IL-21 Polypeptides And Targeted Constructs

Abstract

The present invention relates to a fusion protein comprising: an interleukin-21 (IL-21) polypeptide, and an antibody or antigen binding fragment that binds at least one of CD8a, CD8aa or CD8aß having

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to: US Provisional Application No. 63 / 190,669, filed May 19, 2021; US Provisional Application No. 63 / 223,684, filed July 20, 2021; PCT / US2021 / 056312, filed October 22, 2021; PCT / US2021 / 062485, filed December 8, 2021; and US Provisional Application No. 63 / 297,631, filed January 7, 2022. The entire contents of each application is incorporated herein by this reference. BACKGROUND

[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 augment T cell survival and effector function. Interleukin-21 is a type I cytokine and a member of the common cytokine receptor gamma-chain (yc) family that has emerged as a promising immune therapeutic for the treatment of cancer. In some cases, IL-21 that is produced by activated CD4+ T cells and natural killer T (NKT) cells signals via a heterodimeric receptor complex comprised of a discrete IL-21 receptor (IL-21R) subunit together with the yc. In some cases, activation of the IL-21R complex leads to the activation of the JAK / STAT signaling pathway. IL-21R is broadly 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 (Thl7) as well as follicular helper T cells (Tfh) and can antagonize regulatory T cell (Treg) differentiation. Additionally, IL-21 can augment the survival of CD8+ T cells resulting in a less activated but more persistent T cell phenotype that leads to enhanced tumor and viral control. A challenge of cytokine immunotherapy is that in some cases, while activating immune cells to potentiate immune responses, the same cytokine can also activate counter-regulatory pathways as exemplified by IL-2 and IFNy. These counter-regulatory pathways can activate regulatory T cell responses and inhibitory pathways. Because it plays a key role in anti-turn or and anti-viral responses, in addition to exerting major effects on inflammatory responses that lead to the development of autoimmune diseases and inflammatory diseases, IL-21 has been an attractive target for several therapies.

[0003] There remains a need for treatment modalities utilizing IL-21, including modalities combining IL-21 moi eties that direct IL-21 to specific cell types. SUMMARY

[0004] Provided herein, in one embodiment of the disclosure is an IL-21 polypeptide or a functional fragment or a 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, compared to that of a wild-type IL-21 protein having a 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 an improved exposure compared to the wildtype IL-21 protein, as measured by at least about 1.5 times greater under the curve (AUC) for the polypeptide, relative to that of the wild-type IL-21, when administered to a subject, at equivalent concentrations. 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 relative to the human IL-21 polypeptide without the amino acid substitution. In some embodiments, the IL-21 polypeptide comprises at least four amino acid substitutions that reduce the isoelectric point of the IL-21 polypeptide about 0.6 units to about 5 units relative to the human IL-21 polypeptide without the amino acid substitutions. In some embodiments, the IL-21 polypeptide comprises up to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions that reduce the isoelectric point.

[0005] In some embodiments, the disclosure provides an IL-21 polypeptide or a functional fragment or variant thereof, wherein relative to a region of a human IL-21 polypeptide that comprises about 2 to 20 positively charged amino acid residues, the IL-21 polypeptide comprises at least one amino acid substitution of the about 2 to 20 positively charged amino acid residues. In some embodiments, the region of the human IL-21 polypeptide does not include amino acid residues that bind a human IL-21 receptor. In some embodiments, the region of the human IL-21 polypeptide comprises amino acid residues S80 to T92 of the human IL-21 polypeptide comprising SEQ ID NO: 1. In some embodiments, the disclosure provides an IL-21 polypeptide or a functional fragment or variant thereof, wherein a human IL-21 polypeptide comprises at least one positively charged amino acid residue on the surface of the human IL-21 polypeptide in a three-dimensional structure of the human IL-21 polypeptide which does not directly interact with or bind a human IL-21 receptor, and wherein 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 disclosure provides an IL-21 polypeptide that does not comprise an amino acid substitution at G84 of the 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, the IL-21 polypeptide comprises (a) R85G, R86G, K88G and R90E, or (b) S80G, T81G, N82E, A83G, R85G, R86G, Q87G, K88G, H89G, R90E, L91G, and T92G. In some embodiments, the disclosure provides an IL-21 polypeptide that does not comprise an amino acid substitution at G84 of the human IL-21 polypeptide comprising SEQ ID NO: 1.

[0007] In some embodiments, the disclosure provides an IL-21 polypeptide or functional fragment or variant thereof comprising the amino acid sequence: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANT GNNERIINVSIKKLKRKPPXiX2X3X4GX5X6X7X8X9XioXiiXi2CPSCDSYEKKPPKEFLERFKSL LQKMIHQHLSSRTHGSEDS (SEQ ID NO: 380), wherein Xi = G, S; X2 = G, T; X3 = G, E, N; X4 = G, S, E, A; X5= G, E, S, R; Xe= G, E, R; X7 = S, G, E, Q; X8= G, K; X9= G, S, H; Xio = A, E, S, G, R; Xu = S, G, L; and X12 = G, S, T, provided at least one of X5, Xe, X8, X9, and X10 is not the amino acid residue at the identical position set forth in SEQ ID NO: 1, optionally wherein: (i) X5 = G, Xe = G, X8 = G, X10 = A; (ii) X5 = G, X6 = G, X8 = G, Xw = E; (iii) Xi = G, X2= G, X3 = G, X4= S, X5 = E, X6= G, X7 = S, X8= G, X9= G, Xio = S; (iv) Xi = G, X2= G, X3 = G, X4= S, X5= G, X6= G, X7= S, X8= G, X9= G, Xio = E; (v) X5= G, Xe= G, X7= S, X8= G, X9= G, Xio=E; (vi) X5= G, Xe= G, X7= S, X8= G, X9= G, Xio = S; (vii) X5 = G, X6= G, X7= G, X8= G, X9= G, Xio= E; (viii) X5 = G, Xe = G, X7 = G, X8 = G, X9 = G, X10 = G; (ix) X5= G, Xe= G, X7= E, X8= G, X9= G, Xio= G; (x) Xi = G, X2= G, X3 = G, X4= S, X5= G, X6 = G, X7= S, X8= G, X9= G, Xio = G, Xn = S, Xi2=G; (xi) Xi = G, X2= G, X3 = G, X4= G, X5= G, X6= G, X7 = G, X8= G, X9= G, Xw = G, Xn = G, Xn=G; (xii) Xi = G, X2= G, X3 = G, X4= S, X5= G, Xe = G, X7= E, X8= G, X9= G, Xw= G, Xn = S, Xi2=G; (xiii) Xi = G, X2 = G, X3 = G, X4 = E, X5 = G, X6 = G, X7 = E, X8 = G, X9 = G, Xw = G, Xu = S, Xi2=G; (xiv) X3= G, X4= G, X5= S, X6= G, X7= G, X8= G, X9 = S, Xw = G, Xn = G, Xi2= S; (xv) X3 = G, X4= G, X5 = E, X6= G, X7= G, X8= G, X9 = S, Xw= G, Xn = G, Xi2= S; (xvi) Xi = G, X2= G, X3 = G, X4= G, X5 = E, Xe= G, X7= G, X8= G, X9= G, Xw = G, Xu = G, Xi2=G; (xvii) Xi = G, X2= G, X3 = G, X4= S, X5= G, Xe = G, X7= S, X8= G, X9= G, Xw=E, Xu = G, Xi2=G; (xviii) Xi = G, X2 = G, X3 = G, X4 = G, X5 = G, X6 = G, X7 = G, X8 = G, X9 = G, Xw = E, Xu = G, Xi2=G; (xix) X3= G, X4= G, X5= S, X6= G, X7= G, X8= G, X9 = S, Xw = E, Xn = G, Xi2= S; (xx) X3 = G, X4= G, X5 = G, X6= G, X7= G, X8= G, X9= G, Xw = E, Xn= G, Xi2= G; (xxi) X5 = G, Xe = G, X7 = G, X8 = G, X9 = G, Xw = E, Xu = G; (xxii) X3 = G, X4 = G, X5 = G, X6 = G, X7 = G, X8 = G, X9 = G, Xw = E, Xu = G; (xxiii) X3 = G, X4 = G, X5 = G, X6 = E, X7 = G, X8 = G, X9 = G, Xw = E, Xu = G; (xxiv) X3 = G, X4 = G, X5 = E, X6 = G, X7 = G, X8 = G, X9 = G, Xw = E, Xi i = G; (xxv) Xi = G, X2= G, X3 = G, X4 = E, X5= G, Xe= G, X7= G, X8= G, X9= G, Xw = E, Xu = G, Xi2 = G; or (xxvi) Xi = G, X2= G, X3 = E, X4= G, X5= G, Xe= G, X7= G, X8= G, X9= G, Xw = E, Xu = G, Xi2 = G. In some embodiments, the disclosure provides an IL-21 polypeptide that does not comprise an amino acid substitution at G84 of the human IL-21 polypeptide comprising SEQ ID NO: 1.

[0008] In some embodiments, the 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 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 the human IL-21 polypeptide. In some embodiments, the at least one amino acid substitution is at one or more amino acid residues at positions R5,18, R9, Rll, L13,114,116, V17, D18, K72, K73, L74, K75, R76, K77, or KI 17, of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is selected from: R5F, R5A, R5E, R5S, R5T, R5N, R5Q, R5V, R5I, R5L, R5Y, I8E, R9A, R9D, R9E, R9H, R9S, R9T, R9N, R9G, R9V, R9I, R9L, R9Y, RI ID, RI IE, L13F, L13R, I14D, I16A, Il6S, I16R, V17I, VI7A, 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 KI 17A. 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, Rll, Q12,114, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, QI 16, and KI 17 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, 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: 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 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, 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 a sequence selected from SEQ ID NOs: 16-21, 41-98 and 374-379. 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 a sequence selected from SEQ ID NOs: 16-21, 41-93 and 374-379. 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 a sequence selected from SEQ ID NOs: 94-98.

[0010] Provided herein, in one embodiment of the disclosure, is a targeted cytokine construct comprising an IL-21 polypeptide or a functional fragment or a variant thereof according to the present disclosure and an antibody or an antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment thereof specifically binds to a CD8+ T cell. In some embodiments, the antibody or antigen binding fragment thereof specifically binds to at least one of: CD8a, CD8aa, or CD8ap. In some embodiments, the antibody or antigen binding fragment thereof specifically binds to CD8p.

[0011] Provided herein, in one embodiment of the disclosure, is a targeted cytokine construct comprising: a) an IL-21 polypeptide or a functional fragment or a variant thereof comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1; and b) an antibody or an antigen binding fragment thereof that specifically binds to at least one of CD8a, CD8aa, or CD8ap.

[0012] Provided herein, in one embodiment of the disclosure, is a targeted cytokine construct comprising: a) an IL-21 polypeptide or a functional fragment or a variant thereof comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1; and b) an antibody or an antigen binding fragment thereof that specifically binds to CD8p.

[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: Lin some embodiments, the IL-21 polypeptide comprises mutation at a position selected from the group consisting of: R5,18, R9, Rll, Q12,114, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, QI 16, and KI 17 of SEQ ID NO: 1. In some embodiments, the IL-21 polypeptide or a functional fragment or a 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 a functional fragment or a 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 as compared to activation of NK or CD4+ T cells. In some embodiments, the construct activates CD8+ T cells with a potency that is at least about 10-fold to about 100,000-fold greater as compared to activation of NK or CD4+ T cells. In some embodiments, the antibody or antigen binding fragment thereof comprises: i) a first polypeptide, arranged from N-to-C terminus, comprising: a variable light chain (VL) amino acid sequence, and a light chain constant region amino acid sequence (CL1); ii) a second polypeptide, arranged from N-to-C terminus, comprising: a variable heavy chain (VH) amino acid sequence, a heavy chain CHI 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; iii) a third polypeptide, arranged from N-to-C terminus, comprising: 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, arranged from N-to-C terminus, comprises: a variable heavy chain (VH) amino acid sequence, a heavy chain CHI constant region amino acid sequence, a hinge region amino acid sequence, a heavy chain CH2 constant region amino acid sequence, a heavy chain CH3 constant region amino acid sequence, wherein the antibody or antigen binding fragment thereof further comprises: iv) a fourth polypeptide, arranged from N-to-C terminus, comprising: 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 a functional fragment or a variant thereof and the antibody or antigen binding fragment thereof are operably linked to each other. In some embodiments, the IL-21 polypeptide or a functional fragment or a 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 a functional fragment or variant thereof is conjugated to the C-terminus of the second or the third polypeptide. In some embodiments, the targeted cytokine construct comprises at least one molecule of the IL-21 polypeptide. In some embodiments, the Fc domain is a human IgG Fc domain. In some embodiments, the Fc domain is an IgGl, 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 the CH3 domain, and the third polypeptide comprises a hole modification in the CH2 or the CH3 domain; or wherein the third polypeptide comprises a knob modification in the CH2 or the CH3 domain and the second polypeptide comprises a hole modification in the CH2 or the CH3. In some embodiments, at least one of the second and the third polypeptide comprises the following mutations: L234A, L235A, and G237A, numbering according to the EU index.

[0015] Provided herein, in one embodiments of the disclosure, is a targeted cytokine construct comprising: a) an IL-21 polypeptide or a functional fragment or variant thereof comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1; and b) an antibody or an antigen binding fragment thereof comprising a first antigen binding arm and a second antigen binding arm, wherein the first and the second antigen binding arms bind to two different antigens, wherein at least one of the first and the second antigen binding arm specifically binds to CD8a, CD8aa, CD8ap.

[0016] Provided herein, in one embodiments of the disclosure, is a targeted cytokine construct comprising: a) an IL-21 polypeptide or a functional fragment or variant thereof comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1; and b) an antibody or an antigen binding fragment thereof comprising a first antigen binding arm and a second antigen binding arm, wherein the first and the second antigen binding arms bind to two different antigens, wherein at least one of the first and the second antigen binding arm specifically binds to CD8p. In some embodiments, the targeted cytokine construct activates CD8+ T cells with at least about 10-fold or greater potency as compared to activation of NK cells or CD4+ T cells. In some embodiments, targeted cytokine construct activates CD8+ T cells a potency that is at least about 10-fold to about 100,000-fold greater as compared to activation of NK cells or CD4+ T cells. In some embodiments, the IL-21 polypeptide or a functional fragment thereof or a 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 a functional fragment thereof or a variant thereof comprises a mutation at a position selected from the group consisting of: R5,18, R9, Rll, Q12,114, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, QI 16, and KI 17 of SEQ ID NO: 1. In some embodiments, the IL-21 polypeptide or a functional fragment or a 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 a functional fragment or a 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 disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising (i) one or more amino acid substitutions providing a reduced isoelectric point relative 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, wherein 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide; and b) an antibody or an antigen binding fragment thereof that specifically binds to CD8aP or CD8^. In some embodiments, the IL-21 polypeptide does not comprise a G84 substitution. In some embodiments, the at least one amino acid substitution providing reduced binding is R76E or R76Q.

[0018] In some embodiments, the disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising (i) four or more amino acid substitutions providing a reduced isoelectric point relative 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, 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide; and b) an antibody or an antigen binding fragment thereof that specifically binds to CD8aP or CD8£.

[0019] In some embodiments, the disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising (i) four or more amino acid substitutions providing a reduced isoelectric point relative 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, 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide, wherein the at least one amino acid substitution is at a residue selected from R5,18, R9, Rll, L13,114,116, V17, D18, K72, K73, L74, K75, R76, K77, KI 17, and a combination thereof; and b) an antibody or an antigen binding fragment thereof that specifically binds to CD8aP or CD8£.

[0020] In some embodiments, the disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising (i) four or more amino acid substitutions providing a reduced isoelectric point relative 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, 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 selected from: (a) RI ID; (b) RI IE; (c) I14D, D18A and KI 17A; (d) R76E; (e) R5F; (f) R76F; (g) I8E; (h) R5A; (i) R5E; (j) R5S; (k) R5T; (1) 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; (11) R5F and K72A; (mm) R5F and K72E; (nn) R5F and K76A; (oo) R5F and K76E; (pp) K73 A 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) Il6S, V17I, and L74V; (111) I16R, V17I, and L74I; (mmm) L13F, I16A, V17A and L74M; and (nnn) L13R, I16A, V17I and L74I; and b) an antibody or an antigen binding fragment thereof that specifically binds to CD8aP or CD8£.

[0021] In some embodiments, the disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising (i) four or more amino acid substitutions providing a reduced isoelectric point relative to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the four or more 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide, wherein the at least one amino acid substitution is at a residue selected from R5,18, R9, Rll, L13,114,116, V17, D18, K72, K73, L74, K75, R76, K77, KI 17, and a combination thereof; and b) an antibody or an antigen binding fragment thereof that specifically binds to CD8aP or CD8£.

[0022] In some embodiments, the disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising (i) four or more amino acid substitutions providing a reduced isoelectric point relative to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the four or more 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 providing reduced binding to a human IL-21 receptor relative 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 an antigen binding fragment thereof that specifically binds to CD8aP or CD8£.

[0023] In some embodiments, the 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide; and b) an antibody or an antigen binding fragment thereof that specifically binds to CD8aP or CD8£.

[0024] In some embodiments, the 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide, wherein the at least one amino acid substitution is at a residue selected from R5,18, R9, RI 1, L13,114,116, V17, D18, K72, K73, L74, K75, R76, K77, KI 17, and a combination thereof; and b) an antibody or an antigen binding fragment thereof that specifically binds to CD8aP or CD8£.

[0025] In some embodiments, the 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 providing reduced binding to a human IL-21 receptor relative 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 an antigen binding fragment thereof that specifically binds to CD8aP or CD8£.

[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 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, and wherein the HC constant domain comprises a knob modification; c) a third polypeptide comprising a VH chain and a HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the VH chains and VL chains comprise CDRs that specifically bind to CD8aP or CD8£, wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions providing a reduced isoelectric point relative 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, wherein 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0028] In some embodiments, the 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 a HC constant domain, wherein the HC constant domain comprises a knob modification; c) a third polypeptide comprising a VH chain and a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the VH chains and VL chains comprise CDRs that specifically bind to CD8aP or CD8£, wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions providing a reduced isoelectric point relative 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, wherein 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

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

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

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

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

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

[0034] Provided herein, in one embodiment of the 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 disclosure, is a method for selective activation of CD8+ T cells, wherein the method comprises 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, as 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 a potency that is at least about 10-fold to about 100,000-fold greater as 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 of the CD8+ T cells compared to the STAT3 phosphorylation of the NK cells or CD4+ T cells in the population of cells.

[0036] Provided herein, in one embodiment of the disclosure, is a method of treating a disease in a subject, the method comprising administering an IL-21 polypeptide or a functional fragment or a 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 a cancer or a chronic infection. In some embodiments, the disease comprises the cancer and wherein the cancer is acute lymphoblastic leukemia (ALL) (including non T cell ALL), acute myeloid leukemia, B cell prolymphocytic leukemia, B cell acute lymphoid 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 syndrome, non-Hodgkin's lymphoma (NHL), plasma cell proliferative disorder (including asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytomas (including plasma cell dyscrasia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukase syndrome; Takatsuki disease; and PEP syndrome), primary mediastinal large B cell lymphoma (PMBC), small cell- or a large cell-follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T cell acute lymphoid leukemia (“TALL”), T cell lymphoma, transformed follicular lymphoma, or Waldenstrom 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 cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, head and neck cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, thyroid cancer, uterine cancer,, gastrointestinal cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, endometrial carcinoma, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the cervix, carcinoma of the vagina, vulval cancer, 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, carcinoma of the renal pelvis, mesothelioma, bladder cancer, liver cancer, hepatoma, hepatocellular cancer, cervical cancer, salivary gland carcinoma, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, 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; and wherein: (a) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 137, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 138, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 139; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 140, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 141, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; (b) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 149, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 150, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:151; and wherein 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; (c) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 155, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 156, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 157; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 158, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 159, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 160; (d) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 161, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 162, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 163; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 164, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 165, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 166; (e) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 167, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 168, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 169; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 170, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 171, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 172; (f) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 173, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175; and wherein the VL domain 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; (g) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 179, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 180, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 181; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 182, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 183, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 184; (h) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 144, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147, and a 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, wherein XI is S, K, G, N, R, D, T, or G, and wherein X2 is Y, L, H, or F (SEQ ID NO: 185), a CDR-H2 comprising the amino acid sequence of X1X2X3PX4X5X6X7X8X9YX10QKFX11G, wherein XI is G or H, X2 is I or F, X3 is I, N, or M, X4 is G, N, H, S, R, I, or A, X5 is A, N, H, S, T, F, or Y, X6 is A, D, or G, X7 is T, E, K, V, Q, or A, X8 is A or T, X9 is N or K, X10 is A or N, and Xll is Q or T (SEQ ID NO: 186), and a CDR-H3 comprising the amino acid sequence of X1X2X3GX4X5LFX6X7, wherein XI 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: 187); and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of X1X2SX3X4IX5GX6LN, wherein XI 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), a CDR-L2 comprising the amino acid sequence of GX1X2X3LX4X5, wherein XI is A or S, X2 is T, S, E, Q, or D, X3 is N, R, A, E, or H, X4 is Q or A, and X5 is S or D (SEQ ID NO: 189), and a CDR-L3 comprising the amino acid sequence of QX1X2X3X4X5PWT, wherein XI 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 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:200, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:201; and wherein 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:202; (k) the VH domain 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; and wherein the VL domain 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; (1) the VH domain 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; and wherein 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:202; (m) the VH domain comprises a CDR-H1 comprising the amino acid sequence of X1YX2MS, wherein XI is S, D, E, A, or Q and X2 is A, G, or T (SEQ ID NO:208), a CDR-H2 comprising the amino acid sequence of DIX1X2X3GX4X5TX6YADSVKG, wherein XI is T, N, S, Q, E, H, R, or A, X2 is Y, W, F, or H, X3 is A, S, Q, E, or T, X4 is G or E, X5 is S or I, and X6 is A or G (SEQ ID NO:209), and a CDR-H3 comprising the amino acid sequence of X1X2X3YX4WX5X6AX7DX8, wherein XI 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); and wherein 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: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 wherein 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:220, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:260, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:222; and wherein 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 a CDR-H1 comprising the amino acid sequence of SEQ ID NO:252, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:253, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:254; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:255, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:256, and a 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 wherein: (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 wherein 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 a CDR-H1 comprising the amino acid sequence of SEQ ID NO:228, 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: 157; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 158, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 159, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 160; (c) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:223, 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: 163; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 164, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 165, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 166; (d) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:229, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:227, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 169; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 170, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 171, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 172; (e) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:230, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:231, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175; and wherein the VL domain 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; (f) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:230, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:232, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 181; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 182, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 183, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 184; (g) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:223, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:224, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:225; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 140, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 141, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; (h) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:233, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:234, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147, and a 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 GX1X2FX3X4X5, wherein XI is G, Y, S, or A, X2 is T, S, G, R, N, or H, X3 is S, T, R, H, Y, G, or P, X4 is S, K, G, N, R, D, T, or G, and X5 is Y, L, H, or F (SEQ ID NO:235), a CDR-H2 comprising the amino acid sequence of X1PX2X3X4X5, wherein XI is I, N, or M, X2 is G, N, H, S, R, I, or A, X3 is A, N, H, S, T, F, or Y, X4 is A, D, or G, and X5 is T, E, K, V, Q, or A (SEQ ID NO:236), and a CDR-H3 comprising the amino acid sequence of X1X2X3GX4X5LFX6X7, wherein XI 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); and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of X1X2SX3X4IX5GX6LN, wherein XI 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), a CDR-L2 comprising the amino acid sequence of GX1X2X3LX4X5, wherein XI is A or S, X2 is T, S, E, Q, or D, X3 is N, R, A, E, or H, X4 is Q or A, and X5 is S or D (SEQ ID NO: 189), and a CDR-L3 comprising the amino acid sequence of QX1X2X3X4X5PWT, wherein XI 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 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:242, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:204; and wherein 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:202; (k) the VH domain 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; and wherein the VL domain 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; (1) the VH domain 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; and wherein 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:202; (m) the VH domain comprises a CDR-H1 comprising the amino acid sequence of GFTFX1X2Y, wherein XI is S, D, E, Q, S, or A and X2 is S, D, E, A, or Q (SEQ ID NO:244), a CDR-H2 comprising the amino acid sequence of X1X2X3GX4X5, wherein XI 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), and a CDR-H3 comprising the amino acid sequence of X1X2X3 YX4WX5X6AX7DX8, wherein XI 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:246); and wherein 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 wherein 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 wherein 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 a CDR-H1 comprising the amino acid sequence of SEQ ID NO:223, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:224, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:284; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:285, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:286, and a 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 109, and wherein 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: 110; (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:111, and wherein 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: 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 wherein 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, and wherein 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: 116; (e) 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: 117, and wherein 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: 118; (f) 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: 119, and wherein 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: 120; (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 wherein 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 129, and wherein 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; (i) 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: 131; and wherein 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: 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; and wherein 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, and wherein 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; (1) 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: 133; and wherein 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: 134; (m) 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: 135; and wherein 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: 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 wherein 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 121; and wherein 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 that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:258; and wherein 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:259.

[0040] In some embodiments, (a) the VH domain 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; and wherein the VL domain 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) the VH domain 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; and wherein the VL domain 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; (c) the VH domain 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; and wherein the VL domain 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; or (d) 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 wherein the VL domain 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.

[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 wherein 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 wherein 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 disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising (i) four or more amino acid substitutions providing a reduced isoelectric point relative 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, 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide; and b) an antibody or an antigen binding fragment thereof that specifically binds to CD8aP or CD8 / ?, wherein the antibody or antigen binding fragment thereof comprises: (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 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; (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 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 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.

[0043] In some embodiments, the disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising (i) four or more amino acid substitutions providing a reduced isoelectric point relative 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, 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide, wherein the at least one amino acid substitution is at a residue selected from R5,18, R9, Rll, L13,114,116, V17, D18, K72, K73, L74, K75, R76, K77, KI 17, and a combination thereof; and b) an antibody or an antigen binding fragment thereof that specifically binds to CD8aP or CD8 / ?, wherein the antibody or antigen binding fragment thereof comprises: (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 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; (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 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 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.

[0044] In some embodiments, the disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising (i) four or more amino acid substitutions providing a reduced isoelectric point relative 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, 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 selected from: (a) RI ID; (b) RI IE; (c) I14D, D18A and KI 17A; (d) R76E; (e) R5F; (f) R76F; (g) I8E; (h) R5A; (i) R5E; (j) R5S; (k) R5T; (1) 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; (11) R5F and K72A; (mm) R5F and K72E; (nn) R5F and K76A; (oo) R5F and K76E; (pp) K73 A 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; (fiff) K75A and R76F; (ggg) R76F and K77A; (hhh) K75E and R76F; (iii) V17I and L74I; (jjj) I16A and L74F; (kkk) I16S, V17I, and L74V; (111) I16R, V17I, and L74I; (mmm) L13F, I16A, V17A and L74M; and (nnn) L13R, I16A, V17I and L74I; and b) an antibody or an antigen binding fragment thereof that specifically binds to CD8aP or CD8 / ?, wherein the antibody or antigen binding fragment thereof comprises: (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 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; (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 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 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.

[0045] In some embodiments, the disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising (i) four or more amino acid substitutions providing a reduced isoelectric point relative to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the four or more 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide, wherein the at least one amino acid substitution is at a residue selected from R5,18, R9, Rll, L13,114,116, V17, D18, K72, K73, L74, K75, R76, K77, KI 17, and a combination thereof; and b) an antibody or an antigen binding fragment thereof that specifically binds to CD8aP or CD8 / ?, wherein the antibody or antigen binding fragment thereof comprises: (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 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; (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 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 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.

[0046] In some embodiments, the disclosure provides a fusion protein comprising: a) an IL-21 polypeptide comprising (i) four or more amino acid substitutions providing a reduced isoelectric point relative to a human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the four or more 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 providing reduced binding to a human IL-21 receptor relative 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 an antigen binding fragment thereof that specifically binds to CD8aP or CD8£.

[0047] In some embodiments, the 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide; and b) an antibody or an antigen binding fragment thereof that specifically binds to CD8aP or CD8 / ?, wherein the antibody or antigen binding fragment thereof comprises: (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 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; (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 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 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.

[0048] In some embodiments, the 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide, wherein the at least one amino acid substitution is at a residue selected from R5,18, R9, RI 1, L13,114,116, V17, D18, K72, K73, L74, K75, R76, K77, KI 17, and a combination thereof; and b) an antibody or an antigen binding fragment thereof that specifically binds to CD8aP or CD8 / ?, wherein the antibody or antigen binding fragment thereof comprises: (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 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; (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 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 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.

[0049] In some embodiments, the 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 providing reduced binding to a human IL-21 receptor relative 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 an antigen binding fragment thereof that specifically binds to CD8aP or CD8 / ?, wherein the antibody or antigen binding fragment thereof comprises: (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 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; (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 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 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.

[0050] In some embodiments, the 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the 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 a HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions providing a reduced isoelectric point relative 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, wherein 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0051] In some embodiments, the 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 a 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions providing a reduced isoelectric point relative 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, wherein 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0052] In some embodiments, the 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the 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 a HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions providing a reduced isoelectric point relative 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, wherein 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0053] In some embodiments, the 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 a 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions providing a reduced isoelectric point relative 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, wherein 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0054] In some embodiments, the 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the 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 a HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0055] In some embodiments, the 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 a 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0056] In some embodiments, the 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the 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 a HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0057] In some embodiments, the 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 a 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0058] In some embodiments, the 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the 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 a HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16-21, 41-98, and 374-379.

[0059] In some embodiments, the 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 a 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16-21, 41-98, and 374-379.

[0060] In some embodiments, the 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the 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 a HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16-21, 41-98, and 374-379.

[0061] In some embodiments, the 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 a 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16-21, 41-98, and 374-379.

[0062] In some embodiments, the 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the 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 a HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions providing a reduced isoelectric point relative 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, wherein 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0063] In some embodiments, the 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 a 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions providing a reduced isoelectric point relative 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, wherein 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0064] In some embodiments, the 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the 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 a HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions providing a reduced isoelectric point relative 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, wherein 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0065] In some embodiments, the 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 a 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises (i) one or more amino acid substitutions providing a reduced isoelectric point relative 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, wherein 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0066] In some embodiments, the 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the 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 a HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0067] In some embodiments, the 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 a 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0068] In some embodiments, the 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the 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 a HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0069] In some embodiments, the 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 a 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and 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 providing reduced binding to a human IL-21 receptor relative to binding by the human IL-21 polypeptide.

[0070] In some embodiments, the 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the 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 a HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16-21, 41-98, and 374-379.

[0071] In some embodiments, the 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 a 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the VH chains and VL chains comprise CDRs that specifically bind to CD8aP or CD8£, wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16-21, 41-98, and 374-379.

[0072] In some embodiments, the 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the 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 a HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16-21, 41-98, and 374-379.

[0073] In some embodiments, the 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 a 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 a HC constant domain, wherein an IL-21 polypeptide is operably linked to the C-terminus of the HC constant domain, wherein the HC constant domain comprises a hole modification, and wherein the VH chain 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 chain of the first polypeptide and the fourth polypeptide are the same, and wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16-21, 41-98, and 374-379.

[0074] In some embodiments, the fusion protein comprises four polypeptide chains, 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; 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 polypeptide chains, 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; 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 polypeptide chains, wherein: (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 polypeptide chains, wherein: (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. INCORPORATION BY REFERENCE

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

[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:

[0080] FIGS. 1A-1C show the amino acid sequences of the following polypeptides: mature IL-21 (SEQ ID NO: 1) (FIG. 1A), IL-21R (SEQ ID NO: 381) (FIG. IB), and the common gamma chain (SEQ ID NO: 382) (FIG. IC).

[0081] FIG. 2 shows the amino acid sequences of the mature wild-type IL-21 polypeptide (SEQ ID NO: 1) and the modifications to generate charge variants. “X” denotes the amino acid position substituted in the sequence of wild-type IL-21 polypeptide to generate charge variants.

[0082] FIGS. 3A -3F show ion exchange chromatography traces of fusion proteins of anti-CD8 antibodies 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 vl through v6 are shown in FIG. 3A, as measured by HPLC. Fusion proteins of xhCD8.1 and exemplary IL-21 charge variants v2, or variants v7 through v31 are shown in FIGS. 3B through 3E, and fusion protein of xhCD8.1 and wild type IL-21 is shown in FIG. 3F, as measured by FPLC. In all cases, the peak(s) on the left are undesired products, primarily homodimer, whereas the peak on the right is the desired heterodimer fusion protein, and the percentages of total of each denoted region are shown.

[0083] FIGS. 4A-4D show mouse pharmacokinetic data for fusion proteins. Serum concentrations are shown for Img / kg of a control antibody (xCtrl) with either the wild-type IL-21 or exemplary charge variant IL-21 polypeptide IL-21 v2 dosed either intravenously (FIG. 4A) or subcutaneously (FIG. 4B). In a separate experiment, serum concentrations are additionally shown for Img / kg wild type IL-21, or exemplary charge variant IL-21 polypeptides, IL-21 v2 or IL-21 v31 dosed subcutaneously (FIG. 4C) or intravenously (FIG. 4D).

[0084] FIGS. 5A-5B show the 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) (FIG. 5A) or from whole blood (FIG. 5B). STAT3 activation was measured by flow cytometry, and all cell types were potently activated by wild-type IL-21.

[0085] FIG. 6 is a schematic showing the general mechanism for how targeted fusions of mutant IL-21 polypeptides with CD8 antigen binding molecules and untargeted fusions with mutant IL-21 polypeptides work to stimulate cells expressing or not expressing CD8 antigens.

[0086] FIG. 7 shows activation of STAT3 in human CD8+ T cells by fusion proteins of anti CD8 antibody with either wild-type IL-21, exemplary IL-21 polypeptide charge variants IL-21 vl, IL-21 v2 or IL-21 v6.

[0087] FIGS. 8A-8F show activation of STAT3 in human PBMCs by fusion proteins of an anti-human CD8 antibody, xhCD8, with various attenuated versions of IL-21 v2. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells and B cells. Fusion proteins of xhCD8 with exemplary IL-21 polypeptides comprising attenuation mutations, IL-21 v2.1 (FIG. 8A), IL-21 v2.2 (FIG. 8B), IL-21 v2.3 (FIG. 8C), IL-21 v2.4 (FIG. 8D), IL-21 v2.5 (FIG. 8E) and IL-21 v2.6 (FIG. 8F), all show preferential STAT3 activation of CD8+ T cells over NK, B and CD4+ T cells, as measured by ECso as well as maximal STAT3 activation.

[0088] FIGS. 9A-9H show activation of STAT3 in human PBMCs by fusion proteins of an anti-human CD8 antibody, xhCD8.1, with additional attenuated versions of IL-21 v2 or untargeted Fc fusion to IL-21 v2 as a control. STAT3 activation is shown in CD8+ T cells in FIGS. 9A, 9C, 9E and 9G, and in CD4+ T cells in FIGS. 9B, 9D, 9F, and 9H.

[0089] FIGS. 10A-10F show activation of STAT3 in human whole blood by fusion proteins of an anti-human CD8 antibody, xhCD8.1, with various attenuated versions of IL-21 v31. 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 comprising attenuation mutations, IL-21 v31.4 (FIG. 10A), IL-21 v31.6 (FIG. 10B), IL-21 v31.23 (FIG. 10C), IL-21 v31.48 (FIG. 10D), and IL-21 v31.51 (FIG. 10E), all show preferential STAT3 activation of CD8+ T cells over NK, B and CD4+ T cells, as measured by ECso as well as maximal STAT3 activation. In the case of xhCD8.1-hIL21 v31.6, data from two separate experiments are shown (FIG. 10B). Untargeted, unattenuated Fc-hIL21 v2 is shown in FIG. 10F, with activation observed in all cell types shown.

[0090] FIGS. 11A-11C show activation of STAT3 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 (FIG. 11 A); fusion protein of untargeted control antibody, xCtrl, and mouse IL-21 charge variant mIL-21 vl (FIG. 11B); and fusion protein of antimouse CD8 antibody, xmCD8, and attenuated mouse IL-21 charge variant, mIL21 vl.l (FIG. 11C) For both recombinant wild type mouse IL-21 and xCtrl-mIL21 vl, all cell types are activated, whereas for xmCD8-mIL21 vl.l, there is preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells, as measured by as measured by EC50 as well as maximal STAT3 activation.

[0091] FIG. 12 shows tumor growth curves in an MC38 syngeneic tumor model treated with a single dose of either PBS; 0.3 or 1 mg / kg (mpk) of xCtrl-mIL21 vl, a fusion protein of untargeted control antibody, xCtrl, and unattenuated mouse IL-21 charge variant, IL-21 vl; or 0.1, 0.3 or Impk of xmCD8-mIL21 vl.l, a fusion protein of anti-mouse CD8 antibody, xmCD8, and attenuated mouse IL-21 charge variant, IL21 vl.l. Each subpanel shows tumor growth curves from each individual animal, the fraction showing complete responses (CR), and an arrow denoting the treatment time.

[0092] FIGS. 13A-13D show the impact of FTY720 on the anti-tumor potency of xmCD8-mIL21 vl.l in an MC38 tumor model. FIG. 13A shows the dosing scheme for FTY720 and xmCD8-mIL21 vl.l, and tumor growth curves for 0.1 mpk (FIG. 13B), 0.3 mpk (FIG. 13C), or 1 mpk (FIG. 13D) xmCD8-mIL21 vl.l. Plotted values are the mean tumor volume and error bars show the standard error of the mean, with each group comprising 8 animals.

[0093] FIGS. 14A-14F show activation of STAT3 in human whole blood from additional donors by fusion proteins of an anti-human CD8 antibody, xhCD8.1, with various attenuated versions of IL-21 v31. 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 comprising attenuation mutations, IL-21 v31.4 (FIG. 14A), IL-21 v31.6 (FIG. 14B), IL-21 v31.23 (FIG. 14C), IL-21 v31.48 (FIG. 14D), and IL-21 v31.51 (FIG. 14E), all show preferential STAT3 activation of CD8+ T cells over NK, B and CD4+ T cells, as measured by EC50. In the case of xhCD8.l-hIL21 v31.4 and xhCD8.1-hIL21 v31.23, data from two different donors analyzed in two separate experiments are shown (FIGS. 14A &14C). Untargeted, unattenuated Fc-hIL21 v31 is shown in FIG. 14F, with activation observed in all cell types shown.

[0094] FIGS. 15A-15D show activation of STAT3 in human whole blood by fusion proteins of an anti-human CD8 antibody, xhCD8vl 1, with various attenuated versions of IL-21 v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8vll with exemplary IL-21 polypeptides comprising attenuation mutations, IL-21 v31.4 (FIG. 15A), IL-21 v31.6 (FIG. 15B), IL-21 v31.23 (FIG. 15C), and IL-21 v31.51 (FIG. 15D), all show preferential STAT3 activation of CD8+ T cells over NK, B and CD4+ T cells, as measured by EC50. In the case of xhCD8vl l-hIL21 v31.23, data from two different donors analyzed in two separate experiments are shown (FIG. 15C).

[0095] FIGS. 16A-16C show activation of STAT3 in human whole blood by fusion proteins of an anti-human CD8 antibody, xhCD8.1, with various versions of IL-21 v2 charge variant that also contained additional mutations. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v.l with exemplary IL-21 polypeptides comprising attenuation mutations, IL-21 v2.63 (FIG. 16A), IL-21 v2.64 (FIG. 16B), and IL-21 v2.65 (FIG. 16C) all show preferential STAT3 activation of CD8+ T cells over NK, B and CD4+ T cells, as measured by EC50.

[0096] FIGS. 17A-17C show ion exchange chromatography traces of fusion proteins of anti-CD8 antibodies and IL-21R attenuated IL-21 charge variants after Protein A purification. Traces are shown for xhCD8vll-hIL21 v31.23 (FIG. 17A), xhCD8.1-hIL21 v31.23 (FIG. 17B), and xhCD8. l-hIL21 v31.4 (FIG. 17C), as measured by FPLC. In all cases, the peak(s) to the left of the major peak are undesired products, primarily homodimer, whereas the major peak in the middle is the desired heterodimer fusion protein, and the percentages of total of the desired peak are shown.

[0097] FIG. 18 shows the pharmacokinetic data for fusion proteins in wild-type C57BL6 mice. Serum concentrations are shown for fusion proteins of anti-human CD8 antibodies, xhCD8.1 or xhCD8vl 1, with either hIL-21 v31.4, hIL-21 v31.23, or hIL-21 v0.4 dosed intravenously. The anti-human CD8 antibodies, xhCD8.1 and xhCD8vl 1 are not cross reactive to mouse CD8. Exposures are shown for xhCD8.1-hIL21 v31.4, xhCD8vl l-hIL21 v31.23, xhCD8.1-hIL21 v31.23 and xhCD8.1-hIL21 v0.4.

[0098] FIGS. 19A &19B show the characterization data of recombinant cytokine variants based on wild-type IL-21 or the IL-21 v31 charge variant. SDS-PAGE of material purified by IMAC followed by preparative SEC is shown in FIG. 19A. Analytical SEC of material purified by IMAC followed by preparative SEC is shown in FIG. 19B. Retention times and peak widths are also reported to evaluate column interactions. In summary, recombinant cytokine variants based on the IL-21 v31 charge variant have lower retention times and narrower peak widths compared to variants based on wild-type IL-21, suggesting fewer column interactions and more favorable biophysical properties with IL-21 v31 based constructs.

[0099] FIG. 20 shows the exposure of recombinant (non-fusion) wildtype human IL-21 and IL-21 v31 charge variant in mouse. IL-21 v31 shows increased in vivo exposure compared to wildtype IL-21.

[0100] FIGS. 21A and 21B each provide a structural schematic showing the interaction between wild-type IL-21 (SEQ ID NO: 1) and IL-21R (SEQ ID NO: 381). Residues S80-T92 of SEQ ID NO: 1 form a largely unstructured, flexible loop (circled). FIG. 21A indicates residues P78 and P79 (Pro-Pro), and C93 and P94 (Cys-Pro) surrounding the flexible loop. FIG. 21B shows the charge of wild-type IL-21 indicating the flexible loop is highly charged.

[0101] FIG. 22 shows the activities of IL-21 fusion molecules in cultures of alloreactive T cells and activated dendritic cells isolated from two different human PBMC donors. Molecules tested include two CD8+ T cell targeted fusions, xhCD8.1-hIL21 v31.4 and xhCD8.1-hIL21 v31.23, and an untargeted, unattenuated Fc-hIL21 v31. IL-2 release in culture was measured to determine the level of alloreactive T cell activation. In summary, Fc-hIL21 v31 suppressed activation of alloreactive T cells, while the CD8+ T cell targeted fusions did not.

[0102] FIGS. 23A-23D show in vitro activation of STAT3 in cynomolgus monkey whole blood by fusion proteins of anti-human CD8 antibodies, xhCD8.1 or xhCD8vl 1, with various attenuated versions of IL-21 v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, and B cells. CD8+ T cell targeted fusion proteins, xhCD8.1-IL-21 v31.4 (FIG. 23A), xhCD8.1-IL-21 v31.23 (FIG. 23B) and xhCD8vl 1-IL-21 v31.23 (FIG. 23C), all show preferential STAT3 activation of CD8+ T cells over B and CD4+ T cells, as measured by ECso. Untargeted, unattenuated Fc-hIL21 v31 is shown in FIG. 23D, with activation observed in all cell types shown.

[0103] FIG. 24 shows in vivo activation of STAT3 in cynomolgus monkey by xhCD8.1-IL-21 v31.4. STAT3 activation levels in CD8+ T cells, CD4+ T cells, NK cells and B cells were assessed 20 min post-dose. Results show that xhCD8.1-IL-21 v31.4 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

[0104] This disclosure, in some embodiments, provides IL-21 polypeptides or functional fragments or variants thereof that comprise improved properties relative to a 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 administering the IL-21 polypeptide or a functional fragment or variant thereof to a subject, relative to that following administration of a wild-type IL-21 protein); (ii) attenuated binding to IL-21 receptor; (iii) reduced cytotoxicity, or any combination thereof.

[0105] As described herein, the present disclosure provides IL-21 polypeptides or functional fragments or variants thereof with reduced charge (i. e., isoelectric point) and reduced binding affinity to human IL-21 receptor relative to a human IL-21 polypeptide (SEQ ID NO: 1). In particular, the disclosure provides IL-21 polypeptides with one or more amino acid substitutions in a positively charged region of located within residues S80-T92 of human IL-21. As shown in FIG. 21B, residues within 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, it is believed that residues P78, P79, C93 and P94 of human IL-21 provide structural rigidity surrounding the loop. The present disclosure provides IL-21 polypeptides having one or more amino acid substitutions within the flexible loop with a reduced isoelectric point and improved bioavailability and biological activity. As described herein, the IL-21 polypeptides of the disclosure having reduced isoelectric points relative to the human IL-21 polypeptide demonstrate increased circulatory half-life and reduced polyreactivity (e.g., with positively charged proteins abundant in circulation). As shown in FIGs. 4A-4D, fusion proteins with IL-21 polypeptides having reduced isoelectric points relative to human IL-21 have improved half-life. The disclosure further provides fusion proteins of IL-21 polypeptides to anti-CD8 antibodies which selectively activate CD8+ T cells over CD4+ T cells, NK cells and B cells, including in vivo in non-human primates (see FIG. 24). Without being bound by theory, fusion proteins of the IL-21 polypeptides with antigen binding domains that specifically bind CD8ab or CD8b selectively activate CD8+ T cells relative to NK cells which express CD8aa. In addition, the IL-21 polypeptides with reduced isoelectric points have lower binding affinities to proteins abundant in circulation, such as heparin and hemoglobin (see Table 5). Further, without being bound by theory, IL-21 polypeptides of disclosure having glycine and / or serine substitutions in the flexible loop may have reduced immunogenicity relative to unmodified human IL-21 following administration to a subject.

[0106] The disclosure also provides IL-21 polypeptides having reduced binding to human IL-21 receptors. As shown herein, attenuated binding of the IL-21 polypeptide of the disclosure to its receptor reduces IL-21 mediated suppressive effects on alloreactive T cells and dendritic cells (see FIG. 22). Without wishing to be bound by theory, attenuated binding of the IL-21 polypeptide of the disclosure to its receptor is expected to avoid the sink effect of binding to B cells and NK cells. Also demonstrated herein is the therapeutic efficacy of fusion proteins comprising the IL-21 polypeptides of the disclosure and anti-CD8 targeting antibodies. Without being bound by theory, linking of the IL-21 polypeptide with attenuated binding to an anti-CD8 targeting antibody provides selective restoration of the attenuated IL-21 polypeptide. Specifically, FIG. 12 shows such fusion proteins provide anti-tumor efficacy in vivo. This efficacy results, at least in part, from peripheral activation of CD8+ T cells as blocking lymphocyte trafficking reduces the curative response (see FIGs. 13A-13D). Without being bound by theory, T cells in the tumor microenvironment may be dysfunctional and express inhibitory molecules, and thus peripheral activation of CD8+ T cells by the fusion protein provides the anti-tumor immune response. Accordingly, the present disclosure provides novel IL-21 polypeptides and fusion proteins comprising the IL-21 polypeptides having improved biological properties, as well as methods of using such polypeptides to treat diseases in subjects.

[0107] Various embodiments of the present disclosure provide methods and compositions involving 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 a wild-type IL-21 polypeptide sequence) that are directed or targeted to activate specific cell subsets (e.g., CD8+ T cells), hereby referred to as targeted cytokine constructs. Without wishing to be bound by a certain theory, it is contemplated that being able to selectively activate certain cell types may enable a more robust response as well as the ability to limit the activation of other cell types that would be counter-productive to a successful immune response. In some cases, the methods and compositions disclosed herein allow for targeted delivery of IL-21 polypeptides to desired cell types and for administration of IL-21 at relatively low concentrations to elicit selective activation of CD8+ T cells. In some cases, use of targeted cytokine constructs disclosed herein provide more selective cell (such as a CD8+ T cells) activation compared to activation of other cell types, such as natural killer (NK) cells, CD4+ T cells or B cells. Definitions

[0108] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting.

[0109] Generally, 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 those well-known and commonly used in the art. 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 that are cited and discussed throughout the present specification unless otherwise indicated. 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 those 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.

[0110] 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 “a chimeric transmembrane receptor polypeptide” includes a plurality of chimeric transmembrane receptor polypeptides.

[0111] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend 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, per the 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. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0112] As used herein, “area under the curve” or “AUC” is a pharmacokinetic parameter that refers to the area under the plot of plasma concentration of a drug (e.g., IL-21 polypeptide or fusion protein described herein) versus time after dosage. In some embodiments, the AUC measures a patient’s exposure to a drug and depends on dose, bioavailability, and clearance. Methods for determining the AUC are known to those of skill in the art and include, but is not limited to, the Rectangle Method, the Trapezoidal Rule, and Simpson’s Rule.

[0113] A “cytokine” is a form of immunomodulatory polypeptide that can mediate crosstalk between initiating / primary cells and target / effector cells. It can function as a soluble form or cell-surface associated to bind the “cytokine receptor” on target immune cells to activate signaling. “Cytokine receptor” as used here is the polypeptide on the cell surface that activates intracellular signaling upon binding 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 may be produced by more than one cell type. Cytokines are pleiotropic; since the receptors are expressed on multiple immune cell subsets, one cytokine can activate the signaling pathway in multiple cells. However, depending on the cell type, the signaling events for a cytokine can result in different downstream cellular events such as activation, proliferation, survival, apoptosis, effector function and secretion of other immunomodulatory proteins. A given cytokine, in some embodiments, is a wild-type cytokine polypeptide, a fragment thereof, or a variant thereof, such as a mutated cytokine polypeptide (also referred to herein as a mutein cytokine, e.g., a mutein IL-21 or an IL-21 mutein).

[0114] The term “antigen,” as used herein, refers to a molecule or a fragment thereof capable of being bound by a selective binding agent. As an example, an antigen can be a ligand that can be bound by a selective binding agent such as a receptor. As another example, an antigen can be an antigenic molecule that can be bound by a selective binding agent such as an immunological protein (e.g., an antibody). An antigen can also refer to a molecule or fragment thereof capable of being used in an animal to produce antibodies capable of binding to that antigen.

[0115] The term “antibody”, as used herein, means any antigen-binding molecule comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen. The term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from aminoterminus to carb oxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the disclosure, the FRs of an antibody (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0116] The term “intact antibody” refers to an antibody comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. In one embodiment, the antibody is an intact antibody. In one embodiment, the intact antibody is an intact human IgGl, IgG2 or IgG4 isotype. In certain embodiments, the antibody, or antigen-binding fragment thereof, is a human IgGl, IgG2, or IgG4 isotype.

[0117] The terms “antigen-binding portion” of an antibody, “antigen-binding fragment,” or “antibody-fragment,” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from intact antibody molecules using any suitable standard techniques 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 is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0118] 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 the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.

[0119] The term “variable region” or “variable domain” of an antibody, or fragment thereof, as used herein refers to the portions of the light and heavy chains of antibody molecules that include amino acid sequences of 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 methods used in this disclosure, the amino acid positions assigned to CDRs and FRs may be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)). Amino acid numbering of antibodies or antigen binding fragments is also according to that of Kabat.

[0120] The term “complementarity determining regions” or “CDRs” as used herein refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to 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 coworkers (Chothia et al., J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that certain sub- portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as LI, L2 and L3 or Hl, H2 and H3 where the “L” and the “H” designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping 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 follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs.

[0121] The term “framework regions” (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. The DNA sequence encoding a particular antibody can generally be found following well known methods such as those described in Kabat, et al. 1987 Sequence of Proteins of Immunological Interest, U.S. Department of Health and Human Services, Bethesda MD, which is incorporated herein as a reference. In addition, a general method for cloning functional variable regions from antibodies can be found in Chaudhary, V.K., et al., 1990 Proc. Natl. Acad. Sci. USA 87:1066, which is incorporated herein as a reference.

[0122] A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one “amino acid modification” as herein defined. A variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. In one embodiment, a variant Fc region herein can have a sequence that has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity or at least about 99% identity with a native sequence Fc region. According to another embodiment, the variant Fc region herein can have a sequence that has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity or at least about 99% identity with an Fc region of a parent polypeptide.

[0123] The terms “Fc receptor” or “FcR,” as used herein, generally refers to a receptor, or any derivative, variant or fragment thereof, that can bind to the Fc region of an antibody. In certain embodiments, the FcR is one which binds an IgG antibody (a gamma receptor, Fcgamma R) and includes receptors of the Fcgamma RI (CD64), Fcgamma RII (CD32), and Fcgamma RIII (CD 16) subclasses, including allelic variants and alternatively spliced forms of these receptors. Fcgamma RII receptors include Fcgamma RIIA (an “activating receptor”) and Fcgamma RUB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. The term “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus.

[0124] By “effector function” as used herein is meant a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand, which vary with the antibody isotype. Effector functions include but are not limited to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complementdependent cytotoxicity (CDC), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation. “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express FcRs (such as Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC is correlated with binding to FcyRIIIa; increased binding to FcyRIIIa leads to an increase in ADCC activity. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. “ADCP” or antibody dependent cell-mediated phagocytosis as used herein is meant the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcyRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.

[0125] “Fc null” and “Fc null variant” are used interchangeably and used herein to describe a modified Fc which have reduced or abolished effector functions. Such Fc null or Fc null variant have reduced or abolished to FcyRs and / or complement receptors. Preferably, such Fc null or Fc null variant has abolished effector functions. Exemplary methods for the modification include but not limited to chemical alteration, amino acid residue substitution, insertion and deletions. Exemplary amino acid positions on Fc molecules where one or more modifications were introduced to decrease effector function of the resulting variant (numbering based on the EU numbering scheme) at position i) IgGl: 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 having decreased effector function include those having one or more of the following substitutions: i) IgGl: 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, E23 3P / L234V / L23 5 A / G23 6del / S267K, E23 3P / L234V / L23 5 A / G23 6del / S23 9K / A3 27G, E233P / L234V / L235A / G236del / S267K / A327G and E233P / L234V / L235A / G236del, L234A / L235A / G237deleted; ii) IgG2: A330S / A331S, V234A / G237A, V234A / G237A / D265A, D265A / A330S / A331S, V234A / G237A / D265A / A330S / A331S, and H268Q / V309L / A330S / A331S; iii) IgG4: L235A / G237A / E318A, D265A, L235A / G237A / D265A and L23 5 A / G23 7A / D265A / E318 A.

[0126] “Epitope” as used herein refers to a determinant capable of specific binding to the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope. The epitope may comprise amino acid residues directly involved in the binding and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the antigen binding peptide (in other words, the amino acid residue is within the footprint of the antigen binding peptide). Epitopes may be either conformational or linear. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids. Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen, for example "binning".

[0127] “Linker” as used herein refers to a molecule that connect two polypeptide chains. Linker can be a polypeptide linker or a synthetic chemical linker (for example, see disclosed in Protein Engineering, 9(3), 299-305, 1996). The length and sequence of the polypeptide linkers is not particularly limited and can be selected according to the purpose by those skilled in the art. Polypeptide linker comprises one or more amino acids. Preferably, polypeptide linker is a peptide with a length of at least 5 amino acids, preferably with a length of 5 to 100, more preferably of 10 to 50 amino acids. In one embodiment, said peptide linker is G, S, GS, SG, SGG, GGS, and GSG (with G=glycine and S=serine). In another embodiment, said peptide linker is (GGGS)xGn (SEQ ID NO: 294) or (GGGGS)xGn (SEQ ID NO: 295), (GGGGGS)xGn (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), with x=l, 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)xGn with x=2,3, or 4 and n=0 (SEQ ID NO: 372); in some embodiments the linker is (GGGGS)xGn with 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 crosslinking agents that are routinely used to crosslink peptides, for example, N-hydroxy succinimide (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).

[0128] The term “nucleotide,” as used herein, generally refers to a base-sugar-phosphate combination. A nucleotide can comprise a synthetic nucleotide. A nucleotide can comprise a synthetic nucleotide analog. Nucleotides can be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can 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 can include, for example, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein, in some examples, refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide can be unlabeled or detectably labeled by well-known techniques. Labeling can also be carried out with quantum dots. Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Fluorescent labels of nucleotides can include but are not limited 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-l-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides can 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.; Fluorescein-15-dATP, Fluorescein-12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein-12-UTP, and Fluorescein-15-2'-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, B0DIPY-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 available from Molecular Probes, Eugene, Oreg. Nucleotides can also be labeled or marked by chemical modification. A chemically-modified single nucleotide can be 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).

[0129] 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, either in single-, double-, or multistranded 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 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 comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase). 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 acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (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 can be interrupted by non-nucleotide components.

[0130] The term “gene,” as used herein, refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript. The term as used herein with reference to genomic DNA includes intervening, noncoding regions as well as regulatory regions and can include 5’ and 3’ ends. In some uses, the term encompasses the transcribed sequences, including 5’ and 3’ untranslated regions (5’-UTR and 3’-UTR), exons and introns. In some genes, the transcribed region will contain “open reading frames” that encode polypeptides. In some uses of the term, a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region”) necessary for encoding a polypeptide. In some cases, genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only the transcribed sequences, but in addition, also includes 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 not normally found in the host organism but which is introduced into the host organism by gene transfer. A non-native gene , in some cases, refers to a gene not in its natural location in the genome of an organism. A non-native gene, in some cases, also refers to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and / or deletions (e.g., non-native sequence).

[0131] The term “regulating” with reference to expression or activity, as used herein, refers to altering the level of expression or activity. Regulation can occur at the transcription level and / or translation level.

[0132] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer can 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 an amino acid polymer that has been 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 non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogues. Modified amino acids can include natural amino acids and non-natural amino acids, which have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid. Amino acid analogues can refer to amino acid derivatives. The term “amino acid” includes both D-amino acids and L-amino acids.

[0133] The terms “derivative,” “variant,” and “fragment,” when used herein with reference to a polypeptide, refers to a polypeptide related to a wild type polypeptide, for example either 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 comprise one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wild type polypeptide.

[0134] The term “residue” as used herein means a position in a protein and its associated amino acid identity. For example, Leu 234 (also referred to as Leu234 or L234) is a residue at position 234 in the human antibody IgGl.

[0135] The term “wild-type" as used herein means an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A wild-type protein has an amino acid sequence or a nucleotide sequence that has not been intentionally modified.

[0136] The terms “substitution” or “mutation” refers to a change to the polypeptide backbone wherein an amino acid occurring naturally in the wild-type sequence of a polypeptide is substituted to another amino acid not naturally occurring at the same position in the said polypeptide. In some cases, a mutation or mutations is introduced to modify a polypeptide’s affinity to its receptor thereby altering its activity such that it becomes different from the affinity and activity of the wild-type cognate polypeptide. Mutations can also improve a polypeptide’s biophysical properties. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful.

[0137] The terms “affinity” or “binding affinity” refer to the strength of the sum total 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 indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (koff and kon, respectively). Thus, equivalent affinities may comprise 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) technologies (e.g., BIAcore), BioLayer Interferometry (BLI) technologies (e.g. Octet) and other traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002). In some examples, binding affinity is determined by Scatchard analysis, which comprises generating a Scatchard plot, which is a plot of the ratio of concentrations of bound ligand to unbound ligand versus the bound ligand concentration.

[0138] The terms “binding” or “specific binding” as used here, can refer to the ability of a polypeptide or an antigen binding domain to selectively interact with the receptor for the polypeptide or target antigen, respectively, and this specific interaction can be distinguished from non-targeted or undesired or non-specific interactions. Examples of specific binding can include but are not limited to IL-21 cytokine binding to its specific receptors (e.g., IL-21R and common gamma chain) and an antigen binding domain binding to a specific antigen (e.g., CD8 or PD-1).

[0139] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0140] The terms “treatment” and “treating,” as used herein, refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic benefit. For example, a treatment can comprise administering a system or cell population disclosed herein. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.

[0141] The terms “effective amount,” or “therapeutically effective amount,” or “effective dose,” or “effective dosage,” refer to the quantity of a composition, for example a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and / or NK cells), which can be combined with a targeted cytokine construct of the present disclosure, that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “therapeutically effective” refers to that quantity of a composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure.

[0142] As used herein, the term “Percent (%) amino acid sequence identity” with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, 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 appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alignment for purposes of determining percent amino acid sequence identity can for example be achieved using publicly available sequence comparison computer program ALIGN-2. The source code for the ALIGN-2 sequence comparison computer program is available with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program can be compiled for use on a UNIX operating system, such as a digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0143] For all amino acid positions discussed in the present disclosure, in the context of antibodies or antigen binding fragments thereof, numbering is according to the EU index. The “EU index” or “EU index as in Kabat et al.” or “EU numbering scheme” refers to the numbering of the EU antibody (See Edelman et al., 1969; Kabat et al., 1991). Interleukin-21 Polypeptides

[0144] Interleukin-21 (IL-21) is a cytokine that can be expressed by T cells, B cells, NK cells and myeloid cells, and regulates the activity of both innate and adaptive immune cells and improves T cell survival and effector function. Several Phase I and II clinical trials include IL-21 as the investigational product for the treatment of cancers, 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.

[0145] IL-21 has a four-helix bundle structure and exists as a monomer. In humans, two isoforms of IL-21 are known, each of which are derived from a precursor molecule. The first IL-21 isoform comprises 162 amino acids (aa), the first 29 of which make up the signal peptide; and the second IL-21 isoform comprises 153 aa, the first 29 of which make up the signal peptide as in the first isoform.

[0146] IL-21 binds to the heterodimeric IL-21 receptor complex, comprising of an IL-21 receptor (IL-21R) and common gamma chain (yc). IL-21 receptor complex is expressed on the surface of T, B, and NK cells.IL-21 receptor complex is similar in structure to the IL-2 receptor complex, in that each of these cytokine receptor complex comprises a yc.

[0147] When IL-21 binds to IL-21 receptor complex, the JAK / STAT signaling pathway is activated to activate target genes. While IL-21-induced signaling may be therapeutically desirable, careful consideration of the timing and the location of the signaling is needed, given IL-21's broad expression profile and due to the fact that IL-21 has the ability to potentiate CD8+ T cell responses as well as to suppress antigen presentation and T cell priming.

[0148] The present disclosure provides, in some embodiments, IL-21 polypeptides or functional fragments or variants thereof, comprising at least one amino acid substitution, relative to the wild-type IL-21 amino acid sequence, which is provided herein as SEQ ID NO: 1. Unless noted otherwise, the terms “wild-type IL-21,” “wild-type IL-21 polypeptide,” “human IL-21,” and “human IL-21 polypeptide” are used interchangeably and can refer to the amino acid sequence of SEQ ID NO: 1. Such IL-21 polypeptides comprising at least one amino acid substitution relative to SEQ ID NO: 1 are also referred to herein as IL-21 muteins. In exemplary aspects, an IL-21 polypeptide or functional fragment or variant thereof, as described herein, comprises at least one and not more than X amino acid substitutions, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or greater. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof, as 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, as described herein, comprises an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 10 amino acids, 15 amino acids, 20 amino acids, or 25 amino acids. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof, as described herein, comprises an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by no more than 16 amino acids. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof, as described herein, comprises an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids. In exemplary embodiments, an IL-21 polypeptide or functional fragment or variant thereof, as described herein, comprises an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by no more than 7 amino acids or no more than 5 amino acids. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof, as described herein, comprises an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 10, 11, 12, 13, 14, 15 or 16 amino acids. In exemplary embodiments, an IL-21 polypeptide or functional fragment or variant thereof, as described herein, comprises an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 3, 4, 5, or 6 amino acids. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof, as described herein, comprises an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 5 to 16. In exemplary embodiments, an IL-21 polypeptide or functional fragment or variant thereof, as described herein, comprises an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 3 to 6 amino acids or 1 to 5 amino acids. In exemplary embodiments, an IL-21 polypeptide or functional fragment or variant thereof, as described herein, comprises an amino acid sequence which differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by one or two amino acids. Altered Isoelectric Point or Charge Distribution

[0149] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises at least one mutation that alters the isoelectric point with or without affecting its binding to IL-21R. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant therefor comprises at least one mutation that alters the 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, non-specific binding, and the blood or circulatory halflife for a given protein. In some embodiments, the mutations in the IL-21 polypeptide or a functional fragment or a variant thereof decreases the theoretical isoelectric point by a given unit measured by a publicly available database ProtParam (SwissProt), which is hereby incorporated by reference.

[0150] In some embodiments, the IL-21 polypeptide or a functional fragment or a 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, the IL- 21 polypeptide or a functional fragment or a variant thereof has an isoelectric point of about 6.0 to about 9.0. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a theoretical isoelectric point of about 6 to about 9. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a theoretical isoelectric point of at least about 6. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a theoretical isoelectric point of at most about 9. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a theoretical isoelectric point 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 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 7.7, about 8.1 to about 7.5, about 8.1 to about 7.3, about 8.1 to about 7, about 8.1 to about 6.5, about 8.1 to about 6.0, about 7.9 to about 7.7, about 7.9 to about 7.5, about 7.9 to about 7.3, about 7.9 to about 7, about 7.9 to about 6.5, about 7.9 to about 6.0, about 7.7 to about 7.5, about 7.7 to about 7.3, about 7.7 to about 7, about 7.5 to about 7.3, about 7.5 to about 7, or about 7.3 to about 7, about 7.3 to about 6.5, about 7.3 to about 6.0, about 7.0 to about 6.5, about 7.0 to about 6.0, or about 6.5 to about 6.0. In some embodiments, the IL-21 polypeptide or a functional fragment or a 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, the IL-21 polypeptide or a functional fragment or a variant thereof has a theoretical isoelectric point of less than 9.0. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof has a theoretical isoelectric point of less than 8.9, less than 8.8, less than 8.7, less than 8.5, less than 8.3, less than 8.1, less than 7.9, less than 7.7, less than 7.5, less than 7.3, less than 7.2, less than 7.0, less than 6.5 or less than 6.2.

[0151] In some instances, an altered isoelectric point can increase the protein yield of IL-21 polypeptide or a functional fragment or a variant thereof during the purification compared to wildtype IL-21. In some embodiments, the increased protein yield is at least 5% greater than wild-type IL-21. In some embodiments, the increased protein yield is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% greater than wild-type IL-21. In some embodiments, the increased protein yield is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% greater than wild-type IL-21. In some embodiments, the increased protein yield is 5% to 10 %, 5 % to 15 %, 5 % to 20 %, 5 % to 25 %, 5 % to 50 %, 5 % to 75 %, 5 % to 100 %, 5 % to 125 %, 5 % to 150 %, 5 % to 175 %, 5 % to 200 %, 5 % to 225%, 5 % to 250 %, 5 % to 275 %, 5 % to 300 %, 10 % to 15 %, 10 % to 20 %, 10 % to 25 %, 10 % to 50 %, 10 % to 75 %, 10 % to 100 %, 10 % to 125 %, 10 % to 150 %, 10 % to 175 %, 10 % to 200 %, 10 % to 225%, 10 % to 250 %, 10 % to 275 %, 10 % to 300 %, 15 % to 20 %, 15 % to 25 %, 15 % to 50 %, 15 % to 75 %, 15 % to 100 %, 15 % to 125 %, 15 % to 150 %, 15 % to 175 %, 15 % to 200 %, 15 % to 225%, 15 % to 250 %, 15 % to 275 %, 15 % to 300 %, 20 % to 25 %, 20 % to 50 %, 20 % to 75 %, 20 % to 100 %, 20 % to 125 %, 20 % to 150 %, 20 % to 175 %, 20 % to 200 %, 20 % to 225%, 20 % to 250 %, 20 % to 275 %, 20 % to 300 %, 25 % to 50 %, 25 % to 75 %, 25 % to 100 %, 25 % to 125 %, 25 % to 150 %, 25 % to 175 %, 25 % to 200 %, 25 % to 225%, 25 % to 250 %, 25 % to 275 %, 25 % to 300 %, 50 % to 75 %, 50 % to 100 %, 50 % to 125 %, 50 % to 150 %, 50 % to 175 %, 50 % to 200 %, 75 % to 100 %, 75 % to 125 %, 75 % to 150 %, 75 % to 175 %, 75 % to 200 %, 75 % to 225%, 75 % to 250 %, 75 % to 275 %, 75 % to 300 %, 100 % to 125 %, 100 % to 150 %, 100 % to 175 %, 100 % to 200 %, 100 % to 225%, 100 % to 250 %, 100 % to 275 %, 100 % to 300 %, 125 % to 150 %, 125 % to 175 %, 125 % to 200 %, 125 % to 225%, 125 % to 250 %, 125 % to 275 %, 125 % to 300 %, 150 % to 175 %, 150 % to 200 %, 175 % to 200 %, 175 % to 225%, 175 % to 250 %, 175 % to 275 %, 175 % to 300 %, 200 % to 225%, 200 % to 250 %, 200 % to 275 %, 200 % to 300 %, 225 % to 250 %, 225 % to 275 %, 225 % to 300 %, 250 % to 275 %, 250 % to 300 %, or 275 % to 300 %. In some embodiments, the increased protein yield is about 5 %, 10 %, 15 %, 20 %, 25 %, 50 %, 75 %, 100 %, 125 %, 150 %, 175 %, 200 %, 225%, 250%, 275%, or 300%.

[0152] In some embodiments, relative to a region of wild-type IL-21 polypeptide that comprises one or more positively charged amino acid residues, the IL-21 polypeptide or a functional fragment or a variant thereof comprises at least one amino acid substitution of the one or more positively charged amino acid residues. In some cases, the region of wild-type IL-21 comprises 2-20 positively charged amino acid residues. In some embodiments, the region in wildtype IL-21 comprises 2-3, 2-4, 2-5, 2-7. 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-14, 4-15, 4-16, 4-17, 4-18, 4-19, 4-20, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 6-20, 7-8, 7-9, 7-10, 7-11, 7-12, 7-13, 7-14, 7-15, 7-16, 7-17, 7-18, 7-19, 7-20, 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 11-12,11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 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 positively charged amino acid residues. In some embodiments, the region of wild-type IL-21 comprises 12 positively charged amino acid residues. In some embodiments, the region of wild-type IL-21 comprises 2-20 amino acid residues, of which at least one amino acid is positively charged. In some embodiments, the region of wild-type IL-21 comprises 2-3, 2-4, 2-5, 2-7. 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-14, 4-15, 4-16, 4-17, 4-18, 4-19, 4-20, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 6-20, 7-8, 7-9, 7-10, 7-11, 7-12, 7-13, 7-14, 7-15, 7-16, 7-17, 7-18, 7-19, 7-20, 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 11-12,11-13, 11-14, 11-15, 11-16, 11-17, Ills, 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 with 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. In some embodiments, the 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, the 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.

[0153] In some embodiments, amino acids within the region of wild-type IL-21 do not bind a human IL-21 receptor. In some embodiments, the region of wild-type IL-21 comprises amino acid residues S80-T92 of SEQ ID NO: 1. In some embodiments, the region of wild-type IL-21 comprises positively charged amino acid residues R85, R86, K88, H89, and R90. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises at least one amino acid substitution in the region of wild-type IL-21. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises at least one amino acid substitution of S80-T92. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises at least four amino acid substitutions of S80-T92. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises at least four amino acid substitutions of S80-T92, provided G84 is not substituted. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises at least one amino acid substitution at R85, R86, K88, H89 or R90. In some embodiments, the IL-21 polypeptide or a functional fragment or a 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, the IL-21 polypeptide or a functional fragment or a variant thereof comprises amino acid substitutions at S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91 and T92.

[0154] In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof that comprises an altered isoelectric point relative to wild-type IL-21 (SEQ ID NO: 1) comprises a mutation in 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, the IL-21 polypeptide or a functional fragment or a variant thereof that comprises an altered isoelectric point relative to wild-type IL-21 (SEQ ID NO: 1) comprises a mutation in 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, the IL-21 polypeptide or a functional fragment or a variant thereof that comprises an altered isoelectric point relative to wild-type IL-21 (SEQ ID NO: 1) comprises a mutation in 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, the IL-21 polypeptide or a functional fragment or a variant thereof that comprises an altered isoelectric point relative to wild-type IL-21 (SEQ ID NO: 1) comprises a mutation in 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.

[0155] In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a mutation at position K56 of SEQ ID NO: 1. In some instances, the mutation comprises K56G, K56S, K56E, K56D, or K56A. In some embodiments, the IL-21 polypeptide or a functional fragment or a 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 a functional fragment or a variant thereof comprises a mutation at position T81 of SEQ ID NO: 1. In some instances, the mutation comprises T81G, T81S, T81E, T81D, or T81A. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a mutation at position N82 of SEQ ID NO: 1. In some instances, the mutation comprises N82G, N82S, N82E, N82D, or N82A. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a mutation at position A83 of SEQ ID NO: 1. In some instances, the mutation comprises A83G, A83S, A83E, or A83D. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a mutation at position G84 of SEQ ID NO: 1. In some instances, the mutation comprises G84A, G84S, G84E, or G84D. In some embodiments, G84 is not mutated. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a mutation at position R85 of SEQ ID NO: 1. In some instances, the mutation comprises R85G, R85S, R85E, R85D, or R85A. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a mutation at position R86 of SEQ ID NO: 1. In some instances, the mutation comprises R86G, R86S, R86E, R86D, or R86A. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a mutation at position Q87 of SEQ ID NO: 1. In some instances, the mutation comprises Q87G, Q87S, Q87E, Q87D, or Q87A. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a mutation at position K88 of SEQ ID NO: 1. In some instances, the mutation comprises K88G, K88S, K88E, K88D, or K88A. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a mutation at position H89 of SEQ ID NO: 1. In some instances, the mutation comprises H89G, H89S, H89E, H89D, or H89A. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a mutation at position R90 of SEQ ID NO: 1. In some instances, the mutation comprises R90G, R90S, R90E, R90D, or R90A. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a mutation at position L91 of SEQ ID NO: 1. In some instances, the mutation comprises L91G, L91S, L91E, L91D, or L91A. In some embodiments, the IL-21 polypeptide or a functional fragment or a variant thereof comprises a mutation at position T92 of SEQ ID NO: 1. In some instances, the mutation comprises T92G, T92S, T92E, T92D, or T92A.

[0156] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof that has an altered isoelectric point relative to the 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 in 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 a functional fragment or a variant thereof that has an altered isoelectric point relative to the 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 in at least one position selected from the group consisting of positions K56 and R90 of SEQ ID NO: 1. The mutation at position K56, in some instances, is K56G, K56S, K56E, K56D, or K56A; the mutation at position R90, in some instances, is R90G, R90S, R90E, R90D, or R90A. The altered isoelectric point, in some examples, is a reduced isoelectric point compared to the IL-21 of SEQ ID NO: 1 (which is about 9.42), e.g., reduced by at least about 0.6 units.

[0157] In some embodiments, an IL-21 polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 and comprises amino acids 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; G80, G81, G82, S83, G85, G86, S87, G88, G89, S90; G85, G86, S87, G88, G89, and E90; G85, G86, S87, G88, G89, and S90; G85, G86, G87, G88, G89, and E90; G85, G86, G87, G88, G89, and G90; or G85, G86, E87, G88, G89, and G90, wherein the position numbers correspond to SEQ ID NO: 1.

[0158] 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, an 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, 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 A56, A75, G85, G86, G88, and E90. In some embodiments, an 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, 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 A56, E75, 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: A56, A75, 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 A56, A75, 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: E56, A75, 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 E56, A75, 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: G80, G81, G82, S83, E85, G86, S87, G88, G89, and S90. 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, S83, E85, G86, S87, G88, G89, and S90. 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, S83, G85, G86, S87, G88, G89, and S90. 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, S83, G85, G86, S87, G88, G89, and S90. 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, S83, G85, G86, S87, G88, G89, 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 G80, G81, G82, S83, G85, G86, S87, G88, G89, and E90. In some embodiments, an 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, 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, S87, G88, G89, and E90. In some embodiments, an 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, 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, S87, G88, G89, and S90. In some embodiments, an 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, 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, G87, G88, G89, and E90. In some embodiments, an 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, 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, G87, G88, G89, and G90. In some embodiments, an 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, 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, E87, G88, G89, and G90. Provided in some embodiments are charge variant IL-21 polypeptides or functional fragments or variants thereof that comprise 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.

[0159] 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, S83, G85, G86, S87, G88, G89, G90, S91 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, S83, G85, G86, S87, G88, G89, G90, S91 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, G90, 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, G90, 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, S83, G85, G86, E87, G88, G89, G90, S91 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, S83, G85, G86, E87, G88, G89, G90, S91 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, E83, G85, G86, E87, G88, G89, G90, S91 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, E83, G85, G86, E87, G88, G89, G90, S91 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, G90, G91 and S92. 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, S85, G86, G87, G88, S89, G90, G91 and S92. 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, S89, G90, G91 and S92. 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, S89, G90, G91 and S92. 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, E85, G86, G87, G88, G89, G90, 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, E85, G86, G87, G88, G89, G90, 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, S83, G85, G86, S87, 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, 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, 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, S85, G86, G87, G88, S89, E90, G91 and S92. In some embodiments, an 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, 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, 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: G85, 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 G85, 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: G82, G83, G85, 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, G85, 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: G82, G83, G85, E86, 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, 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, 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, E84, 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: G80, G81, G82, E83, 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, E83, 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: G80, G81, E82, 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, E82, G83, G85, G86, G87, G88, G89, E90, G91 and G92. Provided in some embodiments are charge variant IL-21 polypeptides or functional fragments or variants thereof that comprise 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. Provided in some embodiments are charge variant IL-21 polypeptides or functional fragments or variants thereof that comprise 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.

[0160] In some embodiments, an 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, an 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, an IL-21 polypeptide comprises at most about 100 % sequence identity to a sequence selected from the group consisting of SEQ IDNOs. 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. In some embodiments, an IL-21 polypeptide comprises about 75 % sequence identity to about 80 % sequence identity, about 75 % sequence identity to about 85 % sequence identity, about 75 % sequence identity to about 90 % sequence identity, about 75 % sequence identity to about 95 % sequence identity, about 75 % sequence identity to about 96 % sequence identity, about 75 % sequence identity to about 97 % sequence identity, about 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 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, 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 % sequence identity to about 98 % sequence identity, about 95 % sequence identity to about 99 % sequence identity, about 95 % sequence identity to about 100 % sequence identity, about 96 % sequence identity to about 97 % sequence identity, about 96 % sequence identity to about 98 % sequence identity, about 96 % sequence identity to about 99 % sequence identity, about 96 % sequence identity to about 100 % sequence identity, about 97 % sequence identity to about 98 % sequence identity, about 97 % sequence identity to about 99 % sequence identity, about 97 % sequence identity to about 100 % sequence identity, about 98 % sequence identity to about 99 % sequence identity, about 98 % sequence identity to about 100 % sequence identity, or about 99 % 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, an 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 IDNOs. 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15.

[0161] In some embodiments, an 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. 23-40. In some embodiments, an 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, an IL-21 polypeptide comprises at most about 100 % sequence identity to a sequence selected from the group consisting of SEQ ID NOs. 23-40. In some embodiments, an IL-21 polypeptide comprises about 75 % sequence identity to about 80 % sequence identity, about 75 % sequence identity to about 85 % sequence identity, about 75 % sequence identity to about 90 % sequence identity, about 75 % sequence identity to about 95 % sequence identity, about 75 % sequence identity to about 96 % sequence identity, about 75 % sequence identity to about 97 % sequence identity, about 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 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, 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 % sequence identity to about 98 % sequence identity, about 95 % sequence identity to about 99 % sequence identity, about 95 % sequence identity to about 100 % sequence identity, about 96 % sequence identity to about 97 % sequence identity, about 96 % sequence identity to about 98 % sequence identity, about 96 % sequence identity to about 99 % sequence identity, about 96 % sequence identity to about 100 % sequence identity, about 97 % sequence identity to about 98 % sequence identity, about 97 % sequence identity to about 99 % sequence identity, about 97 % sequence identity to about 100 % sequence identity, about 98 % sequence identity to about 99 % sequence identity, about 98 % sequence identity to about 100 % sequence identity, or about 99 % sequence identity to about 100 % sequence identity to a sequence selected from the group consisting of SEQ ID NOs. 23-40. In some embodiments, an 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.

[0162] In some embodiments, an 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, an 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, an IL-21 polypeptide comprises at most 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, an IL-21 polypeptide comprises about 75 % sequence identity to about 80 % sequence identity, about 75 % sequence identity to about 85 % sequence identity, about 75 % sequence identity to about 90 % sequence identity, about 75 % sequence identity to about 95 % sequence identity, about 75 % sequence identity to about 96 % sequence identity, about 75 % sequence identity to about 97 % sequence identity, about 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 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, 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 % sequence identity to about 98 % sequence identity, about 95 % sequence identity to about 99 % sequence identity, about 95 % sequence identity to about 100 % sequence identity, about 96 % sequence identity to about 97 % sequence identity, about 96 % sequence identity to about 98 % sequence identity, about 96 % sequence identity to about 99 % sequence identity, about 96 % sequence identity to about 100 % sequence identity, about 97 % sequence identity to about 98 % sequence identity, about 97 % sequence identity to about 99 % sequence identity, about 97 % sequence identity to about 100 % sequence identity, about 98 % sequence identity to about 99 % sequence identity, about 98 % sequence identity to about 100 % sequence identity, or about 99 % 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, an 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.

[0163] In some embodiments, an IL-21 polypeptide or a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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 a functional fragment or a 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, an IL-21 polypeptide or a functional fragment or a 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.

[0164] In some embodiments, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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, an 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.

[0165] In some embodiments, an IL-21 polypeptide comprises QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWS AF SCFQKAQLXi SAN TGNNERIINVSIKKLX2RKPPX3X4X5X6X7X8X9XioXiiXi2Xi3Xi4Xi5CPSCDSYEKKPPKEFLER FKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 383), wherein Xi = A, E, K; X2 = A, E, K; X3 = G, S; X4= G, T; X5 = G, E, N; X6= G, S, E, A; X7= E, G; X8= G, E, S, R; X9= G, E, R; Xio = S, G, E, Q; Xu = G, K; Xi2 = G, S, H; Xi3 = A, E, S, G, R; Xi4 = S, G, L; Xi5 = G, S, T; and wherein at least one amino acid residue is not the amino acid residue at the identical position set forth in SEQ ID NO: 1. In some embodiments, at least one of X8,X9,Xn,Xi2, and Xnis not the amino acid residue at the identical position set forth in SEQ ID NO: 1.

[0166] In some embodiments, the IL-21 polypeptide comprises the amino acid sequence: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANT GNNERIINVSIKKLKRKPPXiX2X3X4GX5X6X7X8X9XioXiiXi2CPSCDSYEKKPPKEFLERFKSL LQKMIHQHLSSRTHGSEDS (SEQ ID NO: 380), wherein Xi = G, S; X2 = G, T; X3 = G, E, N; X4 = G, S, E, A; X5= G, E, S, R; Xe= G, E, R; X7= S, G, E, Q; X8= G, K; X9= G, S, H; Xio = A, E, S, G, R; Xu = S, G, L; and X12 = G, S, T, provided at least one of X5, Xe, X8, X9, and X10 is not the amino acid residue at the identical position set forth in SEQ ID NO: 1. In some embodiments, X5 = G, Xe = G, X8 = G, X10 = A. In some embodiments, X5 = G, Xe = G, X8 = G, X10 = E. In some embodiments, Xi = G, X2 = G, X3 = G, X4 = S, X5 = E, X6 = G, X7 = S, X8 = G, X9 = G, X10 = S. In some embodiments, Xi = G, X2 = G, X3 = G, X4 = S, X5 = G, Xe = G, X7 = S, X8 = G, X9 = G, X10 = E. In some embodiments, X5 = G, Xe = G, X7 = S, X8 = G, X9 = G, X10 = E. In some embodiments, X5 = G, X6 = G, X7 = S, X8 = G, X9 = G, X10 = S. In some embodiments, X5 = G, X6 = G, X7 = G, X8 = G, X9 = G, X10 = E. In some embodiments, X5 = G, Xe = G, X7 = G, X8 = G, X9 = G, X10 = G. In some embodiments, X5 = G, Xe = G, X7 = E, X8 = G, X9 = G, X10 = G. In some embodiments, Xi = G, X2= G, X3= G, X4= S, X5 = G, X6= G, X7= S, X8= G, X9= G, Xio = G, Xn = S, Xi2= G. In some embodiments, Xi = G, X2 = G, X3 = G, X4 = G, X5 = G, X6 = G, X7 = G, X8 = G, X9 = G, Xw = G, Xu = G, X12 = G. In some embodiments, Xi = G, X2 = G, X3 = G, X4 = S, X5 = G, Xe = G, X7 = E, X8 = G, X9 = G, Xio = G, Xu = S, Xi2 = G. In some embodiments, Xi = G, X2 = G, X3 = G, X4 = E, X5 = G, X6 = G, X7 = E, X8 = G, X9 = G, Xio = G, Xu = S, X12 = G. In some embodiments, X3 = G, X4= G, X5= S, Xe= G, X7= G, X8= G, X9 = S, Xio = G, Xn= G, Xi2 = S. In some embodiments, X3 = G, X4 = G, X5 = E, X6 = G, X7 = G, X8 = G, X9 = S, Xio = G, Xu = G, X12 = S. In some embodiments, Xi = G, X2 = G, X3 = G, X4 = G, X5 = E, X6 = G, X7 = G, X8 = G, X9 = G, Xio = G, Xu = G, Xi2 = G. In some embodiments, Xi = G, X2 = G, X3 = G, X4 = S, X5 = G, Xe = G, X7 = S, X8 = G, X9 = G, Xio = E, Xu = G, Xi2 = G. In some embodiments, Xi = G, X2 = G, X3 = G, X4 = G, X5 = G, Xe = G, X7 = G, X8 = G, X9 = G, Xw = E, Xu = G, Xi2 = G. In some embodiments, X3 = G, X4 = G, X5 = S, X6 = G, X7 = G, X8 = G, X9 = S, Xw = E, Xu = G, X12 = S. In some embodiments, X3= G, X4= G, X5 = G, Xe= G, X7= G, X8= G, X9= G, Xw= E, Xn = G, X12= G. In some embodiments, X5 = G, Xe = G, X7 = G, X8 = G, X9 = G, Xw = E, Xu = G. In some embodiments, X3 = G, X4 = G, X5 = G, Xe = G, X7 = G, X8 = G, X9 = G, Xw = E, Xu = G. In some embodiments, X3 = G, X4 = G, X5 = G, X6 = E, X7 = G, X8 = G, X9 = G, Xw = E, Xu = G. In some embodiments, X3 = G, X4 = G, X5 = E, X6 = G, X7 = G, X8 = G, X9 = G, Xw = E, Xu = G. In some embodiments, Xi = G, X2= G, X3= G, X4 = E, X5 = G, X6= G, X7= G, X8= G, X9= G, Xw = E, Xn = G, X12= G. In some embodiments, Xi = G, X2 = G, X3 = E, X4 = G, X5 = G, X6 = G, X7 = G, X8 = G, X9 = G, Xw = E, Xh = G,Xi2=G.

[0167] In some embodiments, the IL-21 polypeptide comprises an amino acid sequence starting at position 78 as defined by SEQ ID NO: 1, wherein the amino acid sequence is PPX1X2X3X4GX5X6X7X8X9X1oX11X12CP (SEQ ID NO: 386), wherein Xi = 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; Xw= A, E, S, G, R; Xu = S, G, L; and Xi2 = G, S, T, provided at least one of X5, Xe, X8, X9, and Xw is not the amino acid residue at the identical position set forth in SEQ ID NO: 1.

[0168] In some embodiments, the IL-21 polypeptide comprises an amino acid sequence starting at position 80 as defined by SEQ ID NO: 1, wherein the amino acid sequence is XiX2X3X4GX5X6X7X8X9XwXiiXi2, wherein Xi = 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; Xw= A, E, S, G, R; Xn = S, G, L; and Xi2 = G, S, T, provided at least one of X5, Xe, X8, X9, and Xw is not the amino acid residue at the identical position set forth in SEQ ID NO: 1. Reduced IL-21R binding

[0169] In some embodiments, the disclosure provides an IL-21 polypeptide or a functional fragment or a variant thereof with reduced binding affinity to IL-21R. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises at least one mutation that reduces its binding affinity to IL-21R. Such mutations are in some embodiments in one or more positions selected from the group consisting of: R5,18, R9, Rll, Q12,114, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, QI 16, and KI 17, wherein the position numbering is number according to the amino acid sequence of SEQ ID NO: 1. In some embodiments, such mutations are in one or more positions selected from the group consisting of: R5,18, R9, Rll, L13, 114,116, V17, D18, K72, K73, L74, K75, R76, K77, and KI 17, wherein the position numbering is number according to the amino acid sequence of SEQ ID NO: l.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, 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 RI 1 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 LI3 comprises an amino acid substitution selected from the group consisting of F or R. In some embodiments, the mutation at position 114 comprises an amino acid substitution selected from the group consisting of D or E. In some embodiments, the mutation at position DI5 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 116 comprises an amino acid substitution selected from the group consisting of A, S or R. In some embodiments, the mutation at position VI7 comprises an amino acid substitution selected from the group consisting of I or A. In some embodiments, the mutation at position DI 8 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 QI 16 comprises an amino acid substitution is Y. In some embodiments, the mutation at position KI 17 comprises an amino acid substitution selected from the group consisting of A, D, or E.

[0170] In some cases, an IL-21 polypeptide or a functional fragment or a variant thereof comprises at least one amino acid substitution selected from: R5F, R5A, R5E, R5S, R5T, R5N, R5Q, R5V, R5I, R5L, R5Y, I8E, R9A, R9D, R9E, R9H, R9S, R9T, R9N, R9G, R9V, R9I, R9L, R9Y, RI ID, RUE, L13F, L13R, I14D, I16A, I16S, I16R, V17I, V17A, D18A, K72A, K72E, K73A, K73E, K75A, K75E, L74I, L74F, L74M, L74V, R76E, R76F, R76A, R76N, R76D, R76S, R76T, R76Q, R76V, R76I, R76L, R76Y, R76M, K77A, K77E, and KI 17A. In some cases, an IL-21 polypeptide or a functional fragment or a variant thereof comprises amino acid substitution is R76E or R76Q.

[0171] An example sequence for an IL-21 polypeptide or a functional fragment or a variant thereof of this disclosure is provided as follows: QGQDXiHMX2X3MX4X5LX6X7lVX8X9LKNXioVNDLVPEFLPAPEDVETNCEWSAFSCFQKA QLKSANTGNNEXiiIINVXi2lXi3Xi4LXi5Xi6Xi7PPXi8TNAGRRQKHRLTCPSCDSYEKKPPKE FLERFKSLLX19X20MIHQHLSSRTHGSEDS (SEQ ID NO: 22). In some embodiments, Xi = 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, Xe = 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, X10 = Y or E. In some embodiments, Xu = R, G, S, E, D, or A. In some embodiments, Xi2= S, H, Y, L, V, or F. In some embodiments, X13 = K, G, S, E, D, or A. In some embodiments, X14 = K, A, Y, L, F, G, S, T, E, A, or D. In some embodiments, X15 = K, G, S, E, D, or A. In some embodiments, Xi6 = R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, Xi7 = K, G, S, E, D, or A. In some embodiments, Xi8 = S, H, A, G, E, or D. In some embodiments, X19 = Q or Y. In some embodiments, X20 = K, A, D, or E.

[0172] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof with reduced binding to IL-21R comprises the sequence: QGQDX1HMX2X3MX4QX5X6DX7X8X9QLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKA QLKSANTGNNERimVSIXioXiiXnXisXuXisPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLE RFKSLLQ Xi6MIHQHLSSRTHGSEDS (SEQ ID NO: 384), wherein Xi = 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; Xio = A, E, K; Xu = A, E, K; X12 = I, F, M, L; X13 = A, K, E; Xi4 = E, F, A, N, D, S, T, Q, V, I, L, Y, M, R; X15 = A, E, K; Xi6= A, K; provided at least one of X1-X16 is not the amino acid residue at the identical position set forth in SEQ ID NO: 1.

[0173] In some embodiments, the 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 disclosure provides an IL-21 polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 313-371. Combination of Mutations

[0174] An IL-21 polypeptide or a functional fragment of a variant thereof, as described herein, comprises, in some embodiments, a combination of (a) mutations that alter its isoelectric point relative to a wild-type IL-21 (SEQ ID NO: 1) and (b) mutations that attenuate its binding to IL-21R, relative to the wild-type IL-21. In some embodiments, a mutation in group (a) is at a position selected from positions: K56, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92; and a mutation in group (b) is at a position selected from the group consisting of: R5,18, R9, Rll, Q12,114, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, QI 16, and KI 17, wherein the position numbering is according to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a mutation in group (a) is at a position selected from positions: S80, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92; and a mutation in group (b) is at a position selected from the group consisting of: R5,18, R9, Rll, L13,114,116, V17, D18, K72, K73, L74, K75, R76, K77, and KI 17, wherein the position numbering is according to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a mutation in group (a) is at a position selected from positions: S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, and T92; and a mutation in group (b) is at a position selected from the group consisting of: R5,18, R9, Rll, L13, 114,116, V17, D18, K72, K73, L74, K75, R76, K77, and KI 17, wherein the position numbering is according to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a mutation in group (a) is at a position selected from positions: S80, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92; and a mutation in group (b) is at position R76, wherein the position numbering is according to the amino acid sequence of SEQ ID NO: 1.

[0175] In some embodiments, an IL-21 polypeptide comprises a consensus sequence as follows: QGQDX1HMX2X3MX4X5LX6X7IVX8X9LKNX10VNDLVPEFLPAPEDVETNCEWSAFSCFQKA QLXllSANTGNNEX12lINVX13lX14Xl5LX16X17X18PPXl9X2oX21X22X23X24X25X26X27X28X29X3oX3 1CPSCDSYEKKPPKEFLERFKSLLX32X33MIHQHLSSRTHGSEDS (SEQ ID NO: 3). In some embodiments, Xi = 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, Xe = I or D or E. In some embodiments, X7 = D, R, K, H, L, Y, or F. In some embodiments, Xs = D, A, K, or R. In some embodiments, X9 = Q, L, or Y. In some embodiments, X10 = Y or E. Xu = G, S, E, D, or A. In some embodiments, X12 = R, G, S, E, D, or A. In some embodiments, Xi3= S, H, Y, L, V, or F. In some embodiments, X14 = K, G, S, E, D, or A. In some embodiments, X15 = K, A, Y, L, F, G, S, T, E, A, or D. In some embodiments, Xi6 = K, G, S, E, D, or A. In some embodiments, X17 = R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, Xis = K, G, S, E, D, or A. In some embodiments, X19 = S, H, A, G, E, or D. In some embodiments, X20 = G, S, E, D, or A. In some embodiments, X21 = G, S, E, D, or A. In some embodiments, X22 = G, S, E, or D. In some embodiments, X23 = A, S, E, or D. In some embodiments, X24 = G, S, E, D, or A. In some embodiments, X25 = G, S, E, D, or A. In some embodiments, X26 = G, S, E, D, or A. In some embodiments, X27 = G, S, E, D, or A. In some embodiments, X28 = G, S, E, D, or A. In some embodiments, X29 = G, S, E, D, or A. In some embodiments, X30 = G, S, E, D, or A. In some embodiments, X31 = G, S, E, D, or A. In some embodiments, X32 = Y. In some embodiments, X33 = A, D, or E.

[0176] In some embodiments, an IL-21 polypeptide comprises a consensus sequence as follows: QGQDX1HMX2X3MX4QX5X6DX7X8X9QLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKA QLX1oSANTGNNERIINVSIX11X12X13X14X15X16PPX17X18X19X2oX21X22X23X24X25X26X27X28X29C PSCDSYEKKPPKEFLERFKSLLQ X30MIHQHLSSRTHGSEDS (SEQ ID NO: 385), wherein Xi = 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; Xe = D, I; X7= A, S, R, I; X8= I, A, V; X9= A, D; Xio = A, E, K; Xu = A, E, K; X12 = A, E, K; X13 = I, F, M, L; Xi4 = A, K, E; X15 = E, F, A, N, D, S, T, Q, V, I, L, Y, M, R; Xi6 = A, E, K; X17 = G, S; Xi8 = G, T; X19 = G, E, N; X20 = G, S, E, A; X21 = E, G; X22 = G, E, S, R; X23 = G, E, R; X24 = S, G, E, Q; X25 = G, K; X26 = G, S, H; X27 = A, E, S, G, R; X28 = S, G, L; X29 = G, S, T; X30 = A, K, provided at least one of X1-X9 Xu-Xie and X30 is not the amino acid residue at the identical position set forth in SEQ ID NO: 1 and reduces binding to IL-21R, and provided at least one of Xio and X17-X29 is not the amino acid residue at the identical position set forth in SEQ ID NO: 1 and reduces the isoelectric point relative to human IL-21 (SEQ ID NO: 1).

[0177] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprising a combination of mutations comprises at least one mutation in a position of SEQ ID NO: 2. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprising a combination of mutations comprises at least one mutation in a position selected from the group consisting of positions: R5,18, R9, Rll, Q12,114, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, QI 16, and KI 17, wherein the position numbering is according to SEQ ID NO: 2. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprising a combination of mutations comprises at least one mutation in a position selected from the group consisting of positions: R5,18, R9, Rll, L13,114,116, V17, D18, K72, K73, L74, K75, R76, K77, and KI 17, wherein the position numbering is according to SEQ ID NO: 2.

[0178] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprising a combination of mutations comprises at least one mutation in a position of SEQ ID NO: 40. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprising a combination of mutations comprises at least one mutation in a position selected from the group consisting of positions: R5,18, R9, Rll, Q12,114, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, QI 16, and KI 17, wherein the position numbering is according to SEQ ID NO: 40. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprising a combination of mutations comprises at least one mutation in a position selected from the group consisting of positions: R5,18, R9, Rll, L13,114, 116, V17, D18, K72, K73, L74, K75, R76, K77, and KI 17, wherein the position numbering is according to SEQ ID NO: 40.

[0179] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises (i) four or more amino acid substitutions providing a reduced isoelectric point relative 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, 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 selected from (a) RI ID; (b) RI IE; (c) I14D, D18A and KI 17A; (d) R76E; (e) R5F; (f) R76F; (g) I8E; (h) R5A; (i) R5E; (j) R5S; (k) R5T; (1) R5N; (m) R5Q; (n) R5V; (0) 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; (11) R5F and K72A; (mm) R5F and K72E; (nn) R5F and K76A; (oo) R5F and K76E; (pp) K73 A 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) Il6S, V17I, and L74V; (111) I16R, V17I, and L74I; (mmm) L13F, I16A, V17A and L74M; and (nnn) L13R, I16A, V17I and L74I.

[0180] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises (i) an amino acid sequence selected from SEQ ID Nos: 2, 4-15 and 23-40, and (ii) at least one amino acid substitution selected from (a) RI ID; (b) RI IE; (c) I14D, D18A and KI 17A; (d) R76E; (e) R5F; (f) R76F; (g) I8E; (h) R5A; (i) R5E; (j) R5S; (k) R5T; (1) 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; (11) R5F and K72A; (mm) R5F and K72E; (nn) R5F and K76A; (oo) R5F and K76E; (pp) K73 A 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) VI71 and L74I; (jjj) I16A and L74F; (kkk) Il6S, V17I, and L74V; (111) I16R, V17I, and L74I; (mmm) L13F, I16A, V17A and L74M; and (nnn) L13R, I16A, V17I and L74I.

[0181] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a RI ID mutation and comprises about 75 % sequence identity to about 100 % sequence identity to SEQ ID NO: 16. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a RI ID mutation and comprises at least about 75 % sequence identity SEQ ID NO: 16. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a RI ID mutation and comprises at most about 100 % sequence identity SEQ ID NO: 16. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a RI ID mutation and comprises about 75 % sequence identity to about 80 % sequence identity, about 75 % sequence identity to about 85 % sequence identity, about 75 % sequence identity to about 90 % sequence identity, about 75 % sequence identity to about 95 % sequence identity, about 75 % sequence identity to about 96 % sequence identity, about 75 % sequence identity to about 97 % sequence identity, about 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 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, 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 % sequence identity to about 98 % sequence identity, about 95 % sequence identity to about 99 % sequence identity, about 95 % sequence identity to about 100 % sequence identity, about 96 % sequence identity to about 97 % sequence identity, about 96 % sequence identity to about 98 % sequence identity, about 96 % sequence identity to about 99 % sequence identity, about 96 % sequence identity to about 100 % sequence identity, about 97 % sequence identity to about 98 % sequence identity, about 97 % sequence identity to about 99 % sequence identity, about 97 % sequence identity to about 100 % sequence identity, about 98 % sequence identity to about 99 % sequence identity, about 98 % sequence identity to about 100 % sequence identity, or about 99 % sequence identity to about 100 % sequence identity, to SEQ ID NO: 16. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a RI ID 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.

[0182] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a RI IE mutation and comprises about 75 % sequence identity to about 100 % sequence identity to SEQ ID NO: 17. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a RI IE mutation and comprises at least about 75 % sequence identity SEQ ID NO: 17. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a RI IE mutation and comprises at most about 100 % sequence identity SEQ ID NO: 17. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a RI IE mutation and comprises about 75 % sequence identity to about 80 % sequence identity, about 75 % sequence identity to about 85 % sequence identity, about 75 % sequence identity to about 90 % sequence identity, about 75 % sequence identity to about 95 % sequence identity, about 75 % sequence identity to about 96 % sequence identity, about 75 % sequence identity to about 97 % sequence identity, about 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 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, 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 % sequence identity to about 98 % sequence identity, about 95 % sequence identity to about 99 % sequence identity, about 95 % sequence identity to about 100 % sequence identity, about 96 % sequence identity to about 97 % sequence identity, about 96 % sequence identity to about 98 % sequence identity, about 96 % sequence identity to about 99 % sequence identity, about 96 % sequence identity to about 100 % sequence identity, about 97 % sequence identity to about 98 % sequence identity, about 97 % sequence identity to about 99 % sequence identity, about 97 % sequence identity to about 100 % sequence identity, about 98 % sequence identity to about 99 % sequence identity, about 98 % sequence identity to about 100 % sequence identity, or about 99 % sequence identity to about 100 % sequence identity, to SEQ ID NO: 17. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a RI IE 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.

[0183] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof or a functional fragment or a variant thereof comprises a I14D, D18A and KI 17A mutation and comprises about 75 % sequence identity to about 100 % sequence identity to SEQ ID NO: 18. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof or a functional fragment or a variant thereof comprises a I14D, D18A and KI 17A mutation and comprises at least about 75 % sequence identity SEQ ID NO: 18. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof or a functional fragment or a variant thereof comprises a I14D, D18A and KI 17A mutation and comprises at most about 100 % sequence identity SEQ ID NO: 18. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof or a functional fragment or a variant thereof comprises a I14D, D18A and KI 17A mutation and comprises about 75 % sequence identity to about 80 % sequence identity, about 75 % sequence identity to about 85 % sequence identity, about 75 % sequence identity to about 90 % sequence identity, about 75 % sequence identity to about 95 % sequence identity, about 75 % sequence identity to about 96 % sequence identity, about 75 % sequence identity to about 97 % sequence identity, about 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 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, 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 % sequence identity to about 98 % sequence identity, about 95 % sequence identity to about 99 % sequence identity, about 95 % sequence identity to about 100 % sequence identity, about 96 % sequence identity to about 97 % sequence identity, about 96 % sequence identity to about 98 % sequence identity, about 96 % sequence identity to about 99 % sequence identity, about 96 % sequence identity to about 100 % sequence identity, about 97 % sequence identity to about 98 % sequence identity, about 97 % sequence identity to about 99 % sequence identity, about 97 % sequence identity to about 100 % sequence identity, about 98 % sequence identity to about 99 % sequence identity, about 98 % sequence identity to about 100 % sequence identity, or about 99 % sequence identity to about 100 % sequence identity to SEQ ID NO: 18. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof or a functional fragment or a variant thereof comprises a I14D, D18A and KI 17A 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: 18.

[0184] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a R76E mutation and comprises about 75 % sequence identity to about 100 % sequence identity to SEQ ID NO: 19. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a R76E mutation and comprises at least about 75 % sequence identity SEQ ID NO: 19. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a R76E mutation and comprises at most about 100 % sequence identity SEQ ID NO: 19. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises about 75 % sequence identity to about 80 % sequence identity, about 75 % sequence identity to about 85 % sequence identity, about 75 % sequence identity to about 90 % sequence identity, about 75 % sequence identity to about 95 % sequence identity, about 75 % sequence identity to about 96 % sequence identity, about 75 % sequence identity to about 97 % sequence identity, about 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 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, 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 % sequence identity to about 98 % sequence identity, about 95 % sequence identity to about 99 % sequence identity, about 95 % sequence identity to about 100 % sequence identity, about 96 % sequence identity to about 97 % sequence identity, about 96 % sequence identity to about 98 % sequence identity, about 96 % sequence identity to about 99 % sequence identity, about 96 % sequence identity to about 100 % sequence identity, about 97 % sequence identity to about 98 % sequence identity, about 97 % sequence identity to about 99 % sequence identity, about 97 % sequence identity to about 100 % sequence identity, about 98 % sequence identity to about 99 % sequence identity, about 98 % sequence identity to about 100 % sequence identity, or about 99 % sequence identity to about 100 % sequence identity, to SEQ ID NO: 19. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a 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.

[0185] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a R5F mutation and comprises about 75 % sequence identity to about 100 % sequence identity to SEQ ID NO: 20. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a R5F mutation and comprises at least about 75 % sequence identity SEQ ID NO: 20. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises at most about 100 % sequence identity SEQ ID NO: 20. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a R5F mutation and comprises about 75 % sequence identity to about 80 % sequence identity, about 75 % sequence identity to about 85 % sequence identity, about 75 % sequence identity to about 90 % sequence identity, about 75 % sequence identity to about 95 % sequence identity, about 75 % sequence identity to about 96 % sequence identity, about 75 % sequence identity to about 97 % sequence identity, about 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 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, 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 % sequence identity to about 98 % sequence identity, about 95 % sequence identity to about 99 % sequence identity, about 95 % sequence identity to about 100 % sequence identity, about 96 % sequence identity to about 97 % sequence identity, about 96 % sequence identity to about 98 % sequence identity, about 96 % sequence identity to about 99 % sequence identity, about 96 % sequence identity to about 100 % sequence identity, about 97 % sequence identity to about 98 % sequence identity, about 97 % sequence identity to about 99 % sequence identity, about 97 % sequence identity to about 100 % sequence identity, about 98 % sequence identity to about 99 % sequence identity, about 98 % sequence identity to about 100 % sequence identity, or about 99 % sequence identity to about 100 % sequence identity, to SEQ ID NO: 29. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a R5F 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: 20.

[0186] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a R76F mutation and comprises about 75 % sequence identity to about 100 % sequence identity to SEQ ID NO: 21. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a R76F mutation and comprises at least about 75 % sequence identity SEQ ID NO: 21. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a R76F mutation and comprises at most about 100 % sequence identity SEQ ID NO: 21. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a R76F mutation and comprises about 75 % sequence identity to about 80 % sequence identity, about 75 % sequence identity to about 85 % sequence identity, about 75 % sequence identity to about 90 % sequence identity, about 75 % sequence identity to about 95 % sequence identity, about 75 % sequence identity to about 96 % sequence identity, about 75 % sequence identity to about 97 % sequence identity, about 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 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, 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 % sequence identity to about 98 % sequence identity, about 95 % sequence identity to about 99 % sequence identity, about 95 % sequence identity to about 100 % sequence identity, about 96 % sequence identity to about 97 % sequence identity, about 96 % sequence identity to about 98 % sequence identity, about 96 % sequence identity to about 99 % sequence identity, about 96 % sequence identity to about 100 % sequence identity, about 97 % sequence identity to about 98 % sequence identity, about 97 % sequence identity to about 99 % sequence identity, about 97 % sequence identity to about 100 % sequence identity, about 98 % sequence identity to about 99 % sequence identity, about 98 % sequence identity to about 100 % sequence identity, or about 99 % sequence identity to about 100 % sequence identity to SEQ ID NO: 21. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises a R76F 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: 21.

[0187] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises an I8E mutation and comprises about 75 % sequence identity to about 100 % sequence identity to SEQ ID NO: 41. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises an I8E mutation and comprises at least about 75 % sequence identity SEQ ID NO: 41. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises an I8E mutation and comprises at most about 100 % sequence identity SEQ ID NO: 41. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises an I8E mutation and comprises about 75 % sequence identity to about 80 % sequence identity, about 75 % sequence identity to about 85 % sequence identity, about 75 % sequence identity to about 90 % sequence identity, about 75 % sequence identity to about 95 % sequence identity, about 75 % sequence identity to about 96 % sequence identity, about 75 % sequence identity to about 97 % sequence identity, about 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 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, 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 % sequence identity to about 98 % sequence identity, about 95 % sequence identity to about 99 % sequence identity, about 95 % sequence identity to about 100 % sequence identity, about 96 % sequence identity to about 97 % sequence identity, about 96 % sequence identity to about 98 % sequence identity, about 96 % sequence identity to about 99 % sequence identity, about 96 % sequence identity to about 100 % sequence identity, about 97 % sequence identity to about 98 % sequence identity, about 97 % sequence identity to about 99 % sequence identity, about 97 % sequence identity to about 100 % sequence identity, about 98 % sequence identity to about 99 % sequence identity, about 98 % sequence identity to about 100 % sequence identity, or about 99 % sequence identity to about 100 % sequence identity, to SEQ ID NO: 41. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises an I8E 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: 41.

[0188] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises an R5A mutation and comprises about 75 % sequence identity to about 100 % sequence identity to SEQ ID NO: 42. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises an R5A mutation and comprises at least about 75 % sequence identity SEQ ID NO: 42. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises an R5A mutation and comprises at most about 100 % sequence identity SEQ ID NO: 42. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises an R5A mutation and comprises about 75 % sequence identity to about 80 % sequence identity, about 75 % sequence identity to about 85 % sequence identity, about 75 % sequence identity to about 90 % sequence identity, about 75 % sequence identity to about 95 % sequence identity, about 75 % sequence identity to about 96 % sequence identity, about 75 % sequence identity to about 97 % sequence identity, about 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 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, 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 % sequence identity to about 98 % sequence identity, about 95 % sequence identity to about 99 % sequence identity, about 95 % sequence identity to about 100 % sequence identity, about 96 % sequence identity to about 97 % sequence identity, about 96 % sequence identity to about 98 % sequence identity, about 96 % sequence identity to about 99 % sequence identity, about 96 % sequence identity to about 100 % sequence identity, about 97 % sequence identity to about 98 % sequence identity, about 97 % sequence identity to about 99 % sequence identity, about 97 % sequence identity to about 100 % sequence identity, about 98 % sequence identity to about 99 % sequence identity, about 98 % sequence identity to about 100 % sequence identity, or about 99 % sequence identity to about 100 % sequence identity, to SEQ ID NO: 42. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises an R5A 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: 42.

[0189] In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises an R5E mutation and comprises about 75 % sequence identity to about 100 % sequence identity to SEQ ID NO: 43. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises an R5E mutation and comprises at least about 75 % sequence identity SEQ ID NO: 43. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises an R5E mutation and comprises at most about 100 % sequence identity SEQ ID NO: 43. In some embodiments, an IL-21 polypeptide or a functional fragment or a variant thereof comprises an R5E mutation and comprises about 75 % sequence identity to about 80 % sequence identity, about 75 % sequence identity to about 85 % sequence identity, about 75 % sequence identity to about 90 % sequence identity, about 75 % sequence identity to about 95 % sequence identity, about 75 % sequence identity to about 96 % sequence identity, about 75 % sequence identity to about 97 % sequence identity, about 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 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, 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 % sequence identity ...

Claims

11 08 251. A fusion protein comprising:a) an antibody or an antigen binding fragment thereof that specifically binds to at least one of CD8cr, CD8aa, or CD8aP, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; and wherein the VH domain 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; and wherein the VL domain 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; andb) an IL-21 polypeptide, wherein the IL-21 polypeptide comprises at least one amino acid substitution at a position selected from the group consisting of: R5,18, R9, RI 1, L13,114,116, V17, D18, K72, K73, L74, K75, R76, K77, and KI 17, numbered according to the sequence of SEQ ID NO: 1.

2. The fusion protein of claim 1, wherein the antibody or antigen binding fragment thereof comprises:i) a first polypeptide comprising the VL domain and a light chain constant domain;ii) a second polypeptide comprising the VH domain and a heavy chain constant region; andiii) a third polypeptide comprising the VH domain and a heavy chain constant region, wherein the heavy chain constant regions of the second and third polypeptides form an Fc domain.

3. The fusion protein of claim 2, wherein the antibody or antigen binding fragment thereof comprises:iv) a fourth polypeptide comprising the VL domain and a light chain constant region.11 08 254. The fusion protein of any one of claims 1-3, wherein the IL-21 polypeptide and theantibody or antigen binding fragment thereof are linked to each other via a linker, optionally wherein the IL-21 polypeptide is linked to the C-terminus of the antibody or antigen binding fragment thereof.

5. The fusion protein of claim 4, wherein the linker comprises (GGGS)xGn (SEQ ID NO:294) or (GGGGS)xGn (SEQ ID NO: 295), (GGGGGS)xGn (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), with x=l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 and n=0, 1, 2 or 3, optionally wherein the linker comprises the sequence SGGGGSGGGGSGGGGS (SEQ ID NO:292), or SGGGGSGGGGSGGGG (SEQ ID NO:293).

6. The fusion protein of any one of claims 2-5, wherein the IL-21 polypeptide is linked tothe C-terminus of the heavy chain constant region of the second polypeptide or the third polypeptide.

7. The fusion protein of any one of claims 2-6, wherein the Fc domain comprises one ormore modifications that promote heterodimerization, wherein the one or more modifications are selected from Y349C, T366W, S345C, T366S, L368A, or Y407V, according to EU numbering.

8. The fusion protein of any one of claims 2-7, wherein the second polypeptide comprises aknob modification in the heavy chain constant region, and the third polypeptide comprises a hole modification in the heavy chain constant region, optionally wherein 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.

9. The fusion protein of any one of claims 2-8, wherein the Fc domain comprises an aminoacid mutation that reduces binding of the Fc domain to an Fc gamma receptor, wherein the Fc domain comprises amino acid substitutions L234A, L235A, and G237A, according to EU numbering.11 08 2510. The fusion protein of any one of claims 1-9, 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 wherein 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.

11. The fusion protein of any one of claims 1-9, wherein the VH domain comprises theamino acid sequence of SEQ ID NO: 129, and wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 130.

12. The fusion protein of any one of claims 1-11, wherein the IL-21 polypeptide comprises an amino acid sequence that is at least 90% identical to a human IL-21 polypeptide comprising the sequence of SEQ ID NO: 1.

13. The fusion protein of any one of claims 1-11, wherein the IL-21 polypeptide comprises an amino acid sequence that differs from a human IL-21 polypeptide comprising the sequence of SEQ ID NO: 1 by 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ammo acids.

14. The fusion protein of any one of claims 1-13, wherein the IL-21 polypeptide does not comprise an amino acid substitution at G84 numbered according to the sequence of SEQ ID NO: 1.

15. The fusion protein of claim 1, wherein the at least one amino acid substitution is selected from the group consisting of: R5F, R5A, R5E, R5S, R5T, R5N, R5Q, R5V, R5I, R5L, R5Y, I8E, R9A, R9D, R9E, R9H, R9S, R9T, R9N, R9G, R9V, R9I, R9L, R9Y, RI ID, RI IE, L13F, L13R, I14D, I16A, I16S, I16R, V17I, V17A, D18A, K72A, K72E, K73A, K73E, K75A, K75E, L74I, L74F, L74M, L74V, R76E, R76F, R76A, R76N, R76D, R76S, R76T, R76Q, R76V, R76I, R76L, R76Y, R76M, K77A, K77E, and KI 17A, relative to the sequence of SEQ ID NO: 1.

16. The fusion protein of claim 15, wherein the at least one amino acid substitution is R76E or R76Q.11 08 2517. A polynucleotide encoding the fusion protein of any one of claims 1-16.

18. A vector comprising the polynucleotide of claim 17.

19. A host cell comprising the vector of claim 18.

20. A pharmaceutical composition comprising the fusion protein of any one of claims 1-16.

21. The pharmaceutical composition of claim 20:(i) for use as a medicament,(ii) for use in treating cancer or a chronic infection in a subject, wherein the use comprises administering the pharmaceutical composition to the subject; or(lii) for use in treating cancer in a subject, wherein the use comprises administering the pharmaceutical composition to the subject, and optionally further comprises administering to the subject a T cell therapy, cancer vaccine, chemotherapeutic agent, or immune checkpoint inhibitor (TCI), optionally wherein the ICI is an inhibitor of PD-1, PD-L1, or CTLA-4; and optionally wherein the T cell therapy comprises a chimeric antigen receptor (CAR)-based T cell therapy, a tumor-infiltrating lymphocyte (TIL)-based therapy, or a therapy with T cells bearing a transduced TCR.

22. The fusion protein of any one of claims 1-16:(i) for use as a medicament;(ii) for use in treating cancer or a chronic infection in a subject, wherein the use comprises administering the fusion protein to the subject; or(lii) for use in treating cancer in a subject, wherein the use comprises administering the fusion protein to the subject, and optionally further comprises administering to the subject a T cell therapy, cancer vaccine, chemotherapeutic agent, or immune checkpoint inhibitor (ICI); optionally wherein the ICT is an inhibitor of PD-1, PD-Ll, or CTLA-4; and optionally wherein the T cell therapy comprises a chimeric antigen receptor (CAR)-based T cell therapy, a tumorinfiltrating lymphocyte (TIL)-based therapy, or a therapy with T cells bearing a transduced TCR.

23. A kit comprising a container comprising the fusion protein of any one of claims 1-16, and a pharmaceutically acceptable carrier, and instructions for administering the fusion protein to a subject with a disease, optionally wherein the disease is a cancer or a chronic infection.

24. A method of making the fusion protein of any one of claims 1-16, comprising culturing the host cell of claim 19, and harvesting the fusion protein from the host cell.11 08 25

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