity, about 85 % sequence identity to about 97% sequence identity, about 85% sequence identity to about 98% sequence identity, about 85% sequence identity to about 99% sequence identity, about 85% sequence identity to about 100% sequence identity, about 90% sequence identity to about 95% sequence identity, about 90% sequence identity to about 96% sequence identity, about 90% sequence identity to about 97% sequence identity, about 90% sequence identity to about 98% sequence identity,Approximately 90% to approximately 99% sequence identity, approximately 90% to approximately 100% sequence identity, approximately 95% to approximately 96% sequence identity, approximately 95% to approximately 97% sequence identity, approximately 95% to approximately 98% sequence identity, approximately 95% to approximately 99% sequence identity, approximately 95% to approximately 100% sequence identity, approximately 96% to approximately 97% sequence identity, approximately 96% to approximately 98% sequence identity about 96% to about 99% sequence identity, about 96% to about 100% sequence identity, about 97% to about 98% sequence identity, about 97% to about 99% sequence identity, about 97% to about 100% sequence identity, about 98% to about 99% sequence identity, about 98% to about 100% sequence identity, or about 99% to about 100% sequence identity. In some embodiments, the IL-21 polypeptide comprises about 75% sequence identity, about 80% sequence identity, about 85% sequence identity, about 90% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99% sequence identity, or about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.

[0138] In some embodiments, the IL-21 polypeptide comprises from about 75% sequence identity to about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 23-40. In some embodiments, the IL-21 polypeptide comprises at least about 75% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 23-40. In some embodiments, the IL-21 polypeptide comprises up to about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 23-40. In some embodiments, the IL-21 polypeptide has about 75% to about 80% sequence identity, about 75% to about 85% sequence identity, about 75% to about 90% sequence identity, about 75% to about 95% sequence identity, about 75% to about 96% sequence identity, about 75% to about 97% sequence identity, about 75% to about 98% sequence identity, about 75% to about 99% sequence identity, about 75% to about 90% sequence identity, about 75% to about 91% sequence identity, about 75% to about 92% sequence identity, about 75% to about 93% sequence identity, about 75% to about 94% sequence identity, about 75% to about 95% sequence identity, about 75% to about 96% sequence identity, about 75% to about 97% sequence identity, about 75% to about 98% sequence identity, about 75% to about 9 ... 8% sequence identity, about 75% to about 99% sequence identity, about 75% to about 100% sequence identity, about 80% to about 85% sequence identity, about 80% to about 90% sequence identity, about 80% to about 95% sequence identity, about 80% to about 96% sequence identity, about 80% to about 97% sequence identity, about 80% to about 98% sequence identity, about 80% sequence identity Sequence identity: about 99% to about 80% to about 100%; sequence identity: about 85% to about 90%; sequence identity: about 85% to about 95%; sequence identity: about 85% to about 96%; sequence identity: about 85% to about 97%; sequence identity: about 85% to about 98%; sequence identity: about 85% to about 99%; sequence identity: about 85% to about 100%; sequence identity: about 90% to about 95% sequence identity, about 90% to about 96% sequence identity, about 90% to about 97% sequence identity, about 90% to about 98% sequence identity, about 90% to about 99% sequence identity, about 90% to about 100% sequence identity, about 95% to about 96% sequence identity, about 95% to about 97% sequence identity, about 95% to about 98% sequence identity,The sequence identity includes about 95% to about 99% sequence identity, about 95% to about 100% sequence identity, about 96% to about 97% sequence identity, about 96% to about 98% sequence identity, about 96% to about 99% sequence identity, about 96% to about 100% sequence identity, about 97% to about 98% sequence identity, about 97% to about 99% sequence identity, about 97% to about 100% sequence identity, about 98% to about 99% sequence identity, about 98% to about 100% sequence identity, or about 99% to about 100% sequence identity. In some embodiments, the IL-21 polypeptide comprises about 75% sequence identity, about 80% sequence identity, about 85% sequence identity, about 90% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99% sequence identity, or about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 23-40.

[0139] In some embodiments, the IL-21 polypeptide comprises about 75% sequence identity to about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. In some embodiments, the IL-21 polypeptide comprises at least about 75% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. In some embodiments, the IL-21 polypeptide comprises up to about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40. In some embodiments, the IL-21 polypeptide has about 75% to about 80% sequence identity, about 75% to about 85% sequence identity, about 75% to about 90% sequence identity, about 75% to about 95% sequence identity, about 75% to about 96% sequence identity, about 75% to about 97% sequence identity, about 75% to about 98% sequence identity, about 75% to about 99% sequence identity, about 80% to about 92% sequence identity, about 85% to about 94% sequence identity, about 85% to about 96% sequence identity, about 85% to about 98% sequence identity, about 85% to about 99% sequence identity, about 99% to about 100% sequence identity, about 99% to about 100% sequence identity, about 99% to about 100% sequence identity, about 99% to about 100% sequence identity, about 99% to about 100% sequence identity, about 99% to about 100% sequence identity, about 10 ... 5% sequence identity to about 98% sequence identity, about 75% sequence identity to about 99% sequence identity, about 75% sequence identity to about 100% sequence identity, about 80% sequence identity to about 85% sequence identity, about 80% sequence identity to about 90% sequence identity, about 80% sequence identity to about 95% sequence identity, about 80% sequence identity to about 96% sequence identity, about 80% sequence identity to about 97% sequence identity, about 80% sequence identity to about 98% sequence identity, about 80% sequence identity to about 99% sequence identity, about 80% sequence identity to about 100% sequence identity, about 85% sequence identity to about 90% sequence identity,Approximately 85% to approximately 95% sequence identity, approximately 85% to approximately 96% sequence identity, approximately 85% to approximately 97% sequence identity, approximately 85% to approximately 98% sequence identity, approximately 85% to approximately 99% sequence identity, approximately 85% to approximately 100% sequence identity, approximately 90% to approximately 95% sequence identity Identity, about 90% sequence identity to about 96% sequence identity, about 90% sequence identity to about 97% sequence identity, about 90% sequence identity to about 98% sequence identity, about 90% sequence identity to about 99% sequence identity, about 90% sequence identity to about 100% sequence identity, about 95% sequence identity to about 96% sequence identity, about 95% sequence identity to about 97% sequence identity, about 95% to about 98% sequence identity, about 95% to about 99% sequence identity, about 95% to about 100% sequence identity, about 96% to about 97% sequence identity, about 96% to about 98% sequence identity, about 96% to about 99% sequence identity, about 96% to about Including 100% sequence identity, about 97% to about 98% sequence identity, about 97% to about 99% sequence identity, about 97% to about 100% sequence identity, about 98% to about 99% sequence identity, about 98% to about 100% sequence identity, or about 99% to about 100% sequence identity. In some embodiments, the IL-21 polypeptide comprises about 75% sequence identity, about 80% sequence identity, about 85% sequence identity, about 90% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99% sequence identity, or about 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40.

[0140] In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 80% sequence identity to SEQ ID NO: 4 and comprises amino acids G85, G86, G88, and A90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 and comprises amino acids G85, G86, G88, and A90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 80% sequence identity to SEQ ID NO: 2 and comprises amino acids G85, G86, G88, and E90. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 and comprises amino acids G85, G86, G88, and E90. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprises at least 80% sequence identity to SEQ ID NO: 5 and comprises amino acids A56, A75, G85, G86, G88, and E90. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 and comprises amino acids A56, A75, G85, G86, G88, and E90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 80% sequence identity to SEQ ID NO: 6 and comprises amino acids A56, E75, G85, G86, G88, and A90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6 and comprises amino acids A56, E75, G85, G86, G88, and A90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 80% sequence identity to SEQ ID NO: 7 and comprises amino acids A56, A75, G85, G86, G88, and A90.In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7 and comprises amino acids A56, A75, G85, G86, G88, and A90. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprises at least 80% sequence identity to SEQ ID NO: 8 and comprises amino acids E56, A75, G85, G86, G88, and A90. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8 and comprises amino acids E56, A75, G85, G86, G88, and A90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 80% sequence identity to SEQ ID NO: 9 and comprises amino acids G80, G81, G82, S83, E85, G86, S87, G88, G89, and S90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 9 and comprises amino acids G80, G81, G82, S83, E85, G86, S87, G88, G89, and S90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 80% sequence identity to SEQ ID NO: 10 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, S90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, S90.In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 80% sequence identity to SEQ ID NO: 10 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, E90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, E90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 80% sequence identity to SEQ ID NO: 11 and comprises amino acids G85, G86, S87, G88, G89, and E90. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 11 and comprises amino acids G85, G86, S87, G88, G89, and E90. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprises at least 80% sequence identity to SEQ ID NO: 12 and comprises amino acids G85, G86, S87, G88, G89, and S90. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12 and comprises amino acids G85, G86, S87, G88, G89, and S90. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprises at least 80% sequence identity to SEQ ID NO: 13 and comprises amino acids G85, G86, G87, G88, G89, and E90. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13 and comprises amino acids G85, G86, G87, G88, G89, and E90.In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 80% sequence identity to SEQ ID NO: 14 and comprises amino acids G85, G86, G87, G88, G89, and G90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 and comprises amino acids G85, G86, G87, G88, G89, and G90. In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises at least 80% sequence identity to SEQ ID NO: 15 and comprises amino acids G85, G86, E87, G88, G89, and G90. In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 15 and comprises amino acids G85, G86, E87, G88, G89, and G90.

[0141] In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 23 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 23 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 24 and comprises amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 24 and comprises amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 25 and comprises amino acids G80, G81, G82, S83, G85, G86, E87, G88, G89, G90, S91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25 and comprises amino acids G80, G81, G82, S83, G85, G86, E87, G88, G89, G90, S91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 26 and comprises amino acids G80, G81, G82, E83, G85, G86, E87, G88, G89, G90, S91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 26 and comprises amino acids G80, G81, G82, E83, G85, G86, E87, G88, G89, G90, S91 and G92.In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 27 and comprises amino acids G82, G83, S85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 27 and comprises amino acids G82, G83, S85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 28 and comprises amino acids G82, G83, E85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 28 and comprises amino acids G82, G83, E85, G86, G87, G88, S89, G90, G91 and S92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 29 and comprises amino acids G80, G81, G82, G83, E85, G86, G87, G88, G89, G90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 29 and comprises amino acids G80, G81, G82, G83, E85, G86, G87, G88, G89, G90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 30 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, E90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 30 and comprises amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, E90, G91 and G92.In some embodiments, an IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 31 and comprises amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, an IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 31 and comprises amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, an IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 32 and comprises amino acids G82, G83, S85, G86, G87, G88, S89, E90, G91, and S92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 32 and comprises amino acids G82, G83, S85, G86, G87, G88, S89, E90, G91 and S92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 33 and comprises amino acids G82, G83, G85, G86, G87, G88, G89, E90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 33 and comprises amino acids G82, G83, G85, G86, G87, G88, G89, E90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 34 and comprises amino acids G85, G86, G87, G88, G89, E90 and G91. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 34 and comprises amino acids G85, G86, G87, G88, G89, E90 and G91. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 35 and comprises amino acids G82, G83, G85, G86, G87, G88, G89, E90, and G91.In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 35 and comprises amino acids G82, G83, G85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 36 and comprises amino acids G82, G83, G85, E86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 36 and comprises amino acids G82, G83, G85, E86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 37 and comprises amino acids G82, G83, E85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 37 and comprises amino acids G82, G83, E85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 38 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 comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 38 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 comprises at least 80% sequence identity to SEQ ID NO: 39 and comprises amino acids G80, G81, G82, E83, G85, G86, G87, G88, G89, E90, G91 and G92.In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 39 and comprises amino acids G80, G81, G82, E83, G85, G86, G87, G88, G89, E90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 40 and comprises amino acids G80, G81, E82, G83, G85, G86, G87, G88, G89, E90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 40 and comprises amino acids G80, G81, E82, G83, G85, G86, G87, G88, G89, E90, G91 and G92.

[0142] In some embodiments, the IL-21 polypeptide is 10 X 11 X 12 X 13 X 14 X 15 CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 383) (where, X1=A, E, K; X2=A, E, K; 10 =S, G, E, Q; X 11 =G,K;X 12 =G, S, H;X 13 =A, E, S, G, R;X 14 =S, G, L;X 15 = G, S, T, and at least one amino acid residue is not the amino acid residue at the same position as shown in SEQ ID NO: 1. In some embodiments, X 8、 X 9、 X 11、 X12、 and X 13 is not the amino acid residue at the same position shown in SEQ ID NO:1.

[0143] In some embodiments, the IL-21 polypeptide has the amino acid sequence: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSCFQKAQLKSANTGNNERIINVSIKKLKRKPPX1X2X3X4GX5X6X7X8X9X 10 X 11 X 12 CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 380) (wherein X1 = G,S; X2 = G,T; X3 = G,E,N; X4 = G,S,E,A; X5 = G,E,S,R; X6 = G,E,R; X7 = S,G,E,Q; X8 = G,K; X9 = G,S,H; X 10 =A, E, S, G, R;X 11 = S, G, L; and X 12 = G, S, T, but X 5、 X 6、 X 8、 X9 and X 10 and at least one of X5=G, X6=G, X8=G, X9=G, X10=G, X11=G, X12=G, X13=G, X14=G, X15=G, X16=G, X17=G, X18=G, X19=G, X20=G, X21=G, X22=G, X23=G, X24=G, X25=G, X26=G, 10 = A. In some embodiments, X5 = G, X6 = G, X8 = G, X 10 In some embodiments, X1 = G, X2 = G, X3 = G, X4 = S, X5 = E, X6 = G, X7 = S, X8 = G, X9 = G, X 10 In some embodiments, X1 = G, X2 = G, X3 = G, X4 = S, X5 = G, X6 = G, X7 = S, X8 = G, X9 = G, X 10 In some embodiments, X5=G, X6=G, X7=S, X8=G, X9=G, X 10 In some embodiments, X5=G, X6=G, X7=S, X8=G, X9=G, X 10 In some embodiments, X5=G, X6=G, X7=G, X8=G, X9=G, X 10In some embodiments, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 In some embodiments, X5=G, X6=G, X7=E, X8=G, X9=G, X 10 In some embodiments, 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 In some embodiments, 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 In some embodiments, 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 In some embodiments, 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 In some embodiments, X3=G, X4=G, X5=S, X6=G, X7=G, X8=G, X9=S, X 10 =G,X 11 =G,X 12 In some embodiments, X3=G, X4=G, X5=E, X6=G, X7=G, X8=G, X9=S, X 10 =G,X 11 =G,X 12 In some embodiments, 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 In some embodiments, 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 12In some embodiments, 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 In some embodiments, X3=G, X4=G, X5=S, X6=G, X7=G, X8=G, X9=S, X 10 =E,X 11 =G,X 12 In some embodiments, X3=G, X4=G, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E,X 11 =G,X 12 In some embodiments, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E,X 11 In some embodiments, X3=G, X4=G, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E,X 11 In some embodiments, X3=G, X4=G, X5=G, X6=E, X7=G, X8=G, X9=G, X 10 =E,X 11 In some embodiments, X3=G, X4=G, X5=E, X6=G, X7=G, X8=G, X9=G, X 10 =E,X 11 In some embodiments, 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 In some embodiments, 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.

[0144] In some embodiments, the IL-21 polypeptide comprises an amino acid sequence beginning at position 78 as defined by SEQ ID NO:1, the amino acid sequence being: PPX1X2X3X4GX5X6X7X8X9X 10 X 11 X12 CP (SEQ ID NO: 386) (wherein, X1=G, S;X2=G, T;X3=G, E, N;X4=G, S, E, A; 10 =A, E, S, G, R;X 11 = S, G, L; and X 12 = G, S, T, but X 5、 X 6、 X 8、 X9 and X 10 at least one of which is not the amino acid residue at the same position shown in SEQ ID NO: 1).

[0145] In some embodiments, the IL-21 polypeptide comprises an amino acid sequence beginning at position 80 as defined by SEQ ID NO: 1, the amino acid sequence being: X1X2X3X4GX5X6X7X8X9X 10 X 11 X 12 (In the formula, X1=G, S;X2=G, T;X3=G, E, N;X4=G, S, E, A; 10 =A, E, S, G, R;X 11 = S, G, L; and X 12 = G, S, T, but X 5、 X 6、 X 8、 X9 and X 10 at least one of which is not the amino acid residue at the same position shown in SEQ ID NO: 1).

[0146] Reduced IL-21R binding In some embodiments, the present disclosure provides an IL-21 polypeptide or functional fragment or variant thereof having reduced binding affinity to IL-21R. In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof comprises at least one mutation that reduces its binding affinity to IL-21R. Such mutation, in some embodiments, is at one or more positions selected from the group consisting of R5, 18, R9, R11, Q12, 114, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117, where the position numbering is according to the amino acid sequence of SEQ ID NO: 1. In some embodiments, such mutations are at one or more positions selected from the group consisting of R5, 18, R9, R11, L13, 114, 116, V17, D18, K72, K73, L74, K75, R76, K77, and K117, where the position numbering is according to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutation at position R5 comprises an amino acid substitution selected from the group consisting of A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, the mutation at position 18 comprises an amino acid substitution selected from the group consisting of Q, H, or E. In some embodiments, the mutation at position R9 comprises an amino acid substitution selected from the group consisting of A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, the mutation at position R11 comprises an amino acid substitution selected from the group consisting of D or E. In some embodiments, the mutation at position Q12 comprises an amino acid substitution selected from the group consisting of L, I, or Y. In some embodiments, the mutation at position L13 comprises an amino acid substitution selected from the group consisting of F or R. In some embodiments, the mutation at position I14 comprises an amino acid substitution selected from the group consisting of D or E. In some embodiments, the mutation at position D15 comprises an amino acid substitution selected from the group consisting of R, K, H, L, Y, or F. In some embodiments, the mutation at position I16 comprises an amino acid substitution selected from the group consisting of A, S, or R.In some embodiments, the mutation at position V17 comprises an amino acid substitution selected from the group consisting of I or A. In some embodiments, the mutation at position D18 comprises an amino acid substitution selected from the group consisting of A, K, or R. In some embodiments, the mutation at position Q19 comprises an amino acid substitution selected from the group consisting of L or Y. In some embodiments, the mutation at position Y23 comprises an amino acid substitution of E. In some embodiments, the mutation at position R65 comprises an amino acid substitution selected from the group consisting of G, S, E, D, or A. In some embodiments, the mutation at position S70 comprises an amino acid substitution selected from the group consisting of H, Y, L, V, or F. In some embodiments, the mutation at position K72 comprises an amino acid substitution selected from the group consisting of G, S, E, D, or A. In some embodiments, the mutation at position K73 comprises an amino acid substitution selected from the group consisting of A, Y, L, F, G, S, T, E, or D. In some embodiments, the mutation at position L74 comprises an amino acid substitution selected from the group consisting of I, F, V, or M. In some embodiments, the mutation at position K75 comprises an amino acid substitution selected from the group consisting of G, S, E, D, or A. In some embodiments, the mutation at position R76 comprises an amino acid substitution selected from the group consisting of A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, the mutation at position K77 comprises an amino acid substitution selected from the group consisting of G, S, E, D, or A. In some embodiments, the mutation at position S80 comprises an amino acid substitution of H, A, G, E, or D. In some embodiments, the mutation at position Q116 comprises an amino acid substitution of Y. In some embodiments, the mutation at position K117 comprises an amino acid substitution selected from the group consisting of A, D, or E.

[0147] In some cases, the IL-21 polypeptide or functional fragment or variant thereof 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, I1 In some cases, the IL-21 polypeptide or functional fragment or variant thereof comprises at least one amino acid substitution selected from 6R, 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 cases, the IL-21 polypeptide or functional fragment or variant thereof comprises the amino acid substitution is R76E or R76Q.

[0148] An exemplary sequence for an IL-21 polypeptide of the disclosure, or a functional fragment or variant thereof, is provided as follows: QGQDX1HMX2X3MX4X5LX6X7IVX8X9LKNX 10 VNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNEX 11 IINVX 12 IX 13 X 14 LX 15 X 16 X 17 PPX 18 TNAGRRQKHRLTTCPSCDSYEKPPPKEFLERFKSLLX 19 X 20MIHQHLSSRTHGSEDS (SEQ ID NO: 22). In some embodiments, X1 = R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X2 = I, Q, H, E. In some embodiments, X3 = R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X4 = R, D, or E. In some embodiments, X5 = Q, L, I, or Y. In some embodiments, X6 = I, D, or E. In some embodiments, X7 = D, R, K, H, L, Y, or F. In some embodiments, X8 = D, A, K, or R. In some embodiments, X9 = Q, L, or Y. In some embodiments, X 10 = Y or E. In some embodiments, X 11 = R, G, S, E, D, or A. In some embodiments, X 12 = S, H, Y, L, V, or F. In some embodiments, X 13 = K, G, S, E, D, or A. In some embodiments, X 14 = K, A, Y, L, F, G, S, T, E, A, or D. In some embodiments, X 15 = K, G, S, E, D, or A. In some embodiments, X 16 = R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X 17 = K, G, S, E, D, or A. In some embodiments, X 18 = S, H, A, G, E, or D. In some embodiments, X 19 =Q or Y. In some embodiments, X 20 =K, A, D, or E.

[0149] In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof with reduced binding to IL-21R has the sequence: QGQDX1HMX2X3MX4QX5X6DX7X8X9QLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIX 10 X 11 X 12 X 13 X14 X 15 PPSTNAGRRQKHRLTTCPSCDSYEKPPPKEFLERFKSLLQX 16 MIHQHLSSRTHGSEDS (SEQ ID NO: 384) (wherein, X1=F, A, E, S, T, N, Q, V, I, L, Y, R; X2=E, I; X3=A, D, E, H, S, T, N, G, V, I, L, Y, R;X4=D, E, R;X5=F, R, L;X6=D, I;X7=A, S, R, I;X8=I, A, V;X9=A, D;X 10 =A,E,K;X 11 =A,E,K;X 12 =I, F, M, L;X 13 =A,K,E;X 14 =E, F, A, N, D, S, T, Q, V, I, L, Y, M, R;X 15 =A,E,K;X 16 = A, K; where X1 to X 16 at least one of which is not the amino acid residue at the same position shown in SEQ ID NO:1).

[0150] In some embodiments, the present disclosure provides an IL-21 polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 313-371. In some embodiments, the present disclosure provides an IL-21 polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 313-371.

[0151] Mutation combinations A functional fragment of an IL-21 polypeptide or variant thereof described herein, in some embodiments, comprises a combination of (a) mutations that alter its isoelectric point compared to wild-type IL-21 (SEQ ID NO: 1) and (b) mutations that attenuate its binding to IL-21R compared to wild-type IL-21. In some embodiments, the mutations in group (a) are at positions selected from positions K56, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92; and the mutations in group (b) are at positions selected from the group consisting of R5, 18, R9, R11, Q12, 114, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117, where the position numbering is according to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutations in group (a) are at positions selected from: S80, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92; and the mutations in group (b) are at positions selected from the group consisting of R5, 18, R9, R11, L13, 114, 116, V17, D18, K72, K73, L74, K75, R76, K77, and K117, where the position numbering is according to the amino acid sequence of SEQ ID NO:1. In some embodiments, the mutations in group (a) are at positions selected from: S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, and T92; and the mutations in group (b) are at positions selected from the group consisting of R5, 18, R9, R11, L13, 114, 116, V17, D18, K72, K73, L74, K75, R76, K77, and K117, where the position numbering is according to the amino acid sequence of SEQ ID NO:1. In some embodiments, the mutations in group (a) are at positions selected from: S80, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92; and the mutations in group (b) are at position R76, where the position numbering is according to the amino acid sequence of SEQ ID NO:1.

[0152] In some embodiments, the IL-21 polypeptide comprises the consensus sequence as follows: QGQDX1HMX2X3MX4X5LX6X7IVX8X9LKNX 10 VNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLX 11 SANTGNNEX 12 IINVX 13 IX 14 X 15 LX 16 X 17 X 18 PPX 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 CPSCDSYEKKPPKEFLERFKSLLX 32 X 33 MIHQHLSSRTHGSEDS (SEQ ID NO: 3). In some embodiments, X1 = R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X2 = I, Q, H, E. In some embodiments, X3 = R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X4 = R, D, or E. In some embodiments, X5 = Q, L, I, or Y. In some embodiments, X6 = I, D, or E. In some embodiments, X7 = D, R, K, H, L, Y, or F. In some embodiments, X8 = D, A, K, or R. In some embodiments, X9 = Q, L, or Y. In some embodiments, X 10 = Y or E. X 11 = G, S, E, D, or A. In some embodiments, X 12 = R, G, S, E, D, or A. In some embodiments, X 13 = S, H, Y, L, V, or F. In some embodiments, X 14 = K, G, S, E, D, or A. In some embodiments, X 15= K, A, Y, L, F, G, S, T, E, A, or D. In some embodiments, X 16 = K, G, S, E, D, or A. In some embodiments, X 17 = R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X 18 = K, G, S, E, D, or A. In some embodiments, X 19 = S, H, A, G, E, or D. In some embodiments, X 20 = G, S, E, D, or A. In some embodiments, X 21 = G, S, E, D, or A. In some embodiments, X 22 = G, S, E, or D. In some embodiments, X 23 = A, S, E, or D. In some embodiments, X 24 = G, S, E, D, or A. In some embodiments, X 25 = G, S, E, D, or A. In some embodiments, X 26 = G, S, E, D, or A. In some embodiments, X 27 = G, S, E, D, or A. In some embodiments, X 28 = G, S, E, D, or A. In some embodiments, X 29 = G, S, E, D, or A. In some embodiments, X 30 = G, S, E, D, or A. In some embodiments, X 31 = G, S, E, D, or A. In some embodiments, X 32 = Y. In some embodiments, X 33 =A, D, or E.

[0153] In some embodiments, the IL-21 polypeptide comprises the consensus sequence: QGQDX1HMX2X3MX4QX5X6DX7X8X9QLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLX 10 SANTGNNERIINVSIX 11 X 12 X 13 X 14 X 15 X16 PPX 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 CPSCDSYEKKPPKEFLERFKSLLQX 30 MIHQHLSSRTHGSEDS (SEQ ID NO: 385) (wherein, X1=F, A, E, S, T, N, Q, V, I, L, Y, R; X2=E, I; X3=A, D, E, H, S, T, N, G, V, I, L, Y, R;X4=D, E, R;X5=F, R, L;X6=D, I;X7=A, S, R, I;X8=I, A, V;X9=A, D;X 10 =A,E,K;X 11 =A,E,K;X 12 =A,E,K;X 13 =I, F, M, L;X 14 =A,K,E;X 15 =E, F, A, N, D, S, T, Q, V, I, L, Y, M, R;X 16 =A,E,K;X 17 =G,S;X 18 =G,T;X 19 =G, E, N;X 20 =G,S,E,A;X 21 =E, G;X 22 =G, E, S, R;X 23 =G,E,R;X 24 =S, G, E, Q; X 25 =G,K;X 26 =G, S, H;X 27 =A, E, S, G, R;X 28 =S, G, L;X 29 =G,S,T;X 30 = A, K, but X1 to X9X 11 ~X 16 and X 30 and at least one of X is not an amino acid residue at the same position as shown in SEQ ID NO: 1, and reduces binding to IL-21R, with the proviso that 10 and X 17 ~X 29at least one of which is not an amino acid residue at the same position shown in SEQ ID NO: 1 and reduces the isoelectric point compared to human IL-21 (SEQ ID NO: 1).

[0154] In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof comprising a combination of mutations comprises at least one mutation at a position of SEQ ID NO: 2. In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof comprising a combination of mutations comprises at least one mutation at a position selected from the group consisting of positions: R5, 18, R9, R11, Q12, 114, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117, wherein the position numbering is according to SEQ ID NO: 2. In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof comprising a combination of mutations comprises at least one mutation at a position selected from the group consisting of positions: R5, 18, R9, R11, L13, 114, 116, V17, D18, K72, K73, L74, K75, R76, K77, and K117, wherein the position numbering is according to SEQ ID NO: 2.

[0155] In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprising a combination of mutations comprises at least one mutation at a position of SEQ ID NO: 40. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof comprising a combination of mutations comprises at least one mutation at a position selected from the group consisting of positions: R5, 18, R9, R11, Q12, 114, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117, wherein the position numbering is according to SEQ ID NO: 40. In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof comprising a combination of mutations comprises at least one mutation at a position selected from the group consisting of positions: R5, 18, R9, R11, L13, 114, 116, V17, D18, K72, K73, L74, K75, R76, K77, and K117, wherein the position numbering is according to SEQ ID NO: 40.

[0156] In some embodiments, an IL-21 polypeptide, or functional fragment or variant thereof, comprises (i) four or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the four or more amino acid substitutions are within S80 to T92 of SEQ ID NO: 1, and at least four amino acid substitutions are at residues R85, R86, K88, H89, R90, or a combination thereof, and (ii) four or more amino acid substitutions that provide a reduced isoelectric point compared to a human IL-21 polypeptide comprising: (a) R11D; (b) R11E; (c) I14D , D18A and K117A;(d)R76E;(e)R5F;(f)R76F;(g)I8E;(h)R5A;(i)R5E;(j)R5S;(k)R5T;(l)R5N;(m)R5Q;(n)R5V;(o)R5I;(p)R5L;(q)R5Y;(r) R76A;(s)R76N;(t)R76D;(u)R76S;(v)R76T;(w)R76Q;(x)R76V;(y)R76I;(z)R76L;(aa)R76Y;(bb)K77A;(cc)K77E;(dd)K72A;(ee)K72E;(ff )K75A;(gg)K75E;(hh)K73A;(ii)K73E;(jj)R5F and K77A;(kk)R5F and K77E;(ll)R5F and K72A;(mm)R5F and K72E;(nn)R5F and K76A;(oo)R5F and K76E;(pp)K73A and K76F;(qq)K73E and K76F;(rr)R9A;(ss)R9D;(tt)R9E;(uu)R9H;(vv)R9S;(ww)R9T;(xx)R9N;(zz)R9G;(aaa)R9V;(bbb)R9I;(cc c) Comprises at least one amino acid substitution selected from R9L; (ddd) R9Y; (eee) K72A and R76F; (fff) K75A and R76F; (ggg) R76F and K77A; (hhh) K75E and R76F; (iii) V17I and L74I; (jjj) I16A and L74F; (kkk) I16S, V17I, and L74V; (lll) I16R, V17I, and L74I; (mmm) L13F, I16A, V17A, and L74M; and (nnn) L13R, I16A, V17I, and L74I.

[0157] In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises (i) an amino acid sequence selected from SEQ ID NOs: 2, 4-15, and 23-40, and (ii) a sequence selected from the group consisting of (a) R11D; (b) R11E; (c) I14D, D18A, and K117A; (d) R76E; (e) R5F; (f) R76F; (g) I8E; (h) R5A; (i) R5E; (j) R5S; (k) R5T; (l) R5N; (m) R5Q; (n) R5V; (o )R5I;(p)R5L;(q)R5Y;(r)R76A;(s)R76N;(t)R76D;(u)R76S;(v)R76T;(w)R76Q;(x)R76V;(y)R76I;(z)R76L;(aa)R76Y; (bb)K77A;(cc)K77E;(dd)K72A;(ee)K72E;(ff)K75A;(gg)K75E;(hh)K73A;(ii)K73E;(jj)R5F and K77A;(kk)R5F and K77E;( ll) R5F and K72A; (mm) R5F and K72E; (nn) R5F and K76A; (oo) R5F and K76E; (pp) K73A and K76F; (qq) K73E and K76F; (rr) R9A; (ss) R9D; (tt) R9E; (uu) R9H; (vv) R9S; (ww) R9T; (xx) R9N; (zz) R9G; (aaa) R9V; (bbb) R9I; (ccc) R9L; (ddd) R9Y; (eee) K72A and R76F; (fff) K75A and R76F; (ggg) R76F and K77A; (hhh) K75E and R76F; (iii) V17I and L74I; (jjj) I16A and L74F; (kkk) I16S, V17I, and L74V; (lll) I16R, V17I, and L74I; (mmm) L13F, I16A, V17A, and L74M; and (nnn) L13R, I16A, V17I, and L74I.

[0158] In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R11D mutation and comprises about 75% to about 100% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R11D mutation and comprises at least about 75% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R11D mutation and comprises up to about 100% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R11D mutation and has about 75% to about 80% sequence identity, about 75% to about 85% sequence identity, about 75% to about 90% sequence identity, about 75% to about 95% sequence identity, about 75% to about 96% sequence identity, about 75% to about 97% sequence identity to SEQ ID NO: 16. sequence identity, about 75% to about 98% sequence identity, about 75% to about 99% sequence identity, about 75% to about 100% sequence identity, about 80% to about 85% sequence identity, about 80% to about 90% sequence identity, about 80% to about 95% sequence identity, about 80% to about 96% sequence identity, about 80% to about 97% sequence identity, about 80 % sequence identity to about 98% sequence identity, about 80% sequence identity to about 99% sequence identity, about 80% sequence identity to about 100% sequence identity, about 85% sequence identity to about 90% sequence identity, about 85% sequence identity to about 95% sequence identity, about 85% sequence identity to about 96% sequence identity, about 85% sequence identity to about 97% sequence identity, about 85% sequence identity to about 98% sequence identity, about 85% sequence identity to about 9 9% sequence identity, about 85% to about 100% sequence identity, about 90% to about 95% sequence identity, about 90% to about 96% sequence identity, about 90% to about 97% sequence identity, about 90% to about 98% sequence identity, about 90% to about 99% sequence identity, about 90% to about 100% sequence identity, about 95% to about 96% sequence identity,Approximately 95% to approximately 97% sequence identity, approximately 95% to approximately 98% sequence identity, approximately 95% to approximately 99% sequence identity, approximately 95% to approximately 100% sequence identity, approximately 96% to approximately 97% sequence identity, approximately 96% to approximately 98% sequence identity, approximately 96% to approximately 99% sequence identity, approximately 96% to about 100% sequence identity, about 97% to about 98% sequence identity, about 97% to about 99% sequence identity, about 97% to about 100% sequence identity, about 98% to about 99% sequence identity, about 98% to about 100% sequence identity, or about 99% to about 100% sequence identity. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R11D mutation and comprises about 75% sequence identity, about 80% sequence identity, about 85% sequence identity, about 90% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99% sequence identity, or about 100% sequence identity to SEQ ID NO: 16.

[0159] In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R11E mutation and comprises about 75% to about 100% sequence identity with SEQ ID NO: 17. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R11E mutation and comprises at least about 75% sequence identity with SEQ ID NO: 17. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R11E mutation and comprises up to about 100% sequence identity with SEQ ID NO: 17. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R11E mutation and has about 75% to about 80% sequence identity, about 75% to about 85% sequence identity, about 75% to about 90% sequence identity, about 75% to about 95% sequence identity, about 75% to about 96% sequence identity, about 75% to about 97% sequence identity to SEQ ID NO: 17. sequence identity, about 75% to about 98% sequence identity, about 75% to about 99% sequence identity, about 75% to about 100% sequence identity, about 80% to about 85% sequence identity, about 80% to about 90% sequence identity, about 80% to about 95% sequence identity, about 80% to about 96% sequence identity, about 80% to about 97% sequence identity, about 80 % sequence identity to about 98% sequence identity, about 80% sequence identity to about 99% sequence identity, about 80% sequence identity to about 100% sequence identity, about 85% sequence identity to about 90% sequence identity, about 85% sequence identity to about 95% sequence identity, about 85% sequence identity to about 96% sequence identity, about 85% sequence identity to about 97% sequence identity, about 85% sequence identity to about 98% sequence identity, about 85% sequence identity to about 9 9% sequence identity, about 85% to about 100% sequence identity, about 90% to about 95% sequence identity, about 90% to about 96% sequence identity, about 90% to about 97% sequence identity, about 90% to about 98% sequence identity, about 90% to about 99% sequence identity, about 90% to about 100% sequence identity, about 95% to about 96% sequence identity,Approximately 95% to approximately 97% sequence identity, approximately 95% to approximately 98% sequence identity, approximately 95% to approximately 99% sequence identity, approximately 95% to approximately 100% sequence identity, approximately 96% to approximately 97% sequence identity, approximately 96% to approximately 98% sequence identity, approximately 96% to approximately 99% sequence identity, approximately 96% to about 100% sequence identity, about 97% to about 98% sequence identity, about 97% to about 99% sequence identity, about 97% to about 100% sequence identity, about 98% to about 99% sequence identity, about 98% to about 100% sequence identity, or about 99% to about 100% sequence identity. In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof comprises an R11E mutation and comprises about 75% sequence identity, about 80% sequence identity, about 85% sequence identity, about 90% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99% sequence identity, or about 100% sequence identity to SEQ ID NO: 17.

[0160] In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, or functional fragment or variant thereof, comprises I14D, D18A and K117A mutations and comprises about 75% sequence identity to about 100% sequence identity with SEQ ID NO: 18. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, or functional fragment or variant thereof, comprises I14D, D18A and K117A mutations and comprises at least about 75% sequence identity with SEQ ID NO: 18. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, or functional fragment or variant thereof, comprises I14D, D18A and K117A mutations and comprises up to about 100% sequence identity with SEQ ID NO: 18. In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof comprises I14D, D18A, and K117A mutations and has about 75% to about 80% sequence identity, about 75% to about 85% sequence identity, about 75% to about 90% sequence identity, about 75% to about 95% sequence identity, about 75% to about 96% sequence identity, about 75% to about 97% sequence identity, about 75% to about 98% sequence identity, about 75% to about 99% sequence identity, about 75% to about 100% sequence identity, about 80% to about 85% sequence identity, or about 80% sequence identity to SEQ ID NO: 18. Sequence identity to about 90%; sequence identity to about 95%; sequence identity to about 96%; sequence identity to about 97%; sequence identity to about 98%; sequence identity to about 99%; sequence identity to about 100%; sequence identity to about 90% about 85% to about 95% sequence identity, about 85% to about 96% sequence identity, about 85% to about 97% sequence identity, about 85% to about 98% sequence identity, about 85% to about 99% sequence identity, about 85% to about 100% sequence identity, about 90% to about 95% sequence identity,Approximately 90% sequence identity to approximately 96% sequence identity, approximately 90% sequence identity to approximately 97% sequence identity, approximately 90% sequence identity to approximately 98% sequence identity, approximately 90% sequence identity to approximately 99% sequence identity, approximately 90% sequence identity to approximately 100% sequence identity, approximately 95% sequence identity to approximately 96% sequence identity, approximately 95% sequence identity to approximately 97% sequence identity, approximately 95% sequence identity to approximately 98% sequence identity, approximately 95% sequence identity to approximately 99% sequence identity, approximately 95% sequence identity to approximately 100% sequence identity, approximately 96% to about 97% sequence identity, about 96% to about 98% sequence identity, about 96% to about 99% sequence identity, about 96% to about 100% sequence identity, about 97% to about 98% sequence identity, about 97% to about 99% sequence identity, about 97% to about 100% sequence identity, about 98% to about 99% sequence identity, about 98% to about 100% sequence identity, or about 99% to about 100% sequence identity. In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof comprises I14D, D18A and K117A mutations and comprises about 75% sequence identity, about 80% sequence identity, about 85% sequence identity, about 90% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99% sequence identity, or about 100% sequence identity to SEQ ID NO: 18.

[0161] In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R76E mutation and comprises about 75% to about 100% sequence identity with SEQ ID NO: 19. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R76E mutation and comprises at least about 75% sequence identity with SEQ ID NO: 19. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R76E mutation and comprises up to about 100% sequence identity with SEQ ID NO: 19. In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof has about 75% to about 80% sequence identity, about 75% to about 85% sequence identity, about 75% to about 90% sequence identity, about 75% to about 95% sequence identity, about 75% to about 96% sequence identity, about 75% to about 97% sequence identity, about 75% to about 98% sequence identity, about 75% to about 99% sequence identity, about 80% to about 85% sequence identity, about 80% to about 98% sequence identity, about 80% to about 99% sequence identity, about 80% to about 99% sequence identity, about 90% to about 95% sequence identity, about 96% to about 99% sequence identity, about 90 ...0% sequence identity, about 90% to about 90% sequence identity, about 90% to about 90% sequence identity, about 90% to about 90% sequence identity, about 90% to about 90% sequence identity, about 90% to about 90% sequence identity, about 90% to about 90% sequence identity, about 90% to about 90% sequence identity, about 9 75% sequence identity to about 98% sequence identity, about 75% sequence identity to about 99% sequence identity, about 75% sequence identity to about 100% sequence identity, about 80% sequence identity to about 85% sequence identity, about 80% sequence identity to about 90% sequence identity, about 80% sequence identity to about 95% sequence identity, about 80% sequence identity to about 96% sequence identity, about 80% sequence identity to about 97% sequence identity, about 80% sequence identity Sequence identity to about 98% sequence identity, about 80% sequence identity to about 99% sequence identity, about 80% sequence identity to about 100% sequence identity, about 85% sequence identity to about 90% sequence identity, about 85% sequence identity to about 95% sequence identity, about 85% sequence identity to about 96% sequence identity, about 85% sequence identity to about 97% sequence identity, about 85% sequence identity to about 98% sequence identity, about 85% sequence identity to about 99% sequence identity, about 85% to about 100% sequence identity, about 90% to about 95% sequence identity, about 90% to about 96% sequence identity, about 90% to about 97% sequence identity, about 90% to about 98% sequence identity, about 90% to about 99% sequence identity, about 90% to about 100% sequence identity, about 95% to about 96% sequence identity,Approximately 95% to approximately 97% sequence identity, approximately 95% to approximately 98% sequence identity, approximately 95% to approximately 99% sequence identity, approximately 95% to approximately 100% sequence identity, approximately 96% to approximately 97% sequence identity, approximately 96% to approximately 98% sequence identity, approximately 96% to approximately 99% sequence identity, approximately 96% to about 100% sequence identity, about 97% to about 98% sequence identity, about 97% to about 99% sequence identity, about 97% to about 100% sequence identity, about 98% to about 99% sequence identity, about 98% to about 100% sequence identity, or about 99% to about 100% sequence identity. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R76E mutation and comprises about 75% sequence identity, about 80% sequence identity, about 85% sequence identity, about 90% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99% sequence identity, or about 100% sequence identity to SEQ ID NO: 19.

[0162] In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R5F mutation and comprises about 75% to about 100% sequence identity to SEQ ID NO: 20. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R5F mutation and comprises at least about 75% sequence identity to SEQ ID NO: 20. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises up to about 100% sequence identity to SEQ ID NO: 20. In some embodiments, the IL-21 polypeptide, or functional fragment or variant thereof, comprises an R5F mutation and has about 75% to about 80% sequence identity, about 75% to about 85% sequence identity, about 75% to about 90% sequence identity, about 75% to about 95% sequence identity, about 75% to about 96% sequence identity, about 75% to about 97% sequence identity to SEQ ID NO:29. Sequence identity, about 75% to about 98% sequence identity, about 75% to about 99% sequence identity, about 75% to about 100% sequence identity, about 80% to about 85% sequence identity, about 80% to about 90% sequence identity, about 80% to about 95% sequence identity, about 80% to about 96% sequence identity, about 80% to about 97% sequence identity, about 80% Sequence identity to about 98% sequence identity, about 80% sequence identity to about 99% sequence identity, about 80% sequence identity to about 100% sequence identity, about 85% sequence identity to about 90% sequence identity, about 85% sequence identity to about 95% sequence identity, about 85% sequence identity to about 96% sequence identity, about 85% sequence identity to about 97% sequence identity, about 85% sequence identity to about 98% sequence identity, about 85% sequence identity to about 9 9% sequence identity, about 85% to about 100% sequence identity, about 90% to about 95% sequence identity, about 90% to about 96% sequence identity, about 90% to about 97% sequence identity, about 90% to about 98% sequence identity, about 90% to about 99% sequence identity, about 90% to about 100% sequence identity, about 95% to about 96% sequence id...

Claims

1. A fusion protein comprising: a) an IL-21 polypeptide comprising an amino acid sequence that is at least 80% identical to a human IL-21 polypeptide comprising the sequence of SEQ ID NO:1, said IL-21 polypeptide comprising one or more amino acid substitutions in the region from position S80 to position T92 numbered according to the sequence of SEQ ID NO:1, said one or more amino acid substitutions providing an isoelectric point that is at least 0.6 units to 5 units lower compared to that of said human IL-21 polypeptide comprising the sequence of SEQ ID NO:1; and b) an antibody or antigen-binding fragment thereof that specifically binds to at least one of CD8α, CD8αα, or CD8αβ; A fusion protein comprising:

2. The fusion protein of claim 1, wherein the IL-21 polypeptide has an isoelectric point of 7.12 to 8.

72.

3. The fusion protein of claim 1, wherein the IL-21 polypeptide does not contain an amino acid substitution at G84 numbered according to the sequence of SEQ ID NO:

1.

4. The fusion protein of claim 1, wherein the IL-21 polypeptide comprises 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions at positions selected from the group consisting of S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, and T92, numbered according to the sequence of SEQ ID NO:

1.

5. The fusion protein of claim 1, 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, compared to the sequence of SEQ ID NO:

1.

6. The IL-21 polypeptide has the amino acid sequence: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFCQKAQLKSANTGNNERIINVSIKKLKRKPPX1, X2, X3, X4, GX5, X6, X7, X8, X9, X10, X11, X12, CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO:380), where X1=G, S, X2=G, T, X3=G, E, N, X4=G, S, E, A, X5=G, E, S, R, X6=G, E, S, R, X = G, E, R, X7 = S, G, E, Q, X8 = G, K, X9 = G, S, H, X10 = A, E, S, G, R, X11 = S, G, L, and X12 = G, S, T, with the proviso that at least one of X5, X6, X8, X9, and X10 is not an amino acid residue at the same position shown in SEQ ID NO: 1 and optionally: (i) X 5 = G, X 6 = G, X 8 = G, X 10 = A, (ii) X 5 = G, X 6 = G, X 8 = G, X 10 = E, (iii) X1 = G, X2 = G, X3 = G, X4 = S, X5 = E, X6 = G, X7 = S, X8 = G, X9 = G, X10 = S, (iv) X1 = G, X2 = G, X3 = G, X4 = S, X5 = G, X6 = G, X7 = S, X8 = G, X9 = G, X10 = E, (v) X 5 = G, X 6 = G, X 7 = S, X 8 = G, X 9 = G, X 10 = E, (vi) X 5 = G, X 6 = G, X 7 = S, X 8 = G, X 9 = G, X 10 = S, (vii) X 5 = G, X 6 = G, X 7 = G, X 8 = G, X 9 = G, X 10 = E, (viii) X 5 = G, X 6 = G, X 7 = G, X 8 = G, X 9 = G, X 10 = G, (ix) X 5 = G, X 6 = G, X 7 = E, X 8 = G, X 9 = G, X 10 = G, (x) X 1 = G, X 2 = G, X 3 = G, X 4 = S, X 5 = G, X 6 = G, (xi)X 1 =G、X 2 =G、X 3 =G、X 4 =G、X 5 =G、X 6 =G、X 7 =G、X 8 =G、X 9 =G、X 10 =G、X 11 =G、X 12 =G、 (xii)X 1 =G、X 2 =G、X 3 =G、X 4 =S、X 5 =G、X 6 =G、X 7 =E、X 8 =G、X 9 =G、X 10 =G、X 11 =S、X 12 =G、 (xiii)X 1 =G、X 2 =G、X 3 =G、X 4 =E、X 5 =G、X 6 =G、X 7 =E、X 8 =G、X 9 =G、X 10 =G、X 11 =S、X 12 =G、 (xiv)X 3 =G、X 4 =G、X 5 =S、X 6 =G、X 7 =G、X 8 =G、X 9 =S、X 10 =G、X 11 =G、X 12 =S、 (xv)X 3 =G、X 4 =G、X 5 =E、X 6 =G、X 7 =G、X 8 =G、X 9 =S、X 10 =G、X 11 =G、X 12 =S、 (xvi)X 1 =G、X 2 =G、X 3 =G、X 4 =G、X 5 =E、X 6 =G、X 7 =G、X 8 =G、X 9 =G、X 10 =G、X 11 =G、X 12 =G、 (xvii)X 1 =G、X 2 =G、X 3 =G、X 4 =S、X 5 =G、X 6 =G、X 7 =S、X 8 =G、X 9 =G、X 10 =E、X 11 =G、X 12 =G、 (xviii)X 1 =G、X 2 =G、X 3 =G、X 4 =G、X 5 =G、X 6 =G、X 7 =G、X 8 =G、X 9 =G、X 10 =E、X 11 =G、X 12 =G、 (xix)X 3 =G、X 4 =G、X 5 =S、X 6 =G、X 7 =G、X 8 =G、X 9 =S、X 10 =E、X 11 =G、X 12 =S、 (xx)X 3 =G、X 4 =G、X 5 =G、X 6 =G、X 7 =G、X 8 =G、X 9 =G、X 10 =E、X 11 =G、X 12 =G、 (xxi)X 5 =G、X 6 =G、X 7 =G、X 8 =G、X 9 =G、X 10 =E、X 11 =G、 (xxii)X 3 =G、X 4 =G、X 5 =G、X 6 =G、X 7 =G、X 8 =G、X 9 =G、X 10 =E、X 11 =G、 (xxiii) X3=G, X4=G, X5=G, X6=E, X7=G, X8=G, X9=G, X10=E, X11=G, (xxiv) X 3 = G, X 4 = G, X 5 = E, X 6 = G, X 7 = G, X 8 = G, X 9 = G, (xxv) X1 = G, X2 = G, X3 = G, X4 = E, X5 = G, X6 = G, X7 = G, X8 = G, X9 = G, X10 = E, X11 = G, X12 = G, or (xxvi) X1 = G, X2 = G, X3 = E, X4 = G, X5 = G, X6 = G, X7 = G, X8 = G, X9 = G, X10 = E, X11 = G, and X12 = G. The fusion protein of claim 1, 7. The fusion protein of claim 1, 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: 2, 4-15, and 23-40.

8. The fusion protein of claim 1, wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15, and 23-40.

9. The fusion protein of claim 1, wherein the IL-21 polypeptide further comprises at least one amino acid substitution that reduces binding to the IL-21 receptor compared to binding by the human IL-21 polypeptide.

10. The fusion protein of claim 9, wherein the at least one amino acid substitution that reduces binding to the IL-21 receptor is in one or more amino acid residues at a position selected from the group consisting of R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, and K117, numbered according to the sequence of SEQ ID NO:

1.

11. The at least one amino acid substitution that reduces binding to the IL-21 receptor 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, 10. The fusion protein of claim 9, selected from the group consisting of I16R, V17I, V17A, D18A, K72A, K72E, K73A, K73E, K75A, K75E, L74I, L74F, L74M, L74V, R76E, R76F, R76A, R76N, R76D, R76S, R76T, R76Q, R76V, R76I, R76L, R76Y, R76M, K77A, K77E, and K117A.

12. The fusion protein of claim 9, wherein the at least one amino acid substitution that reduces binding to the IL-21 receptor is R76E or R76Q.

13. The fusion protein of claim 1, 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: 16-21, 41-98, and 374-379.

14. The fusion protein of claim 1, wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16-21, 41-98, and 374-379.

15. A fusion protein comprising: a) an IL-21 polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:94; and b) an antibody or antigen-binding fragment thereof that specifically binds to at least one of CD8α, CD8αα, or CD8αβ; A fusion protein comprising:

16. The fusion protein of claim 1, wherein the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO:

94.

17. The fusion protein of claim 1, wherein the IL-21 polypeptide and the antibody or antigen-binding fragment thereof are linked to each other via a linker, and optionally, the IL-21 polypeptide is linked to the C-terminus of the antibody or antigen-binding fragment thereof.

18. The antibody or antigen-binding fragment thereof, i) a first polypeptide comprising a light chain variable region and a light chain constant region; ii) a second polypeptide comprising a heavy chain variable region and a heavy chain constant region; and iii) a third polypeptide comprising a heavy chain constant region.

2. The fusion protein of claim 1, wherein the heavy chain constant regions of the second polypeptide and the third polypeptide form an Fc domain.

19. The fusion protein of claim 18, wherein the third polypeptide comprises a heavy chain variable region, and optionally, the antibody or antigen-binding fragment thereof further comprises a fourth polypeptide comprising a light chain variable region and a light chain constant region.

20. The fusion protein of claim 18, wherein the IL-21 polypeptide is linked to the C-terminus of the heavy chain constant region of the second polypeptide or the third polypeptide.

21. The fusion protein of claim 18, wherein the Fc domain contains one or more modifications that promote heterodimerization.

22. The fusion protein of claim 18, wherein the second polypeptide comprises a knob modification in the heavy chain constant region and the third polypeptide comprises a hole modification in the heavy chain constant region, optionally, the knob modification comprises amino acid substitutions Y349C and T366W according to EU numbering and the hole modification comprises amino acid substitutions S354C, T366S, L368A and Y407V according to EU numbering.

23. The fusion protein of claim 18, wherein the Fc domain comprises amino acid mutations that reduce binding of the Fc domain to an Fc gamma receptor, and optionally the Fc domain comprises amino acid substitutions L234A, L235A, and G237A according to EU numbering.

24. The antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; (a) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 226, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 227, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 151; and the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 152, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 153, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 154; (b) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 228, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 227, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 157; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 158, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 159, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 160; (c) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 223, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 227, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 163; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 164, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 165, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 166; (d) 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: 227, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 169; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 170, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 171, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 172; (e) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 230, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 231, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; (f) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 230, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 232, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 181; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 182, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 183, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 184; (g) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 223, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 224, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 225; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 140, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 141, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; (h) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 233, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 234, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148; (i) the VH domain comprises a CDR-H1 having an amino acid sequence of GX 1 X 2 FX 3 X 4 X 5 (wherein X 1 is G, Y, S, or A; X 2 is T, S, G, R, N, or H; X 3 is S, T, R, H, Y, G, or P; X 4 is S, K, G, N, R, D, T, or G; and X 5 is Y, L, H, or F) (SEQ ID NO: 235); X 1 is D or A; X 2 is A, G, E, R, Y, K, N, Q, L, or F; X 3 is A, L, P, or Y; X 4 is I or L; X 5 is R, A, Q, or S; X 6 is A or D; and X 7 is D, E, A, or S; CDR-L1 having an amino acid sequence of LN (wherein X 1 is R or G, X 2 is A or T, X 3 is Q or E, X 4 is E, N, T, S, A, K, D, G, R, or Q, X 5 is Y or S, and X 6 is A or V) (SEQ ID NO: 188); CDR-L2 having an amino acid sequence of GX 1 X 2 X 3 LX 4 X 5 (wherein X 1 is A or S, X 2 is T, S, E, Q, or D, X 3 is N, R, A, E, or H, X 4 is Q or A, and X 5 is S or D) (SEQ ID NO: 189); and CDR-L3 having an amino acid sequence of QX 1 X 2 X 3 X 4 X 5 PWT, wherein X 1 is S, N, D, Q, A, or E, X 2 is T, I, or S, X 3 is Y, L, or F, X 4 is D, G, T, E, Q, A, or Y, and X 5 isis A, T, R, S, K, or Y) (SEQ ID NO: 190); (j) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 242, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 152, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 153, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 202; (k) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207; (l) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 152, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 153, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 202; (m) the VH domain comprises a CDR-H1 comprising the amino acid sequence of GFTFX 1 X 2 Y (wherein X 1 is S, D, E, Q, S, or A, and X 2 is S, D, E, A, or Q) (SEQ ID NO: 244); a CDR-H2 comprising the amino acid sequence of X 1 X 2 X 3 GX 4 X 5 (wherein X 1 is T, N, S, Q, E, H, R, or A, X 2 is Y, W, F, or H, X 3 is A, S, Q, E, or T, X 4 is G or E, and X 5 is S or I) (SEQ ID NO: 245); X 2 is S or A, X 2 is N, H, A, D, L, Q, Y, or R, X 3 is A, N, S, or G, X 4 is A, V, R, E, or S, X 5 is D or S, X 6 is D, N, Q, E, S, T, or L, X 7 is L, F, or M, and X 8 is I, Y, or V (SEQ ID NO:246); and the VL domain comprises a CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO:176), a CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:177), and a CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO:178). (n) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; and the VL domain comprises a CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 176), a CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 177), and a CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 178); (o) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 261, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; and the VL domain comprises a CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 176), a CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 177), and a CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 178); or (p) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 223, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 224, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 284; and the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 285, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 286, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 287; The fusion protein of claim 1.

25. The fusion protein of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, the VH domain comprising CDR-H1 having the amino acid sequence of SEQ ID NO:241, CDR-H2 having the amino acid sequence of SEQ ID NO:243, and CDR-H3 having the amino acid sequence of SEQ ID NO:204, and the VL domain comprising CDR-L1 having the amino acid sequence of SEQ ID NO:205, CDR-L2 having the amino acid sequence of SEQ ID NO:206, and CDR-L3 having the amino acid sequence of SEQ ID NO:

207.

26. The antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; (a) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 109, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 110; (b) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 111, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 112; (c) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 113, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 114; (d) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 115, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 116; (e) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 117, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 118; (f) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 119, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 120; (g) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 123, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 124; (h) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 129, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 130; (i) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 131, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 132; (j) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 125, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 126; (k) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 127, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 128; (l) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 133, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 134; (m) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 135, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 136; (n) 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: 107, and 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: 108; or (o) 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: 121, and 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: 122; or (p) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 258, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 259; The fusion protein of claim 1.

27. The fusion protein of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein 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:129, and 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:

130.

28. The fusion protein of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, the VH domain comprising the amino acid sequence of SEQ ID NO: 129 and the VL domain comprising the amino acid sequence of SEQ ID NO:

130.

29. A method for the preparation of a polypeptide comprising the steps of: (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:262, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:263, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:264, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:262; (b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:266, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:267, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:268, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:266; (c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:270, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:271, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:272, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

270. (d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:274, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:275, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:276, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

274. (e) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:278, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:279, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:280, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:278; (f) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:262, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:263, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:265, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

262. (g) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:266, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:267, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:269, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:266; (h) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:270, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:271, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:273, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

270. (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:274, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:275, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:277, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:274; (j) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:278, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:279, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:281, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:278; (k) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:297, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:298, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:299, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:297; (l) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:301, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:302, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:303, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:301; (m) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 305, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 306, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 307, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

305. (n) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 309, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 310, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 311, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 309; (o) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:297, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:298, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:300, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:297; (p) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 301, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 302, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 304, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 301; (q) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 305, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 306, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 308, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 305; or (r) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 309, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 310, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 312, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 309; The fusion protein of claim 1.

30. The fusion protein of claim 1, comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:262, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:263, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:264, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

262.

31. The fusion protein of claim 1, comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:262, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:263, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:265, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

262.

32. A polynucleotide encoding a fusion protein according to any one of claims 1 to 31, optionally comprising a nucleotide sequence encoding the IL-21 polypeptide and at least one nucleotide sequence encoding the antibody or antigen-binding fragment thereof.

33. A vector comprising the polynucleotide described in claim 32.

34. A host cell comprising the vector described in claim 33.

35. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 31.

36. The pharmaceutical composition of claim 35 for use in activating a population of CD8+ T cells, wherein the use comprises contacting the population of CD8+ T cells with the pharmaceutical composition.

37. The pharmaceutical composition of claim 35 for use in selective activation of a population of CD8+ T cells, the use comprising contacting a population of cells comprising CD8+ T cells, CD4+ T cells and NK cells with said pharmaceutical composition, optionally wherein said CD8+ T cells are activated with at least 10-fold to 100,000-fold greater potency compared to activation of said NK cells or said CD4+ T cells in said population of cells by said pharmaceutical composition.

38. A fusion protein according to any one of claims 1 to 31 for use as a drug.

39. A fusion protein according to any one of claims 1 to 31 for use in treating cancer or a chronic infection in a subject, the use comprising administering the fusion protein to the subject.

40. A fusion protein according to any one of claims 1 to 31 for use in treating cancer in a subject, the use comprising administering the fusion protein to the subject, optionally further comprising administering to the subject a T cell therapy, a cancer vaccine, a chemotherapeutic agent, or an immune checkpoint inhibitor (ICI), optionally the ICI is an inhibitor of PD-1, PD-L1, or CTLA-4, and optionally the T cell therapy comprises chimeric antigen receptor (CAR)-based T cell therapy, tumor infiltrating lymphocyte (TIL)-based therapy, or therapy using T cells with a transduced TCR.

41. A kit comprising a container containing a fusion protein according to any one of claims 1 to 31 and optionally a pharma- ceutically acceptable carrier, and instructions for administering the fusion protein to a subject having a disease, wherein optionally the disease is cancer or a chronic infectious disease.

42. A method for producing a fusion protein according to any one of claims 1 to 31, comprising culturing a host cell according to claim 34, and optionally further comprising harvesting the fusion protein from the host cell.

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