IL-21 polypeptide and targeted construct

Abstract

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

Description

[Technical Field] 【0001】 Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 190,669 filed on 19 May 2021; U.S. Provisional Application No. 63 / 223,684 filed on 20 July 2021; PCT / US2021 / 056312 filed on 22 October 2021; PCT / US2021 / 062458 filed on 8 December 2021; and U.S. Provisional Application No. 63 / 297,631 filed on 7 January 2022. The entire contents of each application are incorporated herein by reference. [Background technology] 【0002】 Interleukin-21 (IL-21) is a T cell-derived pleomorphic cytokine that regulates the activity of both innate and adoptive immune cells. IL-21 can enhance T cell survival and effector function. Interleukin-21 is a type I cytokine and a member of the common cytokine receptor gamma chain (γc) family, which has emerged as a promising immunotherapeutic agent for cancer treatment. In some cases, IL-21 produced by activated CD4+ T cells and natural killer T (NKT) cells signals a separate IL-21 receptor (IL-21R) subunit with γc via a heterodimeric receptor complex. In some cases, activation of the IL-21R complex leads to activation of the JAK / STAT signaling pathway. IL-21R is widely expressed in hematopoietic cells, including T and B lymphocytes, natural killer (NK) cells, and myeloid cells. IL-21 is a potent mitogen and survival factor for both NK cells and activated T cells. IL-21 can assist the differentiation of CD4+ T helper 17 (Th17) and follicular helper T cells (Tfh), and block the differentiation of regulatory T cells (Treg). Furthermore, IL-21 can enhance the survival of CD8+ T cells, resulting in a less activated but more persistent T cell phenotype, leading to improved tumor and viral control. A challenge of cytokine immunotherapy is that, in some cases, while activating immune cells to enhance the immune response, the same cytokines can also activate antagonistic regulatory pathways, as exemplified by IL-2 and IFNγ. These antagonistic pathways can activate regulatory T cell responses and inhibitory pathways. Because it plays a role in antitumor and antiviral responses, in addition to exerting a major effect against inflammatory responses that lead to the development of autoimmune and inflammatory diseases, IL-21 is an attractive target for several therapies. 【0003】 There is still a need for IL-21-based treatment modalities, including sites that combine IL-21 modalities to induce IL-21 in specific cell types. [Overview of the project] 【0004】 In one embodiment of the present disclosure, an IL-21 polypeptide or a functional fragment or variant thereof is provided, comprising a polypeptide sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein the IL-21 polypeptide has an isoelectric point at least about 0.6 to about 5 units lower than that of the wild-type IL-21 protein having the sequence of SEQ ID NO: 1. In some embodiments, the isoelectric point of SEQ ID NO: 1 is about 9.42. In some embodiments, the IL-21 polypeptide has an isoelectric point of about 7.12 to about 8.72. In some embodiments, the polypeptide provides improved exposure compared to wild-type IL-21 when administered to a subject at equivalent concentrations, as measured by an under-curve (AUC) at least about 1.5 times higher for the polypeptide compared to the wild-type IL-21 protein. In some embodiments, the IL-21 polypeptide comprises at least one amino acid substitution, which reduces the isoelectric point of the IL-21 polypeptide by about 0.6 to about 5 units compared to a human IL-21 polypeptide without an amino acid substitution. In some embodiments, the IL-21 polypeptide contains at least four amino acid substitutions that reduce the isoelectric point of the IL-21 polypeptide by about 0.6 to about 5 units compared to a human IL-21 polypeptide without amino acid substitutions. In some embodiments, the IL-21 polypeptide contains 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 the IL-21 polypeptide comprises at least one amino acid substitution of about 2 to 20 positively charged amino acid residues, compared to a region of a human IL-21 polypeptide comprising about 2 to 20 positively charged amino acid residues. In some embodiments, the region of the human IL-21 polypeptide does not contain an amino acid residue that binds to the 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 the human IL-21 polypeptide comprises at least one positively charged amino acid residue on the surface of the human IL-21 polypeptide in its three-dimensional structure, does not directly interact with or bind to the human IL-21 receptor, and 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 have an amino acid substitution in G84 of the human IL-21 polypeptide comprising SEQ ID NO: 1. 【0006】 In some embodiments, the IL-21 polypeptide contains mutations 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 contains mutations (e.g., amino acid substitutions) 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 includes 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 includes 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 a human IL-21 polypeptide that does not have an amino acid substitution at G84, including SEQ ID NO: 1. 【0007】 In some embodiments, the present disclosure relates to the amino acid sequence: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPX1X2X3X4GX5X6X7X8X9X 10 X 11 X 12CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 380) (where X1 = G, S; X2 = G, T; X3 = G, E, N; X4 = G, S, E, A; X5 = G, E, S, R; X6 = G, E, R; X7 = S, G, E, Q; X8 = G, K; X9 = G, S, H; X 10 = A, E, S, G, R; X 11 = S, G, L; and X 12 = G, S, T, provided that X 5、 X 6、 X 8、 X9, and X 10 at least one of which is not the amino acid residue at the same position shown in SEQ ID NO: 1, and optionally: (i) X5 = G, X6 = G, X8 = G, X 10 = A; (ii) X5 = G, X6 = G, X8 = G, X 10 = E; (iii) X1 = G, X2 = G, X3 = G, X4 = S, X5 = E, X6 = G, X7 = S, X8 = G, X9 = G, X 10 = S; (iv) X1 = G, X2 = G, X3 = G, X4 = S, X5 = G, X6 = G, X7 = S, X8 = G, X9 = G, X 10 = E; (v) X5 = G, X6 = G, X7 = S, X8 = G, X9 = G, X 10 = E; (vi) X5 = G, X6 = G, X7 = S, X8 = G, X9 = G, X 10 = S; (vii) X5 = G, X6 = G, X7 = G, X8 = G, X9 = G, X 10 = E; (viii) X5 = G, X6 = G, X7 = G, X8 = G, X9 = G, X 10 = G; (ix) X5 = G, X6 = G, X7 = E, X8 = G, X9 = G, X 10 = G; (x) X1 = G, X2 = G, X3 = G, X4 = S, X5 = G, X6 = G, X7 = S, X8 = G, X9 = G, X 10 = G, X 11 = S, X 12 = G; (xi)X1=G、X2=G、X3=G、X4=G、X5=G、X6=G、X7=G、X8=G、X9=G、X 10 =G、X 11 =G、X 12 =G; (xii)X1=G、X2=G、X3=G、X4=S、X5=G、X6=G、X7=E、X8=G、X9=G、X 10 =G、X 11 =S、X 12 =G; (xiii)X1=G、X2=G、X3=G、X4=E、X5=G、X6=G、X7=E、X8=G、X9=G、X 10 =G、X 11 =S、X 12 =G; (xiv)X3=G、X4=G、X5=S、X6=G、X7=G、X8=G、X9=S、X 10 =G、X 11 =G、X 12 =S; (xv)X3=G、X4=G、X5=E、X6=G、X7=G、X8=G、X9=S、X 10 =G、X 11 =G、X 12 =S; (xvi)X1=G、X2=G、X3=G、X4=G、X5=E、X6=G、X7=G、X8=G、X9=G、X 10 =G、X 11 =G、X 12 =G; (xvii)X1=G、X2=G、X3=G、X4=S、X5=G、X6=G、X7=S、X8=G、X9=G、X 10 =E、X 11 =G、X 12 =G; (xviii)X1=G、X2=G、X3=G、X4=G、X5=G、X6=G、X7=G、X8=G、X9=G、X 10 =E、X 11 =G、X 12 =G; (xix)X3=G、X4=G、X5=S、X6=G、X7=G、X8=G、X9=S、X 10 =E、X 11 =G、X 12 =S; (xx)X3=G, X4=G, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E, X 11 =G, X 12 =G; (xxi)X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E, X 11 =G; (xxii)X3=G, X4=G, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E, X 11 =G; (xxiii)X3=G, X4=G, X5=G, X6=E, X7=G, X8=G, X9=G, X 10 =E, X 11 =G; (xxiv)X3=G, X4=G, X5=E, X6=G, X7=G, X8=G, X9=G, X 10 =E, X 11 =G; (xxv)X1=G, X2=G, X3=G, X4=E, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E, X 11 =G, X 12 =G; or (xxvi)X1=G, X2=G, X3=E, X4=G, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E, X 11 =G, X 12 The present disclosure provides an IL-21 polypeptide containing (=G) or a functional fragment or variant thereof. In some embodiments, the present disclosure provides a human IL-21 polypeptide containing SEQ ID NO: 1 that does not have an amino acid substitution at G84. 【0008】 In some embodiments, the disclosure provides an IL-21 polypeptide or a functional fragment or variant thereof containing an amino acid sequence that is 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 contains an amino acid sequence selected from SEQ ID NOs. 2, 4-15, and 23-40. In some embodiments, the IL-21 polypeptide contains the amino acid sequence of SEQ ID NO. 40. 【0009】 In some embodiments, the disclosure provides an IL-21 polypeptide or a functional fragment or variant thereof comprising at least one amino acid substitution that reduces binding to the 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, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, or K117 of SEQ ID NO: 1. In some embodiments, at least one amino acid substitution is R5F, R5A, R5E, R5S, R5T, R5N, R5Q, R5V, R5I, R5L, R5Y, I8E, R9A, R9D, R9E, R9H, R9S, R9T, R9N, R9G, R9V, R9I, R9L, R9Y, R11D, R11E, L13F, L13R, I14D, I16A, I16S, The amino acids selected are I16R, V17I, V17A, D18A, K72A, K72E, K73A, K73E, K75A, K75E, L74I, L74F, L74M, L74V, R76E, R76F, R76A, R76N, R76D, R76S, R76T, R76Q, R76V, R76I, R76L, R76Y, R76M, K77A, K77E, and K117A. In some embodiments, at least one amino acid substitution is R76E or R76Q. In some embodiments, the IL-21 polypeptide includes mutations at positions selected from the group consisting of R5, I8, R9, R11, Q12, I14, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117 of SEQ ID NO: 1. In some embodiments, the IL-21 polypeptide includes 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. 94–98. 【0010】 In one embodiment of the present disclosure, a targeted cytokine construct comprising an IL-21 polypeptide or a functional fragment or variant thereof and an antibody or an antigen-binding fragment thereof is provided. In some embodiments, the antibody or the antigen-binding fragment specifically binds to CD8+ T cells. In some embodiments, the antibody or the antigen-binding fragment specifically binds to at least one of CD8α, CD8αα, or CD8αβ. In some embodiments, the antibody or the antigen-binding fragment specifically binds to CD8β. 【0011】 In one embodiment of the present disclosure, 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) a targeted cytokine construct comprising an antibody or an antigen-binding fragment thereof that specifically binds to at least one of CD8α, CD8αα, or CD8αβ. 【0012】 In this specification, in one embodiment of the present disclosure, a) an IL-21 polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 1 or a functional fragment or variant thereof; and b) a targeted cytokine construct comprising an antibody or an antigen-binding fragment thereof that specifically binds to CD8β. 【0013】 In some embodiments, the IL-21 polypeptide contains mutations at one or more amino acid positions in SEQ ID NO: 1. In some embodiments, the IL-21 polypeptide contains mutations 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 in SEQ ID NO: 1. In some embodiments, the IL-21 polypeptide contains mutations at positions selected from the group consisting of R5, I8, R9, R11, Q12, I14, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117 in SEQ ID NO: 1. In some embodiments, the IL-21 polypeptide or a functional fragment or variant thereof contains 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 its functional fragment or variant contains 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 times or more potency compared to the activation of NK or CD4+ T cells. In some embodiments, the construct activates CD8+ T cells with at least about 10 times to about 100,000 times more potency compared to the activation of NK or CD4+ T cells. In some embodiments, the antibody or its antigen-binding fragment comprises: i) a first polypeptide comprising a variable light chain (VL) amino acid sequence and a light chain constant region amino acid sequence (CL1) located from N to the C terminus; ii) a second polypeptide comprising a variable heavy chain (VH) amino acid sequence, a heavy chain CH1 constant region amino acid sequence, a hinge region amino acid sequence, a heavy chain CH2 constant region amino acid sequence, and a heavy chain CH3 constant region amino acid sequence located from N to the C terminus; and iii) a third polypeptide 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 located from N to the C terminus, wherein the CH2 and CH3 domains of the second and third polypeptides respectively form an Fc domain. In some embodiments, the third polypeptide comprises a variable heavy chain (VH) amino acid sequence, a heavy chain CH1 constant region amino acid sequence, a hinge region amino acid sequence, a heavy chain CH2 constant region amino acid sequence, and a heavy chain CH3 constant region amino acid sequence, arranged from N to the C terminus, and the antibody or its antigen-binding fragment further comprises a fourth polypeptide, comprising a variable light chain (VL) amino acid sequence and a light chain constant amino acid sequence (CL1), arranged from N to the C terminus. In some embodiments, the IL-21 polypeptide or its functional fragment or variant and the antibody or its antigen-binding fragment are functionally linked to each other.In some embodiments, the IL-21 polypeptide or a functional fragment or variant thereof is ligated to the N-terminus or C-terminus of an antibody or its antigen-binding fragment. In some embodiments, the IL-21 polypeptide or a functional fragment or variant thereof is conjugated to the C-terminus of a second or third polypeptide. In some embodiments, the targeted cytokine construct comprises at least one molecule of the IL-21 polypeptide. In some embodiments, the Fc domain is a human IgG Fc domain. In some embodiments, the Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the Fc domain comprises one or more modifications that promote heterodimerization. In some embodiments, the second polypeptide comprises a knob modification in the CH2 or CH3 domain and the third polypeptide comprises a hole modification in the CH2 or CH3 domain; or the third polypeptide comprises a knob modification in the CH2 or CH3 domain and the second polypeptide comprises a hole modification in the CH2 or CH3 domain. In some embodiments, at least one of the second and third polypeptides includes the following mutations, numbered according to the EU index: L234A, L235A, and G237A. 【0015】 In one embodiment of the present disclosure, a targeted cytokine construct is provided, comprising: a) an IL-21 polypeptide or a functional fragment or variant thereof having 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 having a first antigen-binding arm and a second antigen-binding arm, wherein the first and second antigen-binding arms bind to two different antigens, and at least one of the first and second antigen-binding arms specifically binds to CD8α, CD8αα, and CD8αβ. 【0016】 In one embodiment of the present disclosure, a targeted cytokine construct is provided, comprising: a) an IL-21 polypeptide or a functional fragment or variant thereof having an amino acid sequence 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 second antigen-binding arms bind to two different antigens, and at least one of the first and second antigen-binding arms specifically binds to CD8β. In some embodiments, the targeted cytokine construct activates CD8+ T cells with at least about 10 times or more potency compared to the activation of NK cells or CD4+ T cells. In some embodiments, the targeted cytokine construct activates CD8+ T cells with at least about 10 times to about 100,000 times more potency compared to the activation of NK cells or CD4+ T cells. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains 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 its functional fragment or variant contains a mutation at a position selected from the group consisting of R5, I8, R9, R11, Q12, I14, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117 of SEQ ID NO: 1. In some embodiments, the IL-21 polypeptide or a functional fragment or variant thereof contains 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 variant thereof contains an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. 【0017】 In some embodiments, the present disclosure is a fusion protein, a) (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide containing SEQ ID NO: 1, wherein one or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least one amino acid substitution is in residues R85, R86, K88, H89, R90, or a combination thereof; and (ii) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide; and b) An antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β. The invention provides a fusion protein containing the following: In some embodiments, the IL-21 polypeptide does not contain the G84 substitution. In some embodiments, at least one amino acid substitution that provides reduced binding is R76E or R76Q. 【0018】 In some embodiments, the present disclosure is a fusion protein, a) (i) four or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide containing SEQ ID NO: 1, wherein the four or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least four of the amino acid substitutions are in residues R85, R86, K88, H89, R90, or a combination thereof; and (ii) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide; and b) An antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β. The invention provides a fusion protein that includes the following: 【0019】 In some embodiments, the present disclosure is a fusion protein, a) (i) an IL-21 polypeptide comprising four or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the four or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least four of the amino acid substitutions are in residues R85, R86, K88, H89, R90, or combinations thereof; and (ii) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide, wherein at least one amino acid substitution is in a residue selected from R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, K117, and combinations thereof; and b) An antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β. The invention provides a fusion protein that includes the following: 【0020】 In some embodiments, the present disclosure is a fusion protein, a)(i) Four or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide containing SEQ ID NO: 1, wherein the four or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least four of the amino acid substitutions are in residues R85, R86, K88, H89, R90, or a combination thereof, and (ii)(a)R11D;(b)R11E;(c)I14D, D18A and K117A;(d)R76E;(e)R5F;(f)R76F ;(g)I8E;(h)R5A;(i)R5E;(j)R5S;(k)R5T;(l)R5N;(m)R5Q;(n)R5V;(o)R5I;(p)R5L;(q)R5Y;(r)R76A;(s)R76N;(t)R76D;(u)R76S;(v )R76T;(w)R76Q;(x)R76V;(y)R76I;(z)R76L;(aa)R76Y;(bb)K77A;(cc)K77E;(dd)K72A;(ee)K72E;(ff)K75A;(gg)K75E;(hh)K73A;(ii )K73E;(jj)R5F and K77A;(kk)R5F and K77E;(ll)R5F and K72A;(mm)R5F and K72E;(nn)R5F and K76A;(oo)R5F and K76E;(pp)K73A and K76F;(qq)K73E and K76F;(rr)R9A;(ss)R9D;(tt)R9E;(uu)R9H;(vv)R9S;(ww)R9T;(xx)R9N;(zz)R9G;(aaa)R9V;(bbb)R9I;(ccc)R9L;(ddd)R9Y;(eee) IL-21 polypeptides comprising at least one amino acid substitution selected from K72A and R76F; (fff)K75A and R76F; (ggg)R76F and K77A; (hhh)K75E and R76F; (iii)V17I and L74I; (jjj)I16A and L74F; (kkk)I16S, V17I, and L74V; (lll)I16R, V17I, and L74I; (mmm)L13F, I16A, V17A, and L74M; and (nnn)L13R, I16A, V17I, and L74I; and b) An antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β. The invention provides a fusion protein that includes the following: 【0021】 In some embodiments, the present disclosure is a fusion protein, a)(i) an IL-21 polypeptide comprising four or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein at least four of the amino acid substitutions are in residues S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, T92, or combinations thereof; and (ii) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide, wherein at least one of the amino acid substitutions is in a residue selected from R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, K117, and combinations thereof; and b) An antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β. The invention provides a fusion protein that includes the following: 【0022】 In some embodiments, the present disclosure is a fusion protein, a)(i) an IL-21 polypeptide comprising four or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein at least four amino acid substitutions are in residues S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, T92, or combinations thereof, and (ii) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide, wherein at least one amino acid substitution is in residue R76; and b) An antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β. The invention provides a fusion protein that includes the following: 【0023】 In some embodiments, the present disclosure is a fusion protein, a) (i) an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and (ii) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide; and b) An antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β. The invention provides a fusion protein that includes the following: 【0024】 In some embodiments, the present disclosure is a fusion protein, a) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by human IL-21 polypeptide, wherein the at least one amino acid substitution is in a residue selected from R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, K117, and combinations thereof; and b) An antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β. The invention provides a fusion protein that includes the following: 【0025】 In some embodiments, the present disclosure is a fusion protein, a) (i) an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and (ii) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide, wherein at least one amino acid substitution is at residue R76; and b) An antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β. The invention provides a fusion protein that includes the following: 【0026】 In some embodiments, the IL-21 polypeptide and the antibody or its antigen-binding fragment 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 its antigen-binding fragment. In some embodiments, the IL-21 polypeptide is linked to the C-terminus of the antibody or its antigen-binding fragment. 【0027】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is functionally ligated to the C-terminus of the HC constant domain, and the HC constant domain comprises a knob modification; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The VH chain and VL chain contain CD8αβ or CD8β-specifically bound CDRs. The IL-21 polypeptide provides a fusion protein comprising (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein one or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least one amino acid substitution is in residues R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0028】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally linked to the C-terminus of the HC constant domain, the HC constant domain includes hole modifications, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The VH chain and VL chain contain CD8αβ or CD8β-specifically bound CDRs. The IL-21 polypeptide provides a fusion protein comprising (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein one or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least one amino acid substitution is in residues R85, R86, K88, H89, R90, or a combination thereof; and (ii) at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0029】 In one embodiment of this disclosure, a polynucleotide encoding an IL-21 polypeptide or a functional fragment or variant thereof as described in any one of claims 1 to 8 is provided. 【0030】 In one embodiment of the present disclosure, a polynucleotide is provided which encodes a targeted cytokine construct comprising an IL-21 polypeptide or a functional fragment or variant thereof and an antibody or an antigen-binding fragment thereof, wherein the polynucleotide comprises a coding sequence for the IL-21 polypeptide and a coding sequence for the antibody or its antigen-binding fragment. 【0031】 In one embodiment of this disclosure, a vector comprising the polynucleotides of this disclosure is provided. 【0032】 In one embodiment of this disclosure, a host cell comprising a polynucleotide or vector of this disclosure is provided. 【0033】 In one embodiment of the present disclosure, a pharmaceutical composition comprising an IL-21 polypeptide or a functional fragment or variant thereof and a pharmaceutically acceptable carrier is provided. 【0034】 In this specification, in one embodiment of the present disclosure, a pharmaceutical composition comprising a targeted cytokine construct and a pharmaceutically acceptable carrier according to the present disclosure is provided. 【0035】 In this specification, in one embodiment of the present disclosure, a method for selective activation of CD8+ T cells is provided, the method comprising contacting a population of cells comprising CD8+ T cells, CD4+ T cells, and NK cells with a targeted cytokine construct according to the present disclosure. In some embodiments, selective activation includes activation of CD8+ T cells at least about 10 times or higher potency compared to activation of NK cells or CD4+ T cells in a population of cells. In some embodiments, selective activation includes activation of CD8+ T cells at least about 10 times to about 100,000 times higher potency compared to activation of NK cells or CD4+ T cells in a population of cells. In some embodiments, selective activation of CD8+ T cells results in increased STAT3 phosphorylation of CD8+ T cells compared to STAT3 phosphorylation of NK cells or CD4+ T cells in a population of cells. 【0036】 In one embodiment of the present disclosure, a method is provided for treating a disease in a subject, the method comprising administering an IL-21 polypeptide or a functional fragment or variant thereof according to the present disclosure, a targeted cytokine construct according to the present disclosure, or a pharmaceutical composition according to the present disclosure. In some embodiments, the method further comprises additional therapeutic agents. In some embodiments, the disease includes cancer or a chronic infection. In some embodiments, the disease includes cancer, and cancer is acute lymphoblastic leukemia (ALL) (including non-T cell ALL), acute myeloid leukemia, B-cell prelymphoblastic leukemia, B-cell acute lymphoblastic leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasm, Burkitt 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 disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal immunoglobulinemia of unknown significance (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma (NHL), plasma cell proliferative disorders (asymptomatic myeloma (including smoldering multiple myeloma or painless myeloma), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, plasmacytoma (including plasma cell cachexia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndromes (Crow-Fukase syndrome; Takatsuki disease;(also known as PEP syndrome), mediastinal large B-cell lymphoma (PMBC), small cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoblastic leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma, or Valdenström 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 cancer (NSCL), bronchioloalveolar cell lung cancer, squamous cell carcinoma, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, head and neck cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, thyroid cancer, uterine cancer, gastrointestinal cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer This includes cancers such as gastric cancer, colon cancer, breast cancer, endometrial cancer, uterine cancer, fallopian tube cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancers, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcomas, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, bladder cancer, liver cancer, hepatocellular carcinoma, cervical cancer, salivary gland cancer, bile duct cancer, neoplasms of the central nervous system (CNS), spinal axial tumors, brainstem gliomas, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma (including refractory versions of any of the above cancers, or combinations of one or more of the above cancers). 【0037】 In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; (a) the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 137, CDR-H2 containing the amino acid sequence of SEQ ID NO: 138, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 139; the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 140, CDR-L2 containing the amino acid sequence of SEQ ID NO: 141, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 142; (b (c) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 149, CDR-H2 containing the amino acid sequence of SEQ ID NO: 150, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 151; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 152, CDR-L2 containing the amino acid sequence of SEQ ID NO: 153, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 154; (c) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 155, CDR-H2 containing the amino acid sequence of SEQ ID NO: 156, (d) The VH domain includes CDR-H3 containing the amino acid sequence of SEQ ID NO: 157; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 158, CDR-L2 containing the amino acid sequence of SEQ ID NO: 159, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 160; (d) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 161, CDR-H2 containing the amino acid sequence of SEQ ID NO: 162, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 163; the VL domain includes CDR containing the amino acid sequence of SEQ ID NO: 164 - The domain includes CDR-L1, CDR-L2 containing the amino acid sequence of SEQ ID NO: 165, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 166; (e) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 167, CDR-H2 containing the amino acid sequence of SEQ ID NO: 168, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 169; The VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 170, CDR-L2 containing the amino acid sequence of SEQ ID NO: 171, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 172;(f) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 173, CDR-H2 containing the amino acid sequence of SEQ ID NO: 174, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 175; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; (g) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 179, CDR-H2 containing the amino acid sequence of SEQ ID NO: 180, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 181. (i) The VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 182, CDR-L2 containing the amino acid sequence of SEQ ID NO: 183, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 184; (h) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 143, CDR-H2 containing the amino acid sequence of SEQ ID NO: 144, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 145; The VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 146, CDR-L2 containing the amino acid sequence of SEQ ID NO: 147, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 148;(i) The VH domain contains the amino acid sequence X1X2AIS (wherein X1 is S, K, G, N, R, D, T, or G, and X2 is Y, L, H, or F) (Sequence ID 185) CDR-H1, X1X2X3PX4X5X6X7X8X9YX10QKFX11G (wherein X1 is G or H, X2 is I or F, X3 is I, N, or M, X4 is G, N, H, S, R, I, or A, X5 is A, N, H, S, T, F, or Y, and X6 is A, D, or G, CDR-H2 containing the amino acid sequence X7 is T, E, K, V, Q, or A, X8 is A or T, X9 is N or K, X10 is A or N, and X11 is Q or T) (Sequence ID 186), and X1X2X3GX4X5LFX6X7 (wherein X1 is D or A, X2 is A, G, E, R, Y, K, N, Q, L, or F, X3 is A, L, P, or Y, X4 is I or L, X5 is R, A, Q, or S, X6 is A or D, X7 CDR-H3 contains the amino acid sequence X1X2SX3X4IX5GX6LN (wherein X1 is R or G, X2 is A or T, X3 is Q or E, X4 is E, N, T, S, A, K, D, G, R, or Q, X5 is Y or S, and X6 is A or V) (Sequence ID 188), GX1X2X3LX4X5 (wherein X1 is A or S, X2 is T, S, CDR-L2 contains the amino acid sequence QX1X2X3X4X5PWT (wherein X1 is S, N, D, Q, A, or E, X2 is T, I, or S, X3 is Y, L, or F, X4 is D, G, T, E, Q, A, or Y, X5 is A, T, R, S, K, or Y) (Sequence ID 190);(j) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 200, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 201; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 152, CDR-L2 containing the amino acid sequence of SEQ ID NO: 153, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 202; (k) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and SEQ ID NO: 20 (1) The VH domain contains CDR-H3 containing the amino acid sequence of 4; the VL domain contains CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (1) The VH domain contains CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; the VL domain contains CDR-L1 containing the amino acid sequence of SEQ ID NO: 152, and the amino acid sequence of SEQ ID NO: 153 CDR-L2 contains the amino acid sequence of SEQ ID NO: 202, and CDR-L3 contains the amino acid sequence of SEQ ID NO: 202; the (m)VH domain contains the amino acid sequence of X1YX2MS (wherein X1 is S, D, E, A, or Q, and X2 is A, G, or T) (SEQ ID NO: 208), CDR-H1 contains the amino acid sequence of DIX1X2X3GX4X5TX6YADSVKG (wherein X1 is T, N, S, Q, E, H, R, or A, X2 is Y, W, F, or H, X3 is A, S, Q, E, or T, X4 is G or E, and X5 is S or I, CDR-H2 comprises the amino acid sequence X6 is A or G (Sequence ID 209), and CDR-H3 comprises the amino acid sequence X1X2X3YX4WX5X6AX7DX8 (wherein X1 is S or A, X2 is N, H, A, D, L, Q, Y, or R, X3 is A, N, S, or G, X4 is A, V, R, E, or S, X5 is D or S, X6 is D, N, Q, E, S, T, or L, X7 is L, F, or M, and X8 is I, Y, or V) (Sequence ID 210);The VL domain includes CDR-L1 containing the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 176), CDR-L2 containing the amino acid sequence of GASSRAT (SEQ ID NO: 177), and CDR-L3 containing the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 178); (n) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 220, CDR-H2 containing the amino acid sequence of SEQ ID NO: 221, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 222; the VL domain includes CDR-L1 containing the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 176), CDR-L2 containing the amino acid sequence of GASSRAT (SEQ ID NO: 177), and CDR-L3 containing the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 178); (o) The VH domain includes the amino acid sequence of SEQ ID NO: 220 The CDR-H1 domain includes CDR-H2 containing the amino acid sequence of SEQ ID NO: 260, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 222; the VL domain includes CDR-L1 containing the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 176), CDR-L2 containing the amino acid sequence of GASSRAT (SEQ ID NO: 177), and CDR-L3 containing the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 178); or the (p)VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 252, CDR-H2 containing the amino acid sequence of SEQ ID NO: 253, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 254; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 255, CDR-L2 containing the amino acid sequence of SEQ ID NO: 256, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 257. 【0038】 In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; and (a) the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 226, CDR-H2 containing the amino acid sequence of SEQ ID NO: 227, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 151; the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 152, CDR-L2 containing the amino acid sequence of SEQ ID NO: 153, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 154; (b) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 228, CDR-H2 containing the amino acid sequence of SEQ ID NO: 227, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 157; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 158, CDR-L2 containing the amino acid sequence of SEQ ID NO: 159, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 160; (c) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 223, and CDR-H2 containing the amino acid sequence of SEQ ID NO: 227 (d) The VL domain includes CDR-H3 containing the amino acid sequence of SEQ ID NO: 163; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 164, CDR-L2 containing the amino acid sequence of SEQ ID NO: 165, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 166; (d) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 227, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 169; the VL domain includes CD containing the amino acid sequence of SEQ ID NO: 170 (e) The VH domain includes R-L1, CDR-L2 containing the amino acid sequence of SEQ ID NO: 171, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 172; (e) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 230, CDR-H2 containing the amino acid sequence of SEQ ID NO: 231, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 175; The VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178;(f) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 230, CDR-H2 containing the amino acid sequence of SEQ ID NO: 232, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 181; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 182, CDR-L2 containing the amino acid sequence of SEQ ID NO: 183, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 184; (g) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 223, CDR-H2 containing the amino acid sequence of SEQ ID NO: 224, and the amino acid sequence of SEQ ID NO: 225 The (h)VH domain includes CDR-H3; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 140, CDR-L2 containing the amino acid sequence of SEQ ID NO: 141, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 142; the (h)VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 233, CDR-H2 containing the amino acid sequence of SEQ ID NO: 234, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 145; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 146, CDR-L2 containing the amino acid sequence of SEQ ID NO: 147, and SEQ ID NO: 14 CDR-L3 containing 8 amino acid sequences; (i) The VH domain contains CDR-H1, X1PX2X3X4X5 (wherein X1 is G, Y, S, or A, X2 is T, S, G, R, N, or H, X3 is S, T, R, H, Y, G, or P, X4 is S, K, G, N, R, D, T, or G, X5 is Y, L, H, or F) (Sequence ID 235) containing the amino acid sequence GX1X2X3X4X5 (wherein X1 is I, N, or M, X2 is G, N, H, S, R, I, or A, X3 is A, N, H, S, CDR-H2 containing the amino acid sequence X1X2X3GX4X5LFX6X7 (wherein X1 is D or A, X2 is A, G, E, R, Y, K, N, Q, L, or F, X3 is A, L, P, or Y, X4 is I or L, X5 is R, A, Q, or S, X6 is A or D, X7 is D, E, A, or S) (Sequence ID 237);The VL domain contains the amino acid sequence X1X2SX3X4IX5GX6LN (wherein X1 is R or G, X2 is A or T, X3 is Q or E, X4 is E, N, T, S, A, K, D, G, R, or Q, X5 is Y or S, and X6 is A or V) (Sequence ID 188) CDR-L1, GX1X2X3LX4X5 (wherein X1 is A or S, X2 is T, S, E, Q, or D, X3 is N, R, A, E, or H, X4 is Q or A, X5 (j) The VH domain includes CDR-L2 containing the amino acid sequence of (SEQ ID NO: 189) (wherein X1 is S or D), and CDR-L3 containing the amino acid sequence of QX1X2X3X4X5PWT (wherein X1 is S, N, D, Q, A, or E, X2 is T, I, or S, X3 is Y, L, or F, X4 is D, G, T, E, Q, A, or Y, and X5 is A, T, R, S, K, or Y) (SEQ ID NO: 190); (j) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, and the amino acid sequence of SEQ ID NO: 242 The VL domain includes CDR-H2 containing the amino acid sequence of SEQ ID NO: 204, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 152; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 153, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 202; the (k)VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; the VL domain includes the amino acid sequence of SEQ ID NO: 205 (l) The VH domain includes CDR-L1, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (l) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; The VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 152, CDR-L2 containing the amino acid sequence of SEQ ID NO: 153, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 202;The (m)VH domain contains the amino acid sequence GFTFX1X2Y (wherein X1 is S, D, E, Q, S, or A, and X2 is S, D, E, A, or Q) (Sequence ID 244) in CDR-H1, X1X2X3GX4X5 (wherein X1 is T, N, S, Q, E, H, R, or A, X2 is Y, W, F, or H, X3 is A, S, Q, E, or T, X4 is G or E, and X5 is S or I) (Sequence ID 245) in CDR-H2, X1X2X3YX4WX5X6AX The CDR-H3 contains the amino acid sequence of 7DX8 (wherein X1 is S or A, X2 is N, H, A, D, L, Q, Y, or R, X3 is A, N, S, or G, X4 is A, V, R, E, or S, X5 is D or S, X6 is D, N, Q, E, S, T, or L, X7 is L, F, or M, and X8 is I, Y, or V) (Sequence No. 246); the VL domain contains CDR-L1, GASSRAT (Sequence No. 176), which contains the amino acid sequence of RASQSVSSNLA (Sequence No. 176); The (n)VH domain includes CDR-L2 containing the amino acid sequence of (SEQ ID NO. 177), and CDR-L3 containing the amino acid sequence of QQYGSSPPVT (SEQ ID NO. 178); the VL domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO. 250, CDR-H2 containing the amino acid sequence of SEQ ID NO. 251, and CDR-H3 containing the amino acid sequence of SEQ ID NO. 288; the VL domain includes CDR-L1 containing the amino acid sequence of RASQSVSSNLA (SEQ ID NO. 176), CDR-L2 containing the amino acid sequence of GASSRAT (SEQ ID NO. 177), and QQYGSSP (o) The VH domain contains CDR-L3 containing the amino acid sequence of PVT (SEQ ID NO: 178); (o) The VH domain contains CDR-H1 containing the amino acid sequence of SEQ ID NO: 250, CDR-H2 containing the amino acid sequence of SEQ ID NO: 261, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 288; The VL domain contains CDR-L1 containing the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 176), CDR-L2 containing the amino acid sequence of GASSRAT (SEQ ID NO: 177), and CDR-L3 containing the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 178);Alternatively, the (p)VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 223, CDR-H2 containing the amino acid sequence of SEQ ID NO: 224, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 284; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 285, CDR-L2 containing the amino acid sequence of SEQ ID NO: 286, and CDR-L3 containing 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 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 the VL domain comprises at least the sequence of SEQ ID NO: 112 (c) The VH domain contains an amino acid sequence that is 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 113, and the VL domain contains an amino acid sequence that is 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 114; (d) The VH domain contains an amino acid sequence that is 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 115 (e) The VL domain contains 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; (f) The VH domain contains 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 the VL domain contains 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; (g) The VH domain contains an amino acid sequence that is at least 90%, (g) The VL domain contains an amino acid sequence that is at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 120; (g) The VH domain contains 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; the VL domain contains 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 contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 129, and the VL domain contains 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 contains 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; the VL domain contains an amino acid sequence that is at least 90%, at least identical to the sequence of SEQ ID NO: 132 (j) The VH domain contains an amino acid sequence that is 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 125; the VL domain contains 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 contains 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 (i) The VL domain contains 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; (i) The VH domain contains 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; the VL domain contains 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 contains 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 Both contain amino acid sequences that are 95%, at least 99%, or 100% identical; the VL domain contains amino acid sequences that are at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 136; the (n)VH domain contains amino acid sequences that are at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 107; the VL domain contains amino acid sequences that are at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 108;(o) The VH domain contains 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; the VL domain contains 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 contains 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; the VL domain contains 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 CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (b) The VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (c) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 220, CDR-H2 containing the amino acid sequence of SEQ ID NO: 221, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 222; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; or (d) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 250, CDR-H2 containing the amino acid sequence of SEQ ID NO: 251, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 288; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178. 【0041】 In some embodiments, (a) the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 129, and the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 130; or (b) the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 127, and the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 128. 【0042】 In some embodiments, the present disclosure is a fusion protein, a) (i) four or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide containing SEQ ID NO: 1, wherein the four or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least four of the amino acid substitutions are in residues R85, R86, K88, H89, R90, or a combination thereof; and (ii) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide; and b) comprising an antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β, wherein the antibody or antigen-binding fragment is (i) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (ii) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (iii) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 220, CDR-H2 containing the amino acid sequence of SEQ ID NO: 221, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 222; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; or (iv) VH domains comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 250, CDR-H2 containing the amino acid sequence of SEQ ID NO: 251, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 288; and VL domains comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178. The invention provides a fusion protein that includes the following: 【0043】 In some embodiments, the present disclosure is a fusion protein, a) (i) an IL-21 polypeptide comprising four or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the four or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least four of the amino acid substitutions are in residues R85, R86, K88, H89, R90, or combinations thereof; and (ii) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide, wherein at least one amino acid substitution is in a residue selected from R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, K117, and combinations thereof; and b) comprising an antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β, wherein the antibody or antigen-binding fragment is (i) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (ii) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (iii) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 220, CDR-H2 containing the amino acid sequence of SEQ ID NO: 221, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 222; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; or (iv) VH domains comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 250, CDR-H2 containing the amino acid sequence of SEQ ID NO: 251, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 288; and VL domains comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178. The invention provides a fusion protein that includes the following: 【0044】 In some embodiments, the present disclosure is a fusion protein, a)(i) Four or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide containing SEQ ID NO: 1, wherein the four or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least four of the amino acid substitutions are in residues R85, R86, K88, H89, R90, or a combination thereof, and (ii)(a)R11D;(b)R11E;(c)I14D, D18A and K117A;(d)R76E;(e)R5F;(f)R76F ;(g)I8E;(h)R5A;(i)R5E;(j)R5S;(k)R5T;(l)R5N;(m)R5Q;(n)R5V;(o)R5I;(p)R5L;(q)R5Y;(r)R76A;(s)R76N;(t)R76D;(u)R76S;(v )R76T;(w)R76Q;(x)R76V;(y)R76I;(z)R76L;(aa)R76Y;(bb)K77A;(cc)K77E;(dd)K72A;(ee)K72E;(ff)K75A;(gg)K75E;(hh)K73A;(ii )K73E;(jj)R5F and K77A;(kk)R5F and K77E;(ll)R5F and K72A;(mm)R5F and K72E;(nn)R5F and K76A;(oo)R5F and K76E;(pp)K73A and K76F;(qq)K73E and K76F;(rr)R9A;(ss)R9D;(tt)R9E;(uu)R9H;(vv)R9S;(ww)R9T;(xx)R9N;(zz)R9G;(aaa)R9V;(bbb)R9I;(ccc)R9L;(ddd)R9Y;(eee) IL-21 polypeptides comprising at least one amino acid substitution selected from K72A and R76F; (fff)K75A and R76F; (ggg)R76F and K77A; (hhh)K75E and R76F; (iii)V17I and L74I; (jjj)I16A and L74F; (kkk)I16S, V17I, and L74V; (lll)I16R, V17I, and L74I; (mmm)L13F, I16A, V17A, and L74M; and (nnn)L13R, I16A, V17I, and L74I; and b) comprising an antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β, wherein the antibody or antigen-binding fragment is (i) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (ii) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (iii) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 220, CDR-H2 containing the amino acid sequence of SEQ ID NO: 221, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 222; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; or (iv) VH domains comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 250, CDR-H2 containing the amino acid sequence of SEQ ID NO: 251, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 288; and VL domains comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178. The invention provides a fusion protein that includes the following: 【0045】 In some embodiments, the present disclosure is a fusion protein, a)(i) an IL-21 polypeptide comprising four or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein at least four of the amino acid substitutions are in residues S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, T92, or combinations thereof; and (ii) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide, wherein at least one of the amino acid substitutions is in a residue selected from R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, K117, and combinations thereof; and b) comprising an antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β, wherein the antibody or antigen-binding fragment is (i) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (ii) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (iii) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 220, CDR-H2 containing the amino acid sequence of SEQ ID NO: 221, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 222; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; or (iv) VH domains comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 250, CDR-H2 containing the amino acid sequence of SEQ ID NO: 251, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 288; and VL domains comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178. The invention provides a fusion protein that includes the following: 【0046】 In some embodiments, the present disclosure is a fusion protein, a)(i) an IL-21 polypeptide comprising four or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein at least four amino acid substitutions are in residues S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, T92, or combinations thereof, and (ii) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide, wherein at least one amino acid substitution is in residue R76; and b) An antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β. The invention provides a fusion protein that includes the following: 【0047】 In some embodiments, the present disclosure is a fusion protein, a) (i) an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and (ii) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide; and b) comprising an antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β, wherein the antibody or antigen-binding fragment is (i) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (ii) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (iii) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 220, CDR-H2 containing the amino acid sequence of SEQ ID NO: 221, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 222; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; or (iv) VH domains comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 250, CDR-H2 containing the amino acid sequence of SEQ ID NO: 251, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 288; and VL domains comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178. The invention provides a fusion protein that includes the following: 【0048】 In some embodiments, the present disclosure is a fusion protein, a) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by human IL-21 polypeptide, wherein the at least one amino acid substitution is in a residue selected from R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, K117, and combinations thereof; and b) comprising an antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β, wherein the antibody or antigen-binding fragment is (i) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (ii) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (iii) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 220, CDR-H2 containing the amino acid sequence of SEQ ID NO: 221, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 222; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; or (iv) VH domains comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 250, CDR-H2 containing the amino acid sequence of SEQ ID NO: 251, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 288; and VL domains comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178. The invention provides a fusion protein that includes the following: 【0049】 In some embodiments, the present disclosure is a fusion protein, a) (i) an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and (ii) an IL-21 polypeptide comprising at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide, wherein at least one amino acid substitution is at residue R76; and b) comprising an antibody or antigen-binding fragment that specifically binds to CD8αβ or CD8β, wherein the antibody or antigen-binding fragment is (i) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (ii) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; (iii) A VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 220, CDR-H2 containing the amino acid sequence of SEQ ID NO: 221, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 222; and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; or (iv) VH domains comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 250, CDR-H2 containing the amino acid sequence of SEQ ID NO: 251, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 288; and VL domains comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178. The invention provides a fusion protein that includes the following: 【0050】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is functionally ligated to the C-terminus of the HC constant domain, the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The fusion protein comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein one or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least one amino acid substitution is in residues R85, R86, K88, H89, R90, or a combination thereof, and (ii) at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0051】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally linked to the C-terminus of the HC constant domain, the HC constant domain includes hole modifications, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The fusion protein comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein one or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least one amino acid substitution is in residues R85, R86, K88, H89, R90, or a combination thereof, and (ii) at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0052】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally ligated to the C-terminus of the HC constant domain, the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 220, CDR-H2 containing the amino acid sequence of SEQ ID NO: 221, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 222; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The fusion protein comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein one or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least one amino acid substitution is in residues R85, R86, K88, H89, R90, or a combination thereof, and (ii) at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0053】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally linked to the C-terminus of the HC constant domain, the HC constant domain includes hole modifications, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The fusion protein comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein one or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least one amino acid substitution is in residues R85, R86, K88, H89, R90, or a combination thereof, and (ii) at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0054】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is functionally ligated to the C-terminus of the HC constant domain, the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and provides a fusion protein that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0055】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally linked to the C-terminus of the HC constant domain, the HC constant domain includes hole modifications, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and provides a fusion protein that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0056】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally ligated to the C-terminus of the HC constant domain, the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 220, CDR-H2 containing the amino acid sequence of SEQ ID NO: 221, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 222; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and provides a fusion protein that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0057】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally linked to the C-terminus of the HC constant domain, the HC constant domain includes hole modifications, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and provides a fusion protein that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0058】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is functionally ligated to the C-terminus of the HC constant domain, the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide provides a fusion protein containing amino acid sequences selected from SEQ ID NOs: 16-21, 41-98, and 374-379. 【0059】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 199, CDR-H2 containing the amino acid sequence of SEQ ID NO: 203, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally linked to the C-terminus of the HC constant domain, the HC constant domain includes hole modifications, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide provides a fusion protein containing amino acid sequences selected from SEQ ID NOs: 16-21, 41-98, and 374-379. 【0060】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally ligated to the C-terminus of the HC constant domain, the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 220, CDR-H2 containing the amino acid sequence of SEQ ID NO: 221, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 222; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide provides a fusion protein containing amino acid sequences selected from SEQ ID NOs: 16-21, 41-98, and 374-379. 【0061】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally linked to the C-terminus of the HC constant domain, the HC constant domain includes hole modifications, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide provides a fusion protein containing amino acid sequences selected from SEQ ID NOs: 16-21, 41-98, and 374-379. 【0062】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is functionally ligated to the C-terminus of the HC constant domain, the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The fusion protein comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein one or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least one amino acid substitution is in residues R85, R86, K88, H89, R90, or a combination thereof, and (ii) at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0063】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally linked to the C-terminus of the HC constant domain, the HC constant domain includes hole modifications, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The fusion protein comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein one or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least one amino acid substitution is in residues R85, R86, K88, H89, R90, or a combination thereof, and (ii) at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0064】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally ligated to the C-terminus of the HC constant domain, the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 250, CDR-H2 containing the amino acid sequence of SEQ ID NO: 251, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 288; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The fusion protein comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein one or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least one amino acid substitution is in residues R85, R86, K88, H89, R90, or a combination thereof, and (ii) at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0065】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally linked to the C-terminus of the HC constant domain, the HC constant domain includes hole modifications, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The fusion protein comprises (i) one or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein one or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least one amino acid substitution is in residues R85, R86, K88, H89, R90, or a combination thereof, and (ii) at least one amino acid substitution that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0066】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is functionally ligated to the C-terminus of the HC constant domain, the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and provides a fusion protein that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0067】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally linked to the C-terminus of the HC constant domain, the HC constant domain includes hole modifications, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and provides a fusion protein that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0068】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally ligated to the C-terminus of the HC constant domain, the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 250, CDR-H2 containing the amino acid sequence of SEQ ID NO: 251, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 288; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and provides a fusion protein that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0069】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally linked to the C-terminus of the HC constant domain, the HC constant domain includes hole modifications, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and provides a fusion protein that provides reduced binding to the human IL-21 receptor compared to binding by the human IL-21 polypeptide. 【0070】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the IL-21 polypeptide is functionally ligated to the C-terminus of the HC constant domain, the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide provides a fusion protein containing amino acid sequences selected from SEQ ID NOs: 16-21, 41-98, and 374-379. 【0071】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 207; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 241, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 204; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally linked to the C-terminus of the HC constant domain, the HC constant domain includes hole modifications, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The VH chain and VL chain contain CD8αβ or CD8β-specifically bound CDRs. The IL-21 polypeptide provides a fusion protein containing amino acid sequences selected from SEQ ID NOs: 16-21, 41-98, and 374-379. 【0072】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally ligated to the C-terminus of the HC constant domain, the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 250, CDR-H2 containing the amino acid sequence of SEQ ID NO: 251, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 288; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a hole modification, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide provides a fusion protein containing amino acid sequences selected from SEQ ID NOs: 16-21, 41-98, and 374-379. 【0073】 In some embodiments, the present disclosure is a fusion protein, a) A first polypeptide comprising a VL chain and a CL1 chain, wherein the VL chain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 178; b) A second polypeptide comprising a VH chain and an HC constant domain, wherein the HC constant domain comprises a knob modification, and the VH chain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 288; c) A third polypeptide comprising a VH chain and an HC constant domain, wherein an IL-21 polypeptide is functionally linked to the C-terminus of the HC constant domain, the HC constant domain includes hole modifications, and the VH chains of the second and third polypeptides are the same; and d) A fourth polypeptide comprising a VL chain and a CL1 chain, wherein the VL chains of the first polypeptide and the fourth polypeptide are the same. The IL-21 polypeptide provides a fusion protein containing amino acid sequences selected from SEQ ID NOs: 16-21, 41-98, and 374-379. 【0074】 In some embodiments, the fusion protein comprises four polypeptides. The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 262, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 263, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 264, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 262; The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 266, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 267, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 268, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 266; The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 270, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 271, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 272, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 270; The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 274, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 275, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 276, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 274; or The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 278, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 279, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 280, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 278. 【0075】 In some embodiments, the fusion protein comprises four polypeptides. The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 262, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 263, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 265, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 262; The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 266, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 267, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 269, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 266; The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 270, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 271, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 273, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 270; The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 274, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 275, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 277, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 274; or The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 278, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 279, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 281, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 278. 【0076】 In some embodiments, the fusion protein comprises four polypeptides. (a) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 297, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 298, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 299, and the fourth polypeptide chain contains 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 contains the amino acid sequence of SEQ ID NO: 309, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 310, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 311, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 309. 【0077】 In some embodiments, the fusion protein comprises four polypeptides. (a) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 297, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 298, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 300, and the fourth polypeptide chain contains 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 contains the amino acid sequence of SEQ ID NO: 309, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 310, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 312, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 309. 【0078】 Built-in by reference All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent that each individual publication, patent, or patent application is specifically and individually incorporated herein by reference. 【0079】 Novel features of the present invention are particularly shown in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are utilized, and to the appended drawings. [Brief explanation of the drawing] 【0080】 [Figure 1] A-C show the amino acid sequences of the following polypeptides: mature IL-21 (SEQ ID NO: 1) (A), IL-21R (SEQ ID NO: 381) (B), and common gamma chain (SEQ ID NO: 382) (C). [Figure 2] The amino acid sequence of the mature wild-type IL-21 polypeptide (SEQ ID NO: 1) and the modifications used to generate the charge variant are shown. "X" indicates the amino acid position substituted in the wild-type IL-21 polypeptide sequence to generate the charge variant. [Figure 3A] This shows ion-exchange chromatography traces of the fusion protein of the anti-CD8 antibody and either wild-type IL-21 or an IL-21 charge variant after protein A purification. The fusion proteins of xhCD8 and either wild-type IL-21 or exemplary IL-21 charge variants v1-v6 are shown as measured by HPLC. In all cases, the left peak(s) represent unwanted products, primarily homodimers, while the right peak represents the desired heterodimer fusion protein, with the percentage of the total region of each shown peak indicated. [Figure 3B]This shows ion-exchange chromatography traces of the fusion protein of the anti-CD8 antibody and either wild-type IL-21 or an IL-21 charge variant after protein A purification. Fusion proteins of xhCD8.1 and exemplary IL-21 charge variant v2, or variants v7-v31, as measured by FPLC, are shown. In all cases, the left peak(s) represent unwanted products, primarily homodimers, while the right peak represents the desired heterodimer fusion protein, with the percentage of the total region of each shown peak indicated. [Figure 3C] This shows ion-exchange chromatography traces of the fusion protein of the anti-CD8 antibody and either wild-type IL-21 or an IL-21 charge variant after protein A purification. Fusion proteins of xhCD8.1 and exemplary IL-21 charge variant v2, or variants v7-v31, as measured by FPLC, are shown. In all cases, the left peak(s) represent unwanted products, primarily homodimers, while the right peak represents the desired heterodimer fusion protein, with the percentage of the total region of each shown peak indicated. [Figure 3D] This shows ion-exchange chromatography traces of the fusion protein of the anti-CD8 antibody and either wild-type IL-21 or an IL-21 charge variant after protein A purification. Fusion proteins of xhCD8.1 and exemplary IL-21 charge variant v2, or variants v7-v31, as measured by FPLC, are shown. In all cases, the left peak(s) represent unwanted products, primarily homodimers, while the right peak represents the desired heterodimer fusion protein, with the percentage of the total region of each shown peak indicated. [Figure 3E]This shows ion-exchange chromatography traces of the fusion protein of the anti-CD8 antibody and either wild-type IL-21 or an IL-21 charge variant after protein A purification. Fusion proteins of xhCD8.1 and exemplary IL-21 charge variant v2, or variants v7-v31, as measured by FPLC, are shown. In all cases, the left peak(s) represent unwanted products, primarily homodimers, while the right peak represents the desired heterodimer fusion protein, with the percentage of the total region of each shown peak indicated. [Figure 3F] This shows ion-exchange chromatography traces of the fusion protein of anti-CD8 antibody and either wild-type IL-21 or an IL-21 charge variant after purification of protein A. The fusion proteins of xhCD8.1 and wild-type IL-21 as measured by FPLC are shown. In all cases, the left peak(s) represent unwanted products, mainly homodimers, while the right peak represents the desired heterodimer fusion protein, with the percentage of the total region of each shown peak indicated. [Figure 4A] The mouse pharmacokinetic data for the fusion protein are shown. Serum concentrations of 1 mg / kg of a control antibody (xCtrl) containing either wild-type IL-21 or an exemplary charged variant IL-21 polypeptide IL-21 v2 are shown, administered intravenously. [Figure 4B] The mouse pharmacokinetic data for the fusion protein are shown. Serum concentrations of 1 mg / kg of a control antibody (xCtrl) containing either wild-type IL-21 or an exemplary charged variant IL-21 polypeptide IL-21v2 are shown, administered subcutaneously. [Figure 4C] The mouse pharmacokinetic data for the fusion protein are shown. In another experiment, serum concentrations of 1 mg / kg wild-type IL-21, or exemplary charge variant IL-21 polypeptides, IL-21v2, or IL-21v31, administered subcutaneously are additionally shown. [Figure 4D]The mouse pharmacokinetic data for the fusion protein are shown. In another experiment, serum concentrations of 1 mg / kg wild-type IL-21, or exemplary charge variant IL-21 polypeptides, IL-21v2, or IL-21v31, administered intravenously are additionally shown. [Figure 5] Images A and B 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) (A) or whole blood (B). STAT3 activation was measured by flow cytometry, and all cell types were strongly activated by wild-type IL-21. [Figure 6] This is a schematic diagram illustrating the general mechanism by which targeted fusions of mutant IL-21 polypeptides and CD8 antigen-binding molecules, as well as untargeted fusions containing mutant IL-21 polypeptides, stimulate cells that express or do not express the CD8 antigen. [Figure 7] This study demonstrates activation of STAT3 in human CD8+ T cells by a fusion protein of an anti-CD8 antibody with wild-type IL-21, or one of the exemplary IL-21 polypeptide charge variants IL-21v1, IL-21v2, or IL-21v6. [Figure 8A] This study demonstrates STAT3 activation in human PBMCs by fusion proteins of the anti-human CD8 antibody xhCD8 with various attenuated versions of IL-21v2. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. All fusion proteins of xhCD8 with exemplary IL-21 polypeptide IL-21v2.1, including attenuated mutations, show preferential STAT3 activation in CD8+ T cells compared to NK, B, and CD4+ T cells, as measured by EC50 and maximal STAT3 activation. [Figure 8B]Activation of STAT3 in human PBMCs by fusion proteins of anti-human CD8 antibody xhCD8 and various attenuated versions of IL-21v2 is shown. STAT3 activation has been shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. All fusion proteins of xhCD8 and an exemplary IL-21 polypeptide IL-21v2.2 containing an attenuation mutation show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50 and maximal STAT3 activation. [Figure 8C] Activation of STAT3 in human PBMCs by fusion proteins of anti-human CD8 antibody xhCD8 and various attenuated versions of IL-21v2 is shown. STAT3 activation has been shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. All fusion proteins of xhCD8 and an exemplary IL-21 polypeptide IL-21v2.3 containing an attenuation mutation show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50 and maximal STAT3 activation. [Figure 8D] Activation of STAT3 in human PBMCs by fusion proteins of anti-human CD8 antibody xhCD8 and various attenuated versions of IL-21v2 is shown. STAT3 activation has been shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. All fusion proteins of xhCD8 and an exemplary IL-21 polypeptide IL-21v2.4 containing an attenuation mutation show preferential STAT3 activation of CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50 and maximal STAT3 activation. [Figure 8E]Activation of STAT3 in human PBMCs by fusion proteins of anti-human CD8 antibody xhCD8 and various attenuated versions of IL-21v2 is shown. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. All fusion proteins of xhCD8 and an exemplary IL-21 polypeptide IL-21v2.5 containing an attenuation mutation show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50 and maximal STAT3 activation. [Figure 8F] Activation of STAT3 in human PBMCs by fusion proteins of anti-human CD8 antibody xhCD8 and various attenuated versions of IL-21v2 is shown. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. All fusion proteins of xhCD8 and an exemplary IL-21 polypeptide IL-21v2.6 containing an attenuation mutation show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50 and maximal STAT3 activation. [Figure 9A] Activation of STAT3 in human PBMCs by fusion proteins of anti-human CD8 antibody xhCD8.1 and additional attenuated versions of IL-21v2 or a non-targeted Fc-fusion to IL-21v2 as a control is shown. STAT3 activation in CD8+ T cells is shown. [Figure 9B] Activation of STAT3 in human PBMCs by fusion proteins of anti-human CD8 antibody xhCD8.1 and additional attenuated versions of IL-21v2 or a non-targeted Fc-fusion to IL-21v2 as a control is shown. STAT3 activation in CD4+ T cells is shown. [Figure 9C] Activation of STAT3 in human PBMCs by fusion proteins of anti-human CD8 antibody xhCD8.1 and additional attenuated versions of IL-21v2 or a non-targeted Fc-fusion to IL-21v2 as a control is shown. STAT3 activation in CD8+ T cells is shown. [Figure 9D]This study demonstrates activation of STAT3 in human PBMCs by a fusion protein of the anti-human CD8 antibody xhCD8.1 and an additional attenuated version of IL-21v2, or by a non-targeted Fc fusion to IL-21v2 as a control. STAT3 activation in CD4+ T cells is also shown. [Figure 9E] This study demonstrates activation of STAT3 in human PBMCs by a fusion protein of the anti-human CD8 antibody xhCD8.1 and an additional attenuated version of IL-21v2, or by a non-targeted Fc fusion to IL-21v2 as a control. STAT3 activation in CD8+ T cells is also shown. [Figure 9F] This study demonstrates activation of STAT3 in human PBMCs by a fusion protein of the anti-human CD8 antibody xhCD8.1 and an additional attenuated version of IL-21v2, or by a non-targeted Fc fusion to IL-21v2 as a control. STAT3 activation in CD4+ T cells is also shown. [Figure 9G] This study demonstrates activation of STAT3 in human PBMCs by a fusion protein of the anti-human CD8 antibody xhCD8.1 and an additional attenuated version of IL-21v2, or by a non-targeted Fc fusion to IL-21v2 as a control. STAT3 activation in CD8+ T cells is also shown. [Figure 9H] This study demonstrates activation of STAT3 in human PBMCs by a fusion protein of the anti-human CD8 antibody xhCD8.1 and an additional attenuated version of IL-21v2, or by a non-targeted Fc fusion to IL-21v2 as a control. STAT3 activation in CD4+ T cells is also shown. [Figure 10A]This study demonstrates STAT3 activation in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, B cells, and myeloid cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides including attenuated mutations—IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), IL-21v31.48(D), and IL-21v31.51(E)—all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximal STAT3 activation. For xhCD8.1-hIL21v31.6, data from two separate experiments are shown (B). Untargeted and unattenuated Fc-hIL21v2 is shown in cell F, and activation was observed in all indicated cell types. [Figure 10B] This study demonstrates STAT3 activation in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, B cells, and myeloid cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides including attenuated mutations—IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), IL-21v31.48(D), and IL-21v31.51(E)—all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximal STAT3 activation. For xhCD8.1-hIL21v31.6, data from two separate experiments are shown (B). Untargeted and unattenuated Fc-hIL21v2 is shown in cell F, and activation was observed in all indicated cell types. [Figure 10C]This study demonstrates STAT3 activation in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, B cells, and myeloid cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides including attenuated mutations—IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), IL-21v31.48(D), and IL-21v31.51(E)—all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximal STAT3 activation. For xhCD8.1-hIL21v31.6, data from two separate experiments are shown (B). Untargeted and unattenuated Fc-hIL21v2 is shown in cell F, and activation was observed in all indicated cell types. [Figure 10D] This study demonstrates STAT3 activation in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, B cells, and myeloid cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides including attenuated mutations—IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), IL-21v31.48(D), and IL-21v31.51(E)—all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximal STAT3 activation. For xhCD8.1-hIL21v31.6, data from two separate experiments are shown (B). Untargeted and unattenuated Fc-hIL21v2 is shown in cell F, and activation was observed in all indicated cell types. [Figure 10E]This study demonstrates STAT3 activation in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, B cells, and myeloid cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides including attenuated mutations—IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), IL-21v31.48(D), and IL-21v31.51(E)—all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximal STAT3 activation. For xhCD8.1-hIL21v31.6, data from two separate experiments are shown (B). Untargeted and unattenuated Fc-hIL21v2 is shown in cell F, and activation was observed in all indicated cell types. [Figure 10F] This study demonstrates STAT3 activation in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, B cells, and myeloid cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides including attenuated mutations—IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), IL-21v31.48(D), and IL-21v31.51(E)—all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells, as measured by EC50 and maximal STAT3 activation. For xhCD8.1-hIL21v31.6, data from two separate experiments are shown (B). Untargeted and unattenuated Fc-hIL21v2 is shown in cell F, and activation was observed in all indicated cell types. [Figure 11A]This shows STAT3 activation in mouse splenocytes. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Activation profiles are shown for recombinant wild-type mouse IL-21 (A); fusion protein of untargeted control antibody xCtrl and mouse IL-21 charge variant mIL-21v1 (B); and fusion protein of anti-mouse CD8 antibody xmCD8 and attenuated mouse IL-21 charge variant mIL21v1.1 (C). When measured by EC50 and maximal STAT3 activation, all cell types are activated for both recombinant wild-type mouse IL-21 and xCtrl-mIL21v1, while for xmCD8-mIL21v1.1, there is preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells. [Figure 11B] This shows STAT3 activation in mouse splenocytes. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Activation profiles are shown for recombinant wild-type mouse IL-21 (A); fusion protein of untargeted control antibody xCtrl and mouse IL-21 charge variant mIL-21v1 (B); and fusion protein of anti-mouse CD8 antibody xmCD8 and attenuated mouse IL-21 charge variant mIL21v1.1 (C). When measured by EC50 and maximal STAT3 activation, all cell types are activated for both recombinant wild-type mouse IL-21 and xCtrl-mIL21v1, while for xmCD8-mIL21v1.1, there is preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells. [Figure 11C]This shows STAT3 activation in mouse splenocytes. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Activation profiles are shown for recombinant wild-type mouse IL-21 (A); fusion protein of untargeted control antibody xCtrl and mouse IL-21 charge variant mIL-21v1 (B); and fusion protein of anti-mouse CD8 antibody xmCD8 and attenuated mouse IL-21 charge variant mIL21v1.1 (C). When measured by EC50 and maximal STAT3 activation, all cell types are activated for both recombinant wild-type mouse IL-21 and xCtrl-mIL21v1, while for xmCD8-mIL21v1.1, there is preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells. [Figure 12] The images show tumor growth curves in MC38 syngeneic tumor models treated with a single dose of either PBS; 0.3 or 1 mg / kg (mpk) of the untargeted control antibody xCtrl and the fusion protein xCtrl-mIL21v1 of the unattenuated mouse IL-21 charge variant IL-21v1; or 0.1, 0.3, or 1 mpk of the anti-mouse CD8 antibody xmCD8 and the fusion protein xmCD8-mIL21v1.1 of the attenuated mouse IL-21 charge variant IL21v1.1. Each subpanel shows the tumor growth curve from each individual animal, with fractions indicating complete response (CR) and arrows indicating treatment time. [Figure 13] Figures A-D show the effect of FTY720 on the antitumor efficacy of xmCD8-mIL21v1.1 in the MC38 tumor model. Figure A shows the dosing schemes for FTY720 and xmCD8-mIL21v1.1, and tumor growth curves for xmCD8-mIL21v1.1 at 0.1 mpk (B), 0.3 mpk (C), or 1 mpk (D). The plotted values ​​are mean tumor volumes, the error bars show the mean standard error, and each group contains 8 animals. [Figure 14A]This study demonstrates STAT3 activation in human whole blood from additional donors by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides including attenuated mutations—IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), IL-21v31.48(D), and IL-21v31.51(E)—all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50. For xhCD8.1-hIL21v31.4 and xhCD8.1-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (A and C). Untargeted, unattenuated Fc-hIL21v31 is shown in F, and activation was observed in all cell types shown. [Figure 14B] This study demonstrates STAT3 activation in human whole blood from additional donors by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides including attenuated mutations—IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), IL-21v31.48(D), and IL-21v31.51(E)—all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50. For xhCD8.1-hIL21v31.4 and xhCD8.1-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (A and C). Untargeted, unattenuated Fc-hIL21v31 is shown in F, and activation was observed in all cell types shown. [Figure 14C]This study demonstrates STAT3 activation in human whole blood from additional donors by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides including attenuated mutations—IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), IL-21v31.48(D), and IL-21v31.51(E)—all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50. For xhCD8.1-hIL21v31.4 and xhCD8.1-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (A and C). Untargeted, unattenuated Fc-hIL21v31 is shown in F, and activation was observed in all cell types shown. [Figure 14D] This study demonstrates STAT3 activation in human whole blood from additional donors by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides including attenuated mutations—IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), IL-21v31.48(D), and IL-21v31.51(E)—all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50. For xhCD8.1-hIL21v31.4 and xhCD8.1-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (A and C). Untargeted, unattenuated Fc-hIL21v31 is shown in F, and activation was observed in all cell types shown. [Figure 14E]This study demonstrates STAT3 activation in human whole blood from additional donors by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides including attenuated mutations—IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), IL-21v31.48(D), and IL-21v31.51(E)—all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50. For xhCD8.1-hIL21v31.4 and xhCD8.1-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (A and C). Untargeted, unattenuated Fc-hIL21v31 is shown in F, and activation was observed in all cell types shown. [Figure 14F] This study demonstrates STAT3 activation in human whole blood from additional donors by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8.1 with exemplary IL-21 polypeptides including attenuated mutations—IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), IL-21v31.48(D), and IL-21v31.51(E)—all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50. For xhCD8.1-hIL21v31.4 and xhCD8.1-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (A and C). Untargeted, unattenuated Fc-hIL21v31 is shown in F, and activation was observed in all cell types shown. [Figure 15A]This study demonstrates the activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v11 with exemplary IL-21 polypeptides including attenuated mutations IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), and IL-21v31.51(D) all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50. For xhCD8v11-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (C). [Figure 15B] This study demonstrates the activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v11 with exemplary IL-21 polypeptides including attenuated mutations IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), and IL-21v31.51(D) all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50. For xhCD8v11-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (C). [Figure 15C]This study demonstrates the activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v11 with exemplary IL-21 polypeptides including attenuated mutations IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), and IL-21v31.51(D) all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50. For xhCD8v11-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (C). [Figure 15D] This study demonstrates the activation of STAT3 in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v11 with exemplary IL-21 polypeptides including attenuated mutations IL-21v31.4(A), IL-21v31.6(B), IL-21v31.23(C), and IL-21v31.51(D) all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50. For xhCD8v11-hIL21v31.23, data from two different donors analyzed in two separate experiments are shown (C). [Figure 16A]This study demonstrates STAT3 activation in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various versions of IL-21v2 charge variants, including additional mutations. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v.1 with exemplary IL-21 polypeptides IL-21v2.63(A), IL-21v2.64(B), and IL-21v2.65(C), including attenuating mutations, all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50. [Figure 16B] This study demonstrates STAT3 activation in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various versions of IL-21v2 charge variants, including additional mutations. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v.1 with exemplary IL-21 polypeptides IL-21v2.63(A), IL-21v2.64(B), and IL-21v2.65(C), including attenuating mutations, all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50. [Figure 16C] This study demonstrates STAT3 activation in human whole blood by fusion proteins of the anti-human CD8 antibody xhCD8.1 with various versions of IL-21v2 charge variants, including additional mutations. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, NK cells, and B cells. Fusion proteins of xhCD8v.1 with exemplary IL-21 polypeptides IL-21v2.63(A), IL-21v2.64(B), and IL-21v2.65(C), including attenuating mutations, all show preferential STAT3 activation in CD8+ T cells over NK, B, and CD4+ T cells when measured by EC50. [Figure 17]A to C show the ion exchange chromatography traces of the fusion proteins of anti-CD8 antibody and IL-21R attenuated IL-21 charge variants after protein A purification. The traces are shown for xhCD8v11-hIL21v31.23 (A), xhCD8.1-hIL21v31.23 (B), and xhCD8.1-hIL21v31.4 (C) as measured by FPLC. In all cases, the peak(s) to the left of the major peak are unwanted products, mainly homodimers, while the major peak in the center is the desired heterodimeric fusion protein, and the percentage of the total of the desired peaks is shown. [Figure 18] Pharmacokinetic data for the fusion proteins in wild-type C57BL6 mice are shown. Serum concentrations are shown for the fusion proteins of anti-human CD8 antibody xhCD8.1 or xhCD8v11 and either hIL-21v31.4, hIL-21v31.23, or hIL-21v0.4 administered intravenously. The anti-human CD8 antibodies xhCD8.1 and xhCD8v11 are not cross-reactive with mouse CD8. Exposure is shown for xhCD8.1-hIL21v31.4, xhCD8v11-hIL21v31.23, xhCD8.1-hIL21v31.23, and xhCD8.1-hIL21v0.4. [Figure 19A] Characterization data of recombinant cytokine variants based on wild-type IL-21 or IL-21v31 charge variants are shown. SDS-PAGE of the material purified by IMAC followed by preparative SEC is shown. Analytical SEC of the material purified by IMAC followed by preparative SEC is shown. The retention time and peak width are also reported to evaluate the column interaction. In summary, the recombinant cytokine variants based on the IL-21v31 charge variant have a shorter retention time and a narrower peak width compared to the variants based on wild-type IL-21, suggesting less column interaction and more desirable biophysical properties with the IL-21v31-based construct. [Figure 19B]Characterization data for recombinant cytokine variants based on wild-type IL-21 or IL-21v31 charge variants are presented. SDS-PAGE of the material purified by IMAC followed by preparative SEC is shown. Analytical SEC of the material purified by IMAC followed by preparative SEC is shown. Retention times and peak widths are also reported to assess column interactions. In summary, recombinant cytokine variants based on the IL-21v31 charge variant have shorter retention times and narrower peak widths compared to variants based on wild-type IL-21, suggesting less column interaction and more desirable biophysical properties in IL-21v31-based constructs. [Figure 20] This shows the exposure of recombinant (non-fusion) wild-type human IL-21 and IL-21v31 charge variants in mice. IL-21v31 shows increased in vivo exposure compared to wild-type IL-21. [Figure 21A] Each diagram provides a structural schematic illustrating 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 generally unstructured soft loop (circled). Residues P78 and P79 (Pro-Pro), as well as C93 and P94 (Cys-Pro), are shown surrounding the soft loop. [Figure 21B] Each diagram provides 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 generally unstructured soft loop (circled). The diagram shows the charge of wild-type IL-21, indicating that the soft loop is highly charged. [Figure 22]This study demonstrates the activity of IL-21 fusion molecules in cultures of alloreactive T cells and activated dendritic cells isolated from two different human PBMC donors. The molecules tested included two CD8+ T cell-targeted fusions, xhCD8.1-hIL21v31.4 and xhCD8.1-hIL21v31.23, as well as a non-targeted, unattenuated Fc-hIL21v31. IL-2 release in the cultures was measured to determine the level of alloreactive T cell activation. In summary, Fc-hIL21v31 suppressed alloreactive T cell activation, while the CD8+ T cell-targeted fusions did not. [Figure 23A] This study demonstrates in vitro activation of STAT3 in cynomolgus monkey whole blood by fusion proteins of anti-human CD8 antibodies xhCD8.1 or xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, and B cells. The CD8+ T cell-targeted fusion proteins xhCD8.1-IL-21v31.4(A), xhCD8.1-IL-21v31.23(B), and xhCD8v11-IL-21v31.23(C) all show preferential STAT3 activation in CD8+ T cells over B and CD4+ T cells when measured by EC50. A non-targeted, unattenuated Fc-hIL21v31 is shown in D, and activation was observed in all cell types shown. [Figure 23B] This study demonstrates in vitro activation of STAT3 in cynomolgus monkey whole blood by fusion proteins of anti-human CD8 antibodies xhCD8.1 or xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, and B cells. The CD8+ T cell-targeted fusion proteins xhCD8.1-IL-21v31.4(A), xhCD8.1-IL-21v31.23(B), and xhCD8v11-IL-21v31.23(C) all show preferential STAT3 activation in CD8+ T cells over B and CD4+ T cells when measured by EC50. A non-targeted, unattenuated Fc-hIL21v31 is shown in D, and activation was observed in all cell types shown. [Figure 23C] This study demonstrates in vitro activation of STAT3 in cynomolgus monkey whole blood by fusion proteins of anti-human CD8 antibodies xhCD8.1 or xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, and B cells. The CD8+ T cell-targeted fusion proteins xhCD8.1-IL-21v31.4(A), xhCD8.1-IL-21v31.23(B), and xhCD8v11-IL-21v31.23(C) all show preferential STAT3 activation in CD8+ T cells over B and CD4+ T cells when measured by EC50. A non-targeted, unattenuated Fc-hIL21v31 is shown in D, and activation was observed in all cell types shown. [Figure 23D] This study demonstrates in vitro activation of STAT3 in cynomolgus monkey whole blood by fusion proteins of anti-human CD8 antibodies xhCD8.1 or xhCD8v11 with various attenuated versions of IL-21v31. STAT3 activation is shown for CD8+ T cells, CD4+ T cells, and B cells. The CD8+ T cell-targeted fusion proteins xhCD8.1-IL-21v31.4(A), xhCD8.1-IL-21v31.23(B), and xhCD8v11-IL-21v31.23(C) all show preferential STAT3 activation in CD8+ T cells over B and CD4+ T cells when measured by EC50. A non-targeted, unattenuated Fc-hIL21v31 is shown in D, and activation was observed in all cell types shown. [Figure 24] This study demonstrates in vivo activation of STAT3 in cynomolgus monkeys by xhCD8.1-IL-21v31.4. STAT3 activation levels in CD8+ T cells, CD4+ T cells, NK cells, and B cells were evaluated 20 minutes after administration. The results show that xhCD8.1-IL-21v31.4 preferentially activates STAT3 in CD8+ T cells compared to CD4+ T cells, NK cells, and B cells, as measured by STAT3 MFI. [Modes for carrying out the invention] 【0081】 This disclosure provides, in some embodiments, IL-21 polypeptides or functional fragments or variants thereof that exhibit improved properties compared to wild-type IL-21 (SEQ ID NO: 1). Examples of improved properties include, but are not limited to, (i) improved systemic exposure (e.g., measured by an increased area under the curve (AUC) when the IL-21 polypeptide or its functional fragment or variant is administered to a target compared to after administration of wild-type IL-21 protein); (ii) reduced binding to the IL-21 receptor; (iii) reduced cytotoxicity; or any combination thereof. 【0082】 As described herein, this disclosure provides an IL-21 polypeptide or a functional fragment or variant thereof having a reduced charge (i.e., isoelectric point) and reduced binding affinity to the human IL-21 receptor compared to the human IL-21 polypeptide (SEQ ID NO: 1). In particular, this disclosure provides an IL-21 polypeptide having one or more amino acid substitutions in a positively charged region located within residues S80–T92 of human IL-21. As shown in Figure 21B, the residues within S80–T92 of human IL-21 form a soft loop that does not directly interact with the human IL-21 receptor. Not wishing to be bound by theory, residues P78, P79, C93 and P94 of human IL-21 are thought to provide structural rigidity around the loop. This disclosure provides an IL-21 polypeptide having one or more amino acid substitutions within a soft loop having a reduced isoelectric point and improved bioavailability and biological activity. As described herein, the IL-21 polypeptide of this disclosure, having a reduced isoelectric point compared to the human IL-21 polypeptide, demonstrates an increased circulating half-life and reduced multiple reactivity (e.g., with positively charged proteins abundant in circulation). As shown in Figures 4A–4D, fusion proteins having the IL-21 polypeptide with a reduced isoelectric point compared to human IL-21 have an improved half-life. This disclosure further provides fusion proteins of the IL-21 polypeptide to anti-CD8 antibodies that selectively activate CD8+ T cells over CD4+ T cells, NK cells, and B cells, including in vivo in non-human primates (see Figure 24). Without being constrained by theory, fusion proteins of the IL-21 polypeptide with an antigen-binding domain that specifically binds to CD8ab or CD8b selectively activate CD8+ T cells compared to NK cells expressing CD8aa. Furthermore, IL-21 polypeptides with a reduced isoelectric point exhibit lower binding affinity to circulating proteins, such as heparin and hemoglobin (see Table 5).Furthermore, without being bound by theory, the IL-21 polypeptides of this disclosure having glycine and / or serine substitutions in the soft loop may exhibit reduced immunogenicity compared to unmodified human IL-21 after administration to a subject. 【0083】 This disclosure also provides an IL-21 polypeptide having reduced binding to the human IL-21 receptor. As shown herein, the attenuated binding of the IL-21 polypeptide of this disclosure to its receptor reduces the IL-21-mediated inhibitory effect on alloreactive T cells and dendritic cells (see Figure 22). Not wishing to be bound by theory, the attenuated binding of the IL-21 polypeptide of this disclosure to its receptor is expected to avoid the reduced binding effect on B cells and NK cells. The therapeutic efficacy of a fusion protein comprising the IL-21 polypeptide of this disclosure and an anti-CD8 targeted antibody is also demonstrated herein. Not wishing to be bound by theory, ligating an IL-21 polypeptide having attenuated binding to an anti-CD8 targeted antibody provides selective recovery of the attenuated IL-21 polypeptide. Specifically, Figure 12 shows that such a fusion protein provides antitumor efficacy in vivo. This efficacy is at least partially attributable to the peripheral activation of CD8+ T cells, as blocking lymphocyte trafficking reduces the therapeutic response (see Figures 13A-13D). Without being bound by theory, T cells in the tumor microenvironment can be dysfunctional and expressed as inhibitory molecules, and therefore, peripheral activation of CD8+ T cells by the fusion protein provides an anti-tumor immune response. Accordingly, this disclosure provides a novel IL-21 polypeptide with improved biological properties and a fusion protein containing the IL-21 polypeptide, as well as a method of using such polypeptide to treat a disease in a subject. 【0084】 Various embodiments of this disclosure provide methods and compositions involving the use of cytokines (e.g., interleukin-21 (IL-21) or IL-21 polypeptide or a functional fragment or variant thereof containing one or more mutated amino acid residues compared to the wild-type IL-21 polypeptide sequence) that are induced or targeted to activate a specific cell subset (e.g., CD8+ T cells), which are referred to herein as targeted cytokine constructs. While not wishing to be bound by a predetermined theory, the ability to selectively activate a given cell type may enable the ability to limit the activation of other cell types that would be counterproductive to a more robust response and a successful immune response. In some cases, the methods and compositions disclosed herein enable targeted delivery of IL-21 polypeptide to a desired cell type and administration of IL-21 at relatively low concentrations to induce selective activation of CD8+ T cells. In some cases, the use of the targeted cytokine constructs disclosed herein provides more selective cell activation (e.g., CD8+ T cells) compared to the activation of other cell types, such as natural killer (NK) cells, CD4+ T cells, or B cells. 【0085】 definition Unless otherwise defined herein, scientific and technical terms used in conjunction with this disclosure shall have meanings generally understood by those skilled in the art. The meaning and scope of terms should be clear, but in case of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or foreign definitions. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. In this application, the use of “or” shall mean “and / or” unless otherwise stated. Furthermore, the use of the term “including,” as well as other types such as “includes” and “included,” is not limited to these. 【0086】 The cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as the nomenclature and techniques used in conjunction with hybridization, as described herein, are generally well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of this disclosure are generally carried out in accordance with conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as generally achieved in the art or as described herein. The analytical chemistry, synthetic organic chemistry, and nomenclature and experimental procedures and techniques used in conjunction with medical and pharmaceutical chemistry, as described herein, are generally well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, as well as for patient treatment. 【0087】 As used herein and in the claims, the singular forms "a," "an," and "the" include multiple references unless the context otherwise clearly indicates. For example, the term "chimeric transmembrane receptor polypeptide" includes multiple chimeric transmembrane receptor polypeptides. 【0088】 The terms “about” or “approximately” mean an acceptable range of error for a particular value as determined by a person skilled in the art, depending in part to how the value is measured or determined, i.e., the limits of the measuring system. For example, “about” may mean a standard deviation of 1 or more than 1, according to practice in the art. Alternatively, “about” may 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 may mean within one order of magnitude, preferably within five times the value, and more preferably within two times. Where a particular value is described in this application and claims, unless otherwise stated, the term “about” should be assumed to mean an acceptable range of error for that particular value. 【0089】 As used herein, “Area under the curve” or “AUC” is a pharmacokinetic parameter referring to the area under the plot of plasma concentration versus time after administration of a drug (e.g., the IL-21 polypeptide or fusion protein described herein). In some embodiments, AUC measures a patient’s exposure to the drug and is dependent on dose, bioavailability, and clearance. Methods for determining AUC are known to those skilled in the art and include, but are not limited to, the rectangle rule, the trapezoidal rule, and Simpson’s rule. 【0090】 A "cytokine" is a form of immunomodulatory polypeptide that can mediate crosstalk between progenitor / primary cells and target / effector cells. It can function as a soluble form or be associated with the cell surface and bind to "cytokine receptors" on target immune cells to activate signaling. A "cytokine receptor," as used herein, is a polypeptide on the cell surface that activates intracellular signaling when cytokines are bound to it on the extracellular cell surface. Cytokines may 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 multiple cell types. Cytokines are multifaceted; this is because receptors are expressed on multiple subsets of immune cells, and one cytokine may activate signaling pathways in multiple cells. However, depending on the cell type, signaling events for cytokines may result in different downstream cellular events, such as activation, proliferation, survival, apoptosis, effector function, and secretion of other immunomodulatory proteins. In some embodiments, the given cytokine is a wild-type cytokine polypeptide, a fragment thereof, or a variant thereof, for example, a mutated cytokine polypeptide (also referred herein as a mutant cytokine, for example, mutant IL-21 or IL-21 mutant). 【0091】 As used herein, the term “antigen” refers to a molecule or fragment thereof that can be conjugated by a selective binder. For example, an antigen may be a ligand that can be conjugated by a selective binder, such as a receptor. Another example is an antigenic molecule that can be conjugated by a selective binder, such as an immunological protein (e.g., an antibody). An antigen may also refer to a molecule or fragment thereof that can be used in an animal to produce an antibody capable of binding to that antigen. 【0092】 The term “antibody,” as used herein, means any antigen-binding molecule containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. The term “antibody” includes immunoglobulin molecules, as well as their polymers (e.g., IgM), which contain four polypeptide chains interconnected by disulfide bonds, i.e., two heavy (H) chains and two light (L) chains. Each heavy chain contains a heavy chain variable region (hereinafter abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, i.e., CH1, CH2, and CH3. Each light chain contains a light chain variable region (hereinafter abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), into which more conserved regions called framework regions (FRs) are incorporated. Each VH and VL consists of three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of this disclosure, the FRs of the antibody (or its antigen-binding moiety) may be identical to the human germline sequence or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs. 【0093】 The term "intact antibody" refers to an antibody containing four polypeptide chains interconnected by disulfide bonds, namely two heavy (H) chains and two light (L) chains. In one embodiment, the antibody is an intact antibody. In one embodiment, the intact antibody is an intact human IgG1, IgG2, or IgG4 isotype. In a given embodiment, the antibody, or its antigen-binding fragment, is a human IgG1, IgG2, or IgG4 isotype. 【0094】 The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, or “antibody fragment,” as used herein, include any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using, for example, any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated by using chemical or molecular biological techniques to, for example, arrange one or more variable and / or constant domains in a suitable configuration, or introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc. 【0095】 Non-limiting examples of antigen-binding fragments include (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv(scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated complementarity-determining regions (CDRs), e.g., CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. 【0096】 The terms “variable region” or “variable domain” or fragment thereof of an antibody, as used herein, refer to the light and heavy chain portions of an antibody molecule, including the amino acid sequences of the complementarity-determining regions (CDRs; i.e., CDR-1, CDR-2, and CDR-3) and the framework region (FR). 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 herein, the amino acid positions assigned to the CDRs and FRs may be defined according to Kabat’s “Sequences of Proteins of Immunological Interest” (National Institutes of Health, Bethesda, Md., 1987 and 1991). The amino acid numbering of the antibody or antigen-binding fragment also follows that of Kabat. 【0097】 The term “complementarity-determining region” or “CDR” as used herein refers to the complementarity-determining region within the antibody variable sequence. Three CDRs exist in each of the heavy and light chain variable regions, designated as CDR1, CDR2, and CDR3 for each variable region. The term “CDR set” as used herein refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The precise boundaries of these CDRs are defined differently depending on the system. The system described by Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a unique residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia et al., J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) described Kabat We found that certain secondary regions within a CDR adopt nearly identical peptide skeletal conformations despite exhibiting high diversity at the amino acid sequence level. These secondary regions were designated as L1, L2, and L3 or H1, H2, and H3 (where "L" and "H" refer to the light chain and heavy chain regions, respectively). These regions may be referred to as the Chothia CDR, which has boundaries that overlap with the Kabat CDR. Other boundaries defining CDRs that overlap with the Kabat CDR have been described by Padlan (FASEB J.9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)).Furthermore, other definitions of CDR boundaries may not strictly adhere to one of the systems described above, but will nevertheless overlap with Kabat CDRs. However, these may be shortened or extended in terms of predictive or experimental findings that certain residues or groups of residues, or even the entire CDR, do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, but preferred embodiments use CDRs defined by Kabat or Chothia. 【0098】 The term “framework region” (hereinafter, FR), as used herein, refers to the variable domain residues other than the CDR residue. Each variable domain typically has four FRs, which are identified as FR1, FR2, FR3, and FR4. Common structural features between the variable regions of antibodies or their functional fragments are well known in the art. The DNA sequence encoding a particular antibody can usually be found according to well-known methods, e.g., Kabat, et al. 1987 Sequence of Proteins of Immunological Interest, USD Department of Health and Human Services, Bethesda MD (incorporated herein as a reference). A general method for cloning functional variable regions from antibodies can also be found in Chaudhary, VK, et al., 1990 Proc. Natl. Acad. Sci. USA 87:1066 (incorporated herein as a reference). 【0099】 A “variant Fc region” includes an amino acid sequence that differs from that of the native sequence Fc region by at least one “amino acid modification” as defined herein. A variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of the parent polypeptide, for example, about 1 to about 10 amino acid substitutions or about 1 to about 5 amino acid substitutions in the native sequence Fc region or the Fc region of the parent polypeptide. In one embodiment, a variant Fc region as defined herein may have a sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, or at least about 99% identical to the native sequence Fc region. According to another embodiment, a variant Fc region as defined herein may have a sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, or at least about 99% identical to the Fc region of the parent polypeptide. 【0100】 The terms “Fc receptor” or “FcR,” as used herein, typically refer to a receptor, or any derivative, variant, or fragment thereof, that can bind to the Fc region of an antibody. In a given embodiment, FcR is the receptor that binds to an IgG antibody (gamma receptor, FcgammaR), and includes the FcgammaRI(CD64), FcgammaRII(CD32), and FcgammaRIII(CD16) subclass receptors, including allele variants and alternative splicing forms of these receptors. The FcgammaRII receptor includes FcgammaRIIA (“activating receptor”) and FcgammaRIIB (“inhibitory receptor”), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The term “FcR” also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus. 【0101】 As used herein, “effector function” refers to a biochemical event resulting from the interaction between an antibody Fc region and an Fc receptor or ligand, which varies depending on the antibody isotype. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDC), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a cell-mediated response in which nonspecific cytotoxic cells expressing FcRs (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize a bound antibody on a target cell, subsequently causing lysis of the target cell. ADCC correlates with binding to FcγRIIIa; increased binding to FcγRIIIa results in increased ADCC activity. To evaluate the ADCC activity of the target molecule, an in vitro ADCC assay, such as those described in U.S. Patent No. 5,500,362 or 5,821,337, may be performed. "ADCP," or antibody-dependent cell-mediated phagocytosis, as used herein, refers to a cell-mediated response in which nonspecific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently cause phagocytosis of the target cells. 【0102】 "Fc null" and "Fc null variant" are used interchangeably herein to represent modified Fc having reduced or neutralized effector function. Such Fc nulls or Fc null variants have reduced or neutralized FcγR and / or complement receptors. Preferably, such Fc nulls or Fc null variants have neutralized effector function. Exemplary methods for modification include, but are not limited to, chemical changes, amino acid residue substitutions, insertions, and deletions. Exemplary amino acid positions on the Fc molecule where one or more modifications are introduced to reduce the effector function of the resulting variant (numbered according to the EU numbering scheme) are positions i) IgG1: C220, C226, C229, E233, L234, L235, G237, P238, S239, D265, S267, N297, L328, P331, K322, A327 and P329, ii) IgG2: V234, G237, D265, H268, N297, V309, A330, A331, K322 and iii) IgG4: L235, G237, D265 and E318.Exemplary Fc molecules with reduced effector function include those having one or more of the following substitutions: i) IgG1:N297A, N297Q, D265A / N297A, D265A / N297Q, C220S / C226S / C229S / P238S, S267E / L328F, C226S / C229S / E233P / L234V / L235A, L234F / L235E / P331S, L234A / L235A, L234A / L235A / G237A, L234A / L235A / G237A / K322A, L234A / L235A / G237A / A330S / A331S, L234A / L235A / P329G, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G23 6del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A3 27G and E233P / L234V / L235A / G236del, L234A / L235A / G237 deletion; ii) IgG2:A330S / A331S, V234A / G237A, V 234A / G237A / D265A, D265A / A330S / A331S, V234A / G237A / D265A / A330S / A331S, and H268Q / V309L / A330S / A331S;iii)IgG4:L235A / G237A / E318A, D265A, L235A / G237A / D265A and L235A / G237A / D265A / E318A. 【0103】 As used herein, "epitope" refers to a determinant that enables specific binding to a variable region of an antibody molecule, known as a paratope. An epitope is a grouping of molecules, such as amino acids or sugar side chains, which typically possess specific structural and charge characteristics. A single antigen may have multiple epitopes. An epitope may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked by the antigen-binding peptide (i.e., amino acid residues that are within the footprint of the antigen-binding peptide). Epitopes can be conformal or linear. An epitope typically contains at least 3, more commonly, at least 5 or 8-10 amino acids. Antibodies that recognize the same epitope can be validated in a simple immunoassay, such as "binning," which demonstrates the ability of one antibody to block the binding of another antibody to a target antigen. 【0104】 As used herein, “linker” refers to a molecule that connects two polypeptide chains. The linker may be a polypeptide linker or a synthetic chemical linker (see, for example, Protein Engineering, 9(3), 299-305, 1996). The length and sequence of the polypeptide linker are not particularly limited and may be selected according to the purpose by those skilled in the art. The polypeptide linker contains one or more amino acids. Preferably, the polypeptide linker is a peptide having a length of at least 5 amino acids, preferably 5 to 100, more preferably 10 to 50 amino acids. In one embodiment, the peptide linker is G, S, GS, SG, SGG, GGS, and GSG (G = glycine and S = serine). In another embodiment, the peptide linker is (GGGS) x G n (Sequence ID 294) or (GGGGS) x G n (Sequence number 295), (GGGGGS) x G n(Sequence ID 296), S(GGGS)xGn(Sequence ID 289), S(GGGGS)xGn(Sequence ID 290), or S(GGGGGS)xGn(Sequence ID 291) (x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 and n=0, 1, 2, or 3). In some embodiments, the linker is (GGGGS) x G n (x=2, 3, or 4 and n=0) (Sequence ID 372); in some embodiments, the linker is (GGGGS) x G n (x=3 and n=0) (Sequence ID 373). In some embodiments, the linker includes the sequence SGGGGSGGGGSGGGGS (Sequence ID 292) or SGGGGSGGGGSGGGG (Sequence ID 293). Synthetic chemical linkers include crosslinking agents conventionally used to crosslink peptides, such as N-hydroxysuccinimide (NHS), disuccinimidylsverate (DSS), bis(succinimidyl)sverate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(succinimidylpropionate) (DTSSP), ethylene glycol bis(succinimidylsuccinate) (EGS), ethylene glycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). 【0105】 The term “nucleotide,” as used herein, usually refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides can be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside tripphosphates, adenosine tripphosphates (ATP), uridine tripphosphates (UTP), cytosine tripphosphates (CTP), guanosine tripphosphates (GTP), and deoxyribonucleoside tripphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide, in some examples, refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Exemplary examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled by well-known techniques. Labeling may also be performed with quantum dots. Detectable labeling may include, for example, radioisotopes, fluorescent labeling, chemiluminescence labeling, bioluminescence labeling, and enzymatic labeling. Fluorescent labels for nucleotides may include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP; FluoroLink deoxynucleotides available from Amersham, Arlington Heights, Ill.: FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP; Boehringer Fluorescein-15-dATP, fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2′-dATP, available from Mannheim, Indianapolis, Ind., as well as Molecular Chromosome-labeled nucleotides available from Probes, Eugene, and Oreg may include BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP. Nucleotides can also be labeled or marked by chemical modification. A chemically modified single nucleotide can be biotin-dNTP.Some non-limiting examples of biotinylated dNTPs may include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP). 【0106】 The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to polymeric forms of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or their analogues, and whether single-stranded, double-stranded, or multi-stranded. Polynucleotides may be exogenous or endogenous to cells. Polynucleotides may exist in a cell-free environment. Polynucleotides may be genes or fragments thereof. Polynucleotides may be DNA. Polynucleotides may be RNA. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Polynucleotides may contain one or more analogues (e.g., modified backbone, sugar, or nucleic acid base). Modifications to the nucleotide structure, if present, may be conferred before or after polymer assembly. Some non-exclusive examples of analogs include 5-bromouracil, peptide nucleic acids, xeno nucleic acids, morpholino, roq nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., sugar-linked rhodamine or fluorescein), thiol-containing nucleotides, biotin-linked nucleotides, fluorescence-based analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, cuosin, and waiosin. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of a nucleotide may be interrupted by non-nucleotide components. 【0107】 The term “gene,” as used herein, refers to the corresponding nucleotide sequence involved in encoding nucleic acids (e.g., DNA, e.g., genomic DNA and cDNA) and RNA transcripts. When used herein in relation to genomic DNA, the term includes intervening non-coding and regulatory regions, which may include the 5' and 3' ends. In some uses, the term encompasses the transcribed sequence, including the 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region contains an “open reading frame” that encodes a polypeptide. In some uses of the term, “gene” includes only the coding sequence (e.g., the “open reading frame” or “coding region”) necessary to encode a polypeptide. In some cases, a gene does not encode polypeptides, e.g., ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only the transcribed sequence but also, in addition, the non-transcribed region, including upstream and downstream regulatory regions, enhancers, and promoters. In some cases, a gene refers to an “endogenous gene” or a native gene in its natural location in the genome of an organism. In some cases, a gene refers to an “exogenous gene” or a non-native gene. In some cases, a non-native gene refers to a gene that is not normally found in a host organism but is introduced into the host organism by gene transfer. In some cases, a non-native gene refers to a gene that is not in its natural location in the genome of an organism. In some cases, a non-native gene also refers to a naturally occurring nucleic acid or polypeptide sequence (e.g., a non-native sequence) that includes mutations, insertions, and / or deletions. 【0108】 With respect to expression or activity, the term "control" means, as used herein, altering the level of expression or activity. Control can occur at the transcriptional and / or translational levels. 【0109】 The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to polymers of at least two amino acid residues linked by peptide bonds. These terms do not imply a specific length of the polymer, nor are they intended to indicate or distinguish whether peptides are produced using recombinant techniques, chemical or enzymatic synthesis, or naturally occurring. The terms apply to naturally occurring amino acid polymers and amino acid polymers containing at least one modified amino acid. In some cases, polymers may be interrupted by non-amino acids. The terms include amino acid chains of any length, including full-length proteins and proteins with or without secondary and / or tertiary structures (e.g., domains). The terms also encompass amino acid polymers modified by, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other operations, such as conjugation with labeled components. The terms “amino acid” and “amino acids,” as used herein, typically refer to natural and non-natural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids can include both natural and unnatural amino acids, which are chemically modified to include groups or chemical sites not naturally present in the amino acid. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids. 【0110】 When used herein in relation to polypeptides, the terms “derivative,” “variant,” and “fragment” refer to polypeptides related to the wild-type polypeptide, for example, by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Polypeptide derivatives, variants, and fragments may include one or more amino acid variations (e.g., mutations, insertions, and deletions), cleavage, modification, or combinations thereof compared to the wild-type polypeptide. 【0111】 As used herein, the term "residue" refers to the location in a protein and its associated amino acid identity. For example, Leu 234 (also referred to as Leu234 or L234) is the residue at position 234 in the human antibody IgG1. 【0112】 As used herein, the term "wild-type" refers to a naturally occurring amino acid or nucleotide sequence, including allele variations. Wild-type proteins have an amino acid or nucleotide sequence that is not intentionally modified. 【0113】 The term "substitution" or "mutation" refers to a change in the polypeptide backbone in which an amino acid naturally present in the wild-type sequence of a polypeptide is substituted for another amino acid not naturally present at the same position in the polypeptide. In some cases, mutations are introduced to modify the polypeptide's affinity for its receptor, thereby altering its activity to differ from that of the wild-type homologous polypeptide. Mutations can also improve the biophysical properties of the polypeptide. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic manipulation, such as chemical modification of amino acid side chain groups, are also intended to be useful. 【0114】 The term “affinity” or “binding affinity” refers to the strength of the combined non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated herein, “binding affinity” refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). Affinity is usually expressed as the ratio of the dissociation and association rate constants (koff and kon, respectively), which is the dissociation constant (K). D) can be expressed by ). Therefore, equivalent affinity can include different rate constants, as long as the ratio of rate constants remains the same. Affinity can be measured by common methods known in the art, e.g., enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) techniques (e.g., BIAcore), biolayer interferometry (BLI) techniques (e.g., Octet), and other conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In some cases, binding affinity is determined by scatchard analysis, which involves generating a scatchard plot, which is a plot of the ratio of the concentration of bound ligand to the concentration of unbound ligand relative to the concentration of bound ligand. 【0115】 The terms “binding” or “specific binding,” as used herein, may refer to the ability of a polypeptide or antigen-binding domain to selectively interact with a receptor for a polypeptide or target antigen, respectively, and this specific interaction may be distinguished from non-targeted, unwanted, or nonspecific interactions. Examples of specific binding may include, but are not limited to, the binding of the IL-21 cytokine to its particular receptor (e.g., IL-21R and the common gamma chain) and the binding of an antigen-binding domain to a particular antigen (e.g., CD8 or PD-1). 【0116】 The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to vertebrates, preferably mammals, such as humans. Mammals include, but are not limited to, mice, monkeys, humans, farm animals, sports animals, and pets. Tissues, cells, and their offspring of biological entities obtained in vivo or cultured in vitro are also included. 【0117】 The terms “treatment” and “to treat” as used herein refer to an approach to obtain beneficial or desired results, including but not limited to therapeutic and / or preventive benefits. For example, treatment may include administering a system or cell population disclosed herein. Therapeutic benefit means any therapeutically related improvement or effect of one or more diseases, conditions, or symptoms under treatment. Preventive benefit means that a composition may be administered to subjects at risk of developing a particular disease, condition, or symptom, or to subjects reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptoms have not yet manifested. 【0118】 The terms “effective dose,” “therapeutic effective dose,” “effective dosage,” or “effective dosage” refer to an amount of a composition, e.g., immune cells, e.g., lymphocytes (e.g., T lymphocytes and / or NK cells), that can be combined with the targeted cytokine construct of this disclosure to produce the desired activity after administration to a target requiring it. In the context of this disclosure, the term “therapeutic effective” refers to an amount of a composition sufficient to delay the signs of a disorder treated by the method of this disclosure, to halt its progression, or to alleviate or reduce at least one of its symptoms. 【0119】 As used herein, the sequence term “amino acid sequence identity (%)” is defined as the percentage of amino acid residues in a candidate sequence that are identical to an amino acid residue in a given sequence, after aligning sequences and introducing gaps as necessary to achieve maximum sequence identity, without considering any conservative substitutions as part of sequence identity. Alignment for the purpose of determining amino acid sequence identity can be achieved using various methods within the scope of the art, e.g., publicly available computer software, e.g., EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A person skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. Alignment for the purpose of determining amino acid sequence identity can be achieved, for example, using the publicly available sequence comparison computer program ALIGN-2. The source code for the ALIGN-2 sequence comparison computer program is available with user documentation at the U.S. Copyright Office, Washington DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program can be compiled for use on UNIX operating systems, such as Digital UNIX v4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change. 【0120】 For all amino acid positions discussed in this disclosure, the numbering in relation to the antibody or its antigen-binding fragment follows the EU index. "EU index," "EU index in Kabat et al.," or "EU numbering scheme" refers to the numbering of EU antibodies (see Edelman et al., 1969; Kabat et al., 1991). 【0121】 Interleukin-21 polypeptide Interleukin-21 (IL-21) is a cytokine expressed by T cells, B cells, NK cells, and myeloid cells that can regulate the activity of both innate and adaptive immune cells and improve T cell survival and effector function. Several Phase I and Phase II clinical trials have included IL-21 as an investigational product for the treatment of cancer, inflammatory diseases, and autoimmune diseases, including melanoma, renal cell carcinoma, acute myeloid leukemia, non-Hodgkin lymphoma, ovarian cancer, colorectal cancer, systemic lupus erythematosus, Crohn's disease, and rheumatoid arthritis. 【0122】 IL-21 has a 4-helix bundle structure and exists as a monomer. In humans, two isoforms of IL-21 are known, each derived from a precursor molecule. The first IL-21 isoform contains 162 amino acids (aa), of which the first 29 constitute a signal peptide; the second IL-21 isoform contains 153 aa, of which the first 29 constitute a signal peptide, similar to the first isoform. 【0123】 IL-21 binds to the heterodimeric IL-21 receptor complex, which contains the IL-21 receptor (IL-21R) and a common gamma chain (γc). The IL-21 receptor complex is expressed on the surface of T, B, and NK cells. The IL-21 receptor complex is structurally similar to the IL-2 receptor complex in that each of these cytokine receptor complexes contains γc. 【0124】 When IL-21 binds to the IL-21 receptor complex, the JAK / STAT signaling pathway is activated, leading to the activation of target genes. While IL-21-induced signaling may be therapeutically desirable, careful consideration of the timing and location of signaling is necessary, given the broad expression profile of IL-21 and its ability to enhance the CD8+ T cell response and suppress antigen presentation and T cell priming. 【0125】 This disclosure provides, in some embodiments, IL-21 polypeptides or functional fragments or variants thereof that include at least one amino acid substitution compared to the wild-type IL-21 amino acid sequence provided herein as SEQ ID NO: 1. Unless otherwise stated, the terms “wild-type IL-21,” “wild-type IL-21 polypeptide,” “human IL-21,” and “human IL-21 polypeptide” are used interchangeably and may refer to the amino acid sequence of SEQ ID NO: 1. Such IL-21 polypeptides that include at least one amino acid substitution compared to SEQ ID NO: 1 are also referred to herein as IL-21 variants. In exemplary embodiments, the IL-21 polypeptide or functional fragment or variant described herein comprises at least one and X or fewer amino acid substitutions, where X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more. In some embodiments, the IL-21 polypeptide or functional fragment or variant described herein comprises at least 35 amino acid substitutions compared to SEQ ID NO: 1. In exemplary embodiments, the IL-21 polypeptide or functional fragment or variant described herein comprises an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 10, 15, 20, or 25 amino acids. In some embodiments, the IL-21 polypeptide or its functional fragment or variant described herein includes an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 16 amino acids or less. In some embodiments, the IL-21 polypeptide or its functional fragment or variant described herein includes an amino acid sequence that 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, the IL-21 polypeptide or its functional fragment or variant described herein includes an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 7 amino acids or less, or by 5 amino acids or less.In some embodiments, the IL-21 polypeptide or its functional fragment or variant described herein includes an amino acid sequence that differs by 10, 11, 12, 13, 14, 15, or 16 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1). In exemplary embodiments, the IL-21 polypeptide or its functional fragment or variant described herein includes an amino acid sequence that differs by 3, 4, 5, or 6 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1). In some embodiments, the IL-21 polypeptide or its functional fragment or variant described herein includes an amino acid sequence that differs by 5 to 16 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1). In exemplary embodiments, the IL-21 polypeptide or its functional fragment or variant described herein includes an amino acid sequence that differs by 3 to 6 amino acids or 1 to 5 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1). In exemplary embodiments, the IL-21 polypeptide or its functional fragment or variant described herein includes an amino acid sequence that differs by 1 or 2 amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1). 【0126】 Modified isoelectric point or charge distribution In some embodiments, the IL-21 polypeptide or its functional fragment or variant includes at least one mutation that alters its isoelectric point with or without affecting its binding to IL-21R. In some embodiments, the IL-21 polypeptide or its functional fragment or variant includes at least one mutation that alters its isoelectric point without affecting its binding to IL-21R. The altered isoelectric point of a protein can change its surface charge distribution, thereby affecting the protein yield, nonspecific binding, and blood or circulating half-life for a given protein. In some embodiments, the mutation in the IL-21 polypeptide or its functional fragment or variant lowers the theoretical isoelectric point in units of a given, as measured by the publicly available database ProtParam (SwissProt) (incorporated herein by reference). 【0127】In some embodiments, the IL-21 polypeptide or its functional fragment or variant 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 its functional fragment or variant has an isoelectric point of about 6.0 to about 9.0. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has a theoretical isoelectric point of about 6 to about 9. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has a theoretical isoelectric point of at least about 6. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has a theoretical isoelectric point of up to about 9.In some embodiments, the IL-21 polypeptide or its functional fragment or variant is approximately 9 to approximately 8.9, approximately 9 to approximately 8.8, approximately 9 to approximately 8.7, approximately 9 to approximately 8.5, approximately 9 to approximately 8.3, approximately 9 to approximately 8.1, approximately 9 to approximately 7.9, approximately 9 to approximately 7.7, approximately 9 to approximately 7.5, approximately 9 to approximately 7.3, approximately 9 to approximately 7, approximately 9 to approximately 6.5, approximately 9 to approximately 6.0, approximately 8.9 to approximately 8.8, approximately 8.9 to approximately 8.7, approximately 8.9 to approximately 8.5, approximately 8.9 to approximately 8.3, approximately 8.9 to approximately 8.1, approximately 8.9 to approximately 7.9, approximately 8.9 to approximately 7.7, approximately 8.9 to approximately 7.5, approximately 8. 9~approx. 7.3, approx. 8.9~approx. 7, approx. 8.9~approx. 6.5, approx. 8.9~approx. 6.0, approx. 8.8~approx. 8.7, approx. 8.8~approx. 8.5, approx. 8.8~approx. 8.3, approx. 8.8~approx. 8.1, approx. 8.8~approx. 7.9, approx. 8.8~approx. 7.7, approx. 8.8~approx. 7.5, approx. 8.8~approx. 7.3, approx. 8.8~approx. 7, approx. 8.8~approx. 6.5, approx. 8.8~approx. 6.0, approx. 8.7~approx. 8.6, approx. 8.7~approx. 8.5, approx. 8.7~approx. 8.3, approx. 8.7~approx. 8.1, approx. 8.7~approx. 7.9, approx. 8.7~approx. 7.7, approx. 8.7~approx. 7.5, approx. 8.7~approx. 7.3, approx. 8.7~approx. 7 , approximately 8.7~6.5, approximately 8.7~6.0, approximately 8.5~8.3, approximately 8.5~8.4, approximately 8.5~8.39, approximately 8.5~8.1, approximately 8.5~7.9, approximately 8.5~7.7, approximately 8.5~7.5, approximately 8.5~7.3, approximately 8.5~7, approximately 8.5~6.5, approximately 8.5~6.0, approximately 8.39, approximately 8.3~8.1, approximately 8.3~7.9, approximately 8.3~7.7, approximately 8.3~7.5, approximately 8.3~7.3, approximately 8.3~7.5, approximately 8.3~6.5, approximately 8.3~6.0, approximately 8.1~7.9, approximately 8.1~ Includes theoretical isoelectric points of 7.7, approximately 8.1 to 7.5, approximately 8.1 to 7.3, approximately 8.1 to 7, approximately 8.1 to 6.5, approximately 8.1 to 6.0, approximately 7.9 to 7.7, approximately 7.9 to 7.5, approximately 7.9 to 7.3, approximately 7.9 to 7, approximately 7.9 to 6.5, approximately 7.9 to 6.0, approximately 7.7 to 7.5, approximately 7.7 to 7.3, approximately 7.7 to 7, approximately 7.5 to 7.3, approximately 7.5 to 7, or approximately 7.3 to 7, approximately 7.3 to 6.5, approximately 7.3 to 6.0, approximately 7.0 to 6.5, approximately 7.0 to 6.0, or approximately 6.5 to 6.0.In some embodiments, the IL-21 polypeptide or its functional fragment or variant has a theoretical isoelectric point of about 9, about 8.9, about 8.8, about 8.79, about 8.7, about 8.5, about 8.39, about 8.3, about 8.1, about 7.9, about 7.7, about 7.5, about 7.3, about 7, about 6.5, or about 6.0. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has a theoretical isoelectric point of less than 9.0. In some embodiments, the IL-21 polypeptide or its functional fragment or variant 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. 【0128】 In some embodiments, the modified isoelectric point may increase the protein yield of IL-21 polypeptide or its functional fragment or variant during purification compared to wild-type IL-21. In some embodiments, the increased protein yield is at least 5% higher than that of wild-type IL-21. In some embodiments, the increased protein yield is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% higher than that of wild-type IL-21. In some embodiments, the increased protein yield is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% higher than that of wild-type IL-21. In some embodiments, the increased protein yield is 5%~10%, 5%~15%, 5%~20%, 5%~25%, 5%~50%, 5%~75%, 5%~100%, 5%~125%, 5%~150%, 5%~175%, 5%~200%, 5%~225%, 5%~250%, 5%~275%, 5%~300%, 10%~15%, 10%~20%, 10%~ 25%, 10%~50%, 10%~75%, 10%~100%, 10%~125%, 10%~150%, 10%~175%, 10%~200%, 10%~225%, 10%~250%, 10%~275%, 10%~300%, 15%~20%, 15%~25%, 15%~50%, 15%~75%, 15%~100%, 15%~125%, 15%~150% , 15%~175%, 15%~200%, 15%~225%, 15%~250%, 15%~275%, 15%~300%, 20%~25%, 20%~50%, 20%~75%, 20%~100%, 20%~125%, 20%~150%, 20%~175%, 20%~200%, 20%~225%, 20%~250%, 20%~275%, 20%~300% , 25%~50%, 25%~75%, 25%~100%, 25%~125%, 25%~150%, 25%~175%, 25%~200%, 25%~225%, 25%~250%, 25%~275%, 25%~300%, 50%~75%, 50%~100%, 50%~125%, 50%~150%, 50%~175%, 50%~200%, 75%~100%,75%~125%, 75%~150%, 75%~175%, 75%~200%, 75%~225%, 75%~250%, 75%~275%, 75%~300%, 100%~125%, 100%~150%, 100%~175%, 100%~200%, 100%~225%, 100%~250%, 100%~275%, 100%~300%, 125%~150%, 125%~175%, 125%~200%, 125%~225%, 125%~25 The percentages are 0%, 125%~275%, 125%~300%, 150%~175%, 150%~200%, 175%~200%, 175%~225%, 175%~250%, 175%~275%, 175%~300%, 200%~225%, 200%~250%, 200%~275%, 200%~300%, 225%~250%, 225%~275%, 225%~300%, 250%~275%, 250%~300%, or 275%~300%. In some embodiments, the increased protein yield is approximately 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300%. 【0129】 In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains at least one amino acid substitution of one or more positively charged amino acid residues, compared to a region of the wild-type IL-21 polypeptide containing one or more positively charged amino acid residues. In some cases, the region of wild-type IL-21 contains 2 to 20 positively charged amino acid residues.In some embodiments, the region in wild-type IL-21 is 2-3, 2-4, 2-5, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-1 4, 4-15, 4-16, 4-17, 4-18, 4-19, 4-20, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 6-20, 7-8, 7-9, 7-10, 7-11, 7-12, 7-13, 7-14, 7-15, 7-16, 7-17, 7-18, 7-19, 7-20 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 12- Contains 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 contains 12 positively charged amino acid residues.In some embodiments, the region of wild-type IL-21 contains 2 to 20 amino acid residues, of which at least one amino acid is positively charged.In some embodiments, the region of wild-type IL-21 has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 positively charged amino acid residues: 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 , containing 11-18, 11-19, 11-20, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 14-15, 14-16, 14-17, 3-18, 14-19, 14-20, 15-16, 15-17, 15-18, 15-19, 15-20, 16-17, 16-18, 16-19, 16-20, 17-18, 17-19, 17-20, 18-19, 18-20, or 19-20 amino acid residues.In some embodiments, the region of wild-type IL-21 contains 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 contains 12 amino acid residues, of which 2, 3, 4, 5, or 6 amino acid residues are positively charged. 【0130】 In some embodiments, the amino acids in the region of wild-type IL-21 do not bind to the human IL-21 receptor. In some embodiments, the region of wild-type IL-21 includes amino acid residues S80-T92 of SEQ ID NO: 1. In some embodiments, the region of wild-type IL-21 includes positively charged amino acid residues R85, R86, K88, H89, and R90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant includes at least one amino acid substitution in the region of wild-type IL-21. In some embodiments, the IL-21 polypeptide or its functional fragment or variant includes at least one amino acid substitution from S80-T92. In some embodiments, the IL-21 polypeptide or its functional fragment or variant includes at least four amino acid substitutions from S80-T92. In some embodiments, the IL-21 polypeptide or its functional fragment or variant includes at least four amino acid substitutions from S80-T92, except that G84 is not substituted. In some embodiments, the IL-21 polypeptide or its functional fragment or variant includes at least one amino acid substitution at R85, R86, K88, H89, or R90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant includes one or more amino acid substitutions at S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, and T92. In some embodiments, the IL-21 polypeptide or its functional fragment or variant includes amino acid substitutions at S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, and T92. 【0131】 In some embodiments, an IL-21 polypeptide or a functional fragment or variant thereof, which includes a modified isoelectric point compared to wild-type IL-21 (SEQ ID NO: 1), includes a mutation at a position selected from the group consisting of K56, S80, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92 of SEQ ID NO: 1. In some embodiments, an IL-21 polypeptide or a functional fragment or variant containing a modified isoelectric point compared to wild-type IL-21 (SEQ ID NO: 1) includes a mutation at a position selected from the group consisting of S80, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92 in SEQ ID NO: 1. 【0132】 In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains a mutation at position K56 of SEQ ID NO: 1. In some examples, the mutation includes K56G, K56S, K56E, K56D, or K56A. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains a mutation at position S80. In some embodiments, the mutation includes S80G, S80A, S80D, or S80E. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains a mutation at position T81 of SEQ ID NO: 1. In some examples, the mutation includes T81G, T81S, T81E, T81D, or T81A. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains a mutation at position N82 of SEQ ID NO: 1. In some examples, the mutation includes N82G, N82S, N82E, N82D, or N82A. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains a mutation at position A83 of SEQ ID NO: 1. In some examples, the mutation includes A83G, A83S, A83E, or A83D. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains a mutation at position G84 of SEQ ID NO: 1. In some examples, the mutation includes G84A, G84S, G84E, or G84D. In some embodiments, G84 is not mutated. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains a mutation at position R85 ​​of SEQ ID NO: 1. In some examples, the mutation includes R85G, R85S, R85E, R85D, or R85A. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains a mutation at position R86 of SEQ ID NO: 1. In some examples, the mutation includes R86G, R86S, R86E, R86D, or R86A. In some embodiments, the IL-21 polypeptide or a functional fragment or variant thereof contains a mutation at position Q87 of SEQ ID NO: 1. In some examples, the mutation includes Q87G, Q87S, Q87E, Q87D, or Q87A.In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains a mutation at position K88 of SEQ ID NO: 1. In some examples, the mutation includes K88G, K88S, K88E, K88D, or K88A. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains a mutation at position H89 of SEQ ID NO: 1. In some examples, the mutation includes H89G, H89S, H89E, H89D, or H89A. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains a mutation at position R90 of SEQ ID NO: 1. In some examples, the mutation includes R90G, R90S, R90E, R90D, or R90A. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains a mutation at position L91 of SEQ ID NO: 1. In some examples, the mutation includes L91G, L91S, L91E, L91D, or L91A. In some embodiments, the IL-21 polypeptide or a functional fragment or variant thereof contains a mutation at position T92 of SEQ ID NO: 1. In some examples, the mutation includes T92G, T92S, T92E, T92D, or T92A. 【0133】 In some embodiments, an IL-21 polypeptide or a functional fragment or variant having a modified isoelectric point compared to IL-21 of SEQ ID NO: 1 comprises an amino acid sequence that is at least 75% identical to the sequence of SEQ ID NO: 1 and includes a mutation at 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 variant having a modified isoelectric point compared to IL-21 of SEQ ID NO: 1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 1 and includes a mutation at at least one position selected from the group consisting of positions K56 and R90 of SEQ ID NO: 1. The mutation at position K56 is, in some examples, K56G, K56S, K56E, K56D, or K56A; the mutation at position R90 is, in some examples, R90G, R90S, R90E, R90D, or R90A. The modified isoelectric point is, in some examples, a reduced isoelectric point compared to IL-21 of SEQ ID NO: 1 (which is approximately 9.42), for example, reduced by at least approximately 0.6 units. 【0134】 In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1, and includes G85, G86, G88, and A90; G85, G86, G88, and E90; A56, A75, G85, G86, G88, and E90; A56, E75, G85, G86, G88, and A90; E56, A75, G85, G86, G88, and A90; G80, G81, G82, S83, E85, G86, S87, G88, G89, and S90; G8 It contains amino acids selected from the group consisting of 0, 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 (position number corresponds to sequence number 1). 【0135】In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids A56, A75, G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids A56, A75, G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids A56, E75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids A56, E75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids A56, A75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids A56, A75, G85, G86, G88, and A90.In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids E56, A75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids E56, A75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, S83, E85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, S83, E85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, S87, G88, G89, and E90.In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, G87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, G87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, G87, G88, G89, and G90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, G87, G88, G89, and G90. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, E87, G88, G89, and G90. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, E87, G88, G89, and G90.In some embodiments, a charge variant IL-21 polypeptide or a functional fragment or variant thereof is provided, comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. 【0136】 In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, S83, G85, G86, E87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, S83, G85, G86, E87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, E83, G85, G86, E87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, E83, G85, G86, E87, G88, G89, G90, S91, and G92.In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G82, G83, S85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G82, G83, S85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G82, G83, E85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G82, G83, E85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, G83, E85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, G83, E85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, E90, G91, and G92.In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G82, G83, S85, G86, G87, G88, S89, E90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G82, G83, S85, G86, G87, G88, S89, E90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G82, G83, G85, G86, G87, G88, G89, E90, and G91.In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G82, G83, G85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G82, G83, G85, E86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G82, G83, G85, E86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G82, G83, E85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G82, G83, E85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, G83, E84, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, G83, E84, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, E83, G85, G86, G87, G88, G89, E90, G91, and G92.In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, G82, E83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, E82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide contains at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and includes amino acids G80, G81, E82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, a charge variant IL-21 polypeptide or a functional fragment or variant thereof is provided, containing 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 to 40. In some embodiments, a charge variant IL-21 polypeptide or a functional fragment or variant thereof is provided, comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. 【0137】 In some embodiments, the IL-21 polypeptide contains approximately 75% to approximately 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. In some embodiments, the IL-21 polypeptide contains at least approximately 75% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. In some embodiments, the IL-21 polypeptide contains up to approximately 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. In some embodiments, the IL-21 polypeptide has approximately 75% to 80% sequence identity with 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, approximately 75% to 85% sequence identity, approximately 75% to 90% sequence identity, approximately 75% to 95% sequence identity, and approximately 75% sequence identity. Syntax ~ approximately 96% sequence identity, approximately 75% sequence identity ~ approximately 97% sequence identity, approximately 75% sequence identity ~ approximately 98% sequence identity, approximately 75% sequence identity ~ approximately 99% sequence identity, approximately 75% sequence identity ~ approximately 100% sequence identity, approximately 80% sequence identity ~ approximately 85% sequence identity, approximately 80% sequence identity ~ approximately 90% sequence identity, approximately 80% sequence identity ~ approximately 95% sequence identity, approximately 80% distribution Column identity ~ approximately 96% sequence identity, approximately 80% sequence identity ~ approximately 97% sequence identity, approximately 80% sequence identity ~ approximately 98% sequence identity, approximately 80% sequence identity ~ approximately 99% sequence identity, approximately 80% sequence identity ~ approximately 100% sequence identity, approximately 85% sequence identity ~ approximately 90% sequence identity, approximately 85% sequence identity ~ approximately 95% sequence identity, approximately 85% sequence identity ~ approximately 96% sequence identity, approximately 85 % sequence identity ~ approximately 97% sequence identity, approximately 85% sequence identity ~ approximately 98% sequence identity, approximately 85% sequence identity ~ approximately 99% sequence identity, approximately 85% sequence identity ~ approximately 100% sequence identity, approximately 90% sequence identity ~ approximately 95% sequence identity, approximately 90% sequence identity ~ approximately 96% sequence identity, approximately 90% sequence identity ~ approximately 97% sequence identity, approximately 90% sequence identity ~ approximately 98% sequence identity,Approximately 90% sequence identity to approximately 99% sequence identity, approximately 90% sequence identity to approximately 100% sequence identity, approximately 95% sequence identity to approximately 96% sequence identity, approximately 95% sequence identity to approximately 97% sequence identity, approximately 95% sequence identity to approximately 98% sequence identity, approximately 95% sequence identity to approximately 99% sequence identity, approximately 95% sequence identity to approximately 100% sequence identity, approximately 96% sequence identity to approximately 97% sequence identity, approximately 96% sequence identity to approximately 98% sequence identity Includes sequence identity of approximately 96% to 99% sequence identity, approximately 96% to 100% sequence identity, approximately 97% to 98% sequence identity, approximately 97% to 99% sequence identity, approximately 97% to 100% sequence identity, approximately 98% to 99% sequence identity, approximately 98% to 100% sequence identity, or approximately 99% to 100% sequence identity. In some embodiments, the IL-21 polypeptide includes approximately 75% sequence identity, approximately 80% sequence identity, approximately 85% sequence identity, approximately 90% sequence identity, approximately 95% sequence identity, approximately 96% sequence identity, approximately 97% sequence identity, approximately 98% sequence identity, approximately 99% sequence identity, or approximately 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. 【0138】 In some embodiments, the IL-21 polypeptide contains approximately 75% to approximately 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. In some embodiments, the IL-21 polypeptide contains at least approximately 75% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. In some embodiments, the IL-21 polypeptide contains up to approximately 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. In some embodiments, the IL-21 polypeptide exhibits approximately 75% to 80% sequence identity with a sequence selected from the group consisting of sequence numbers 23 to 40, approximately 75% to 85% sequence identity, approximately 75% to 90% sequence identity, approximately 75% to 95% sequence identity, approximately 75% to 96% sequence identity, approximately 75% to 97% sequence identity, and approximately 75% to 9 8% sequence identity, approximately 75% to approximately 99% sequence identity, approximately 75% to approximately 100% sequence identity, approximately 80% to approximately 85% sequence identity, approximately 80% to approximately 90% sequence identity, approximately 80% to approximately 95% sequence identity, approximately 80% to approximately 96% sequence identity, approximately 80% to approximately 97% sequence identity, approximately 80% to approximately 98% sequence identity, approximately 80% sequence identity Sex ~ approximately 99% sequence identity, approximately 80% sequence identity ~ approximately 100% sequence identity, approximately 85% sequence identity ~ approximately 90% sequence identity, approximately 85% sequence identity ~ approximately 95% sequence identity, approximately 85% sequence identity ~ approximately 96% sequence identity, approximately 85% sequence identity ~ approximately 97% sequence identity, approximately 85% sequence identity ~ approximately 98% sequence identity, approximately 85% sequence identity ~ approximately 99% sequence identity, approximately 85% sequence identity ~ approximately 100% sequence identity, approximately 90% Sequence identity of approximately 95% sequence identity, approximately 90% sequence identity to approximately 96% sequence identity, approximately 90% sequence identity to approximately 97% sequence identity, approximately 90% sequence identity to approximately 98% sequence identity, approximately 90% sequence identity to approximately 99% sequence identity, approximately 90% sequence identity to approximately 100% sequence identity, approximately 95% sequence identity to approximately 96% sequence identity, approximately 95% sequence identity to approximately 97% sequence identity, approximately 95% sequence identity to approximately 98% sequence identity,Includes approximately 95% to 99% sequence identity, approximately 95% to 100% sequence identity, approximately 96% to 97% sequence identity, approximately 96% to 98% sequence identity, approximately 96% to 99% sequence identity, approximately 96% to 100% sequence identity, approximately 97% to 98% sequence identity, approximately 97% to 99% sequence identity, approximately 97% to 100% sequence identity, approximately 98% to 99% sequence identity, approximately 98% to 100% sequence identity, or approximately 99% to 100% sequence identity. In some embodiments, the IL-21 polypeptide includes approximately 75% sequence identity, approximately 80% sequence identity, approximately 85% sequence identity, approximately 90% sequence identity, approximately 95% sequence identity, approximately 96% sequence identity, approximately 97% sequence identity, approximately 98% sequence identity, approximately 99% sequence identity, or approximately 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 【0139】 In some embodiments, the IL-21 polypeptide contains approximately 75% to approximately 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. In some embodiments, the IL-21 polypeptide contains up to approximately 100% sequence identity with sequences selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. In some embodiments, the IL-21 polypeptide has approximately 75% to approximately 80% sequence identity with 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, with approximately 75% to approximately 80% sequence identity, approximately 75% to approximately 85% sequence identity, approximately 75% to approximately 90% sequence identity, approximately 75% to approximately 95% sequence identity, approximately 75% to approximately 96% sequence identity, approximately 75% to approximately 97% sequence identity, and approximately 7 5% sequence identity to approximately 98% sequence identity, approximately 75% sequence identity to approximately 99% sequence identity, approximately 75% sequence identity to approximately 100% sequence identity, approximately 80% sequence identity to approximately 85% sequence identity, approximately 80% sequence identity to approximately 90% sequence identity, approximately 80% sequence identity to approximately 95% sequence identity, approximately 80% sequence identity to approximately 96% sequence identity, approximately 80% sequence identity to approximately 97% sequence identity, approximately 80% sequence identity to approximately 98% sequence identity, approximately 80% sequence identity to approximately 99% sequence identity, approximately 80% sequence identity to approximately 100% sequence identity, approximately 85% sequence identity to approximately 90% sequence identity,Approximately 85% sequence identity to approximately 95% sequence identity, approximately 85% sequence identity to approximately 96% sequence identity, approximately 85% sequence identity to approximately 97% sequence identity, approximately 85% sequence identity to approximately 98% sequence identity, approximately 85% sequence identity to approximately 99% sequence identity, approximately 85% sequence identity to approximately 100% sequence identity, approximately 90% sequence identity to approximately 95% sequence identity Identity, approximately 90% sequence identity to approximately 96% sequence identity, approximately 90% sequence identity to approximately 97% sequence identity, approximately 90% sequence identity to approximately 98% sequence identity, approximately 90% sequence identity to approximately 99% sequence identity, approximately 90% sequence identity to approximately 100% sequence identity, approximately 95% sequence identity to approximately 96% sequence identity, approximately 95% sequence identity to approximately 97% sequence identity Sequence identity of approximately 95% to approximately 98% sequence identity, approximately 95% to approximately 99% sequence identity, approximately 95% to approximately 100% sequence identity, approximately 96% to approximately 97% sequence identity, approximately 96% to approximately 98% sequence identity, approximately 96% to approximately 99% sequence identity, approximately 96% to approximately 96% sequence identity Includes 100% sequence identity, approximately 97% to approximately 98% sequence identity, approximately 97% to approximately 99% sequence identity, approximately 97% to approximately 100% sequence identity, approximately 98% to approximately 99% sequence identity, approximately 98% to approximately 100% sequence identity, or approximately 99% to approximately 100% sequence identity. In some embodiments, the IL-21 polypeptide includes approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. 【0140】 In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 80% sequence identity with SEQ ID NO: 4 and includes amino acids G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 4 and includes amino acids G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 80% sequence identity with SEQ ID NO: 2 and includes amino acids G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2 and includes amino acids G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 80% sequence identity with SEQ ID NO: 5 and includes amino acids A56, A75, G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5 and includes amino acids A56, A75, G85, G86, G88, and E90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 80% sequence identity with SEQ ID NO: 6 and includes amino acids A56, E75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 6 and includes amino acids A56, E75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 80% sequence identity with SEQ ID NO: 7 and includes amino acids A56, A75, G85, G86, G88, and A90.In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7 and includes amino acids A56, A75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 80% sequence identity with SEQ ID NO: 8 and includes amino acids E56, A75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8 and includes amino acids E56, A75, G85, G86, G88, and A90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 80% sequence identity with SEQ ID NO: 9 and includes amino acids G80, G81, G82, S83, E85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9 and includes amino acids G80, G81, G82, S83, E85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 80% sequence identity with SEQ ID NO: 10 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and S90.In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 80% sequence identity with SEQ ID NO: 10 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 80% sequence identity with SEQ ID NO: 11 and includes amino acids G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11 and includes amino acids G85, G86, S87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 80% sequence identity with SEQ ID NO: 12 and includes amino acids G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12 and includes amino acids G85, G86, S87, G88, G89, and S90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 80% sequence identity with SEQ ID NO: 13 and includes amino acids G85, G86, G87, G88, G89, and E90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 13 and includes amino acids G85, G86, G87, G88, G89, and E90.In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 80% sequence identity with SEQ ID NO: 14 and includes amino acids G85, G86, G87, G88, G89, and G90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 14 and includes amino acids G85, G86, G87, G88, G89, and G90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has at least 80% sequence identity with SEQ ID NO: 15 and includes amino acids G85, G86, G87, G88, G89, and G90. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 15 and includes amino acids G85, G86, E87, G88, G89, and G90. 【0141】 In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 23 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 23 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 24 and includes amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 24 and includes amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 25 and includes amino acids G80, G81, G82, S83, G85, G86, E87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 25 and includes amino acids G80, G81, G82, S83, G85, G86, E87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 26 and includes amino acids G80, G81, G82, E83, G85, G86, E87, G88, G89, G90, S91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 26 and includes amino acids G80, G81, G82, E83, G85, G86, E87, G88, G89, G90, S91, and G92.In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 27 and includes amino acids G82, G83, S85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27 and includes amino acids G82, G83, S85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 28 and includes amino acids G82, G83, E85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 28 and includes amino acids G82, G83, E85, G86, G87, G88, S89, G90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 29 and includes amino acids G80, G81, G82, G83, E85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29 and includes amino acids G80, G81, G82, G83, E85, G86, G87, G88, G89, G90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 30 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 30 and includes amino acids G80, G81, G82, S83, G85, G86, S87, G88, G89, E90, G91, and G92.In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 31 and includes amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 31 and includes amino acids G80, G81, G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 32 and includes amino acids G82, G83, S85, G86, G87, G88, S89, E90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 32 and includes amino acids G82, G83, S85, G86, G87, G88, S89, E90, G91, and S92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 33 and includes amino acids G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 33 and includes amino acids G82, G83, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 34 and includes amino acids G85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 34 and includes amino acids G85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 35 and includes amino acids G82, G83, G85, G86, G87, G88, G89, E90, and G91.In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 35 and includes amino acids G82, G83, G85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 36 and includes amino acids G82, G83, G85, E86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 36 and includes amino acids G82, G83, G85, E86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 37 and includes amino acids G82, G83, E85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 37 and includes amino acids G82, G83, E85, G86, G87, G88, G89, E90, and G91. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 38 and includes amino acids G80, G81, G82, G83, E84, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 38 and includes amino acids G80, G81, G82, G83, E84, G85, G86, G87, G88, G89, E90, G91, and G92. In some embodiments, the IL-21 polypeptide has at least 80% sequence identity with SEQ ID NO: 39 and includes amino acids G80, G81, G82, E83, G85, G86, G87, G88, G89, E90, G91, and G92.In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 39 and comprises amino acids G80, G81, G82, E83, G85, G86, G87, G88, G89, E90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 80% sequence identity with SEQ ID NO: 40 and comprises amino acids G80, G81, E82, G83, G85, G86, G87, G88, G89, E90, G91 and G92. In some embodiments, the IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 40 and comprises amino acids G80, G81, E82, G83, G85, G86, G87, G88, G89, E90, G91 and G92. 【0142】 In some embodiments, the IL-21 polypeptide is QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLX1SANTGNNERIINVSIKKLX2RKPPX3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 383) (where X1 = A, E, K; X2 = A, E, K; 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; X 10 = S, G, E, Q; X 11 = G, K; X 12 = G, S, H; X 13 = A, E, S, G, R; X 14 = S, G, L; X 15 = G, S, T and at least one amino acid residue is not the amino acid residue at the same position shown in SEQ ID NO: 1). In some embodiments, X 8、 X 9、 X 11、 X12、 and X 13 At least one of them is not an amino acid residue at the same position shown in SEQ ID NO: 1. 【0143】 In some embodiments, the IL-21 polypeptide has the amino acid sequence: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPX1X2X3X4GX5X6X7X8X9X 10 X 11 X 12 CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 380) (where X1 = G, S; X2 = G, T; X3 = G, E, N; X4 = G, S, E, A; X5 = G, E, S, R; X6 = G, E, R; X7 = S, G, E, Q; X8 = G, K; X9 = G, S, H; X 10 = A, E, S, G, R; X 11 = S, G, L; and X 12 = G, S, T, provided that X 5、 X 6、 X 8、 X9, and X 10 At least one of them is not an amino acid residue at the same position shown in SEQ ID NO: 1). In some embodiments, X5 = G, X6 = G, X8 = G, X 10 = A. In some embodiments, X5 = G, X6 = G, X8 = G, X 10 = E. In some embodiments, X1 = G, X2 = G, X3 = G, X4 = S, X5 = E, X6 = G, X7 = S, X8 = G, X9 = G, X 10 = S. In some embodiments, X1 = G, X2 = G, X3 = G, X4 = S, X5 = G, X6 = G, X7 = S, X8 = G, X9 = G, X 10 = E. In some embodiments, X5 = G, X6 = G, X7 = S, X8 = G, X9 = G, X 10 = E. In some embodiments, X5 = G, X6 = G, X7 = S, X8 = G, X9 = G, X 10 = S. In some embodiments, X5 = G, X6 = G, X7 = G, X8 = G, X9 = G, X 10=E. In some embodiments, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =G. In some embodiments, X5=G, X6=G, X7=E, X8=G, X9=G, X 10 =G. In several embodiments, X1=G, X2=G, X3=G, X4=S, X5=G, X6=G, X7=S, X8=G, X9=G, X 10 =G, X 11 =S, Christmas 12 =G. In several embodiments, X1=G, X2=G, X3=G, X4=G, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =G, X 11 =G, X 12 =G. In several embodiments, X1=G, X2=G, X3=G, X4=S, X5=G, X6=G, X7=E, X8=G, X9=G, X 10 =G, X 11 =S, Christmas 12 =G. In several embodiments, X1=G, X2=G, X3=G, X4=E, X5=G, X6=G, X7=E, X8=G, X9=G, X 10 =G, X 11 =S, Christmas 12 =G. In some embodiments, X3=G, X4=G, X5=S, X6=G, X7=G, X8=G, X9=S, X 10 =G, X 11 =G, X 12 =S. In some embodiments, X3=G, X4=G, X5=E, X6=G, X7=G, X8=G, X9=S, X 10 =G, X 11 =G, X 12 =S. In several embodiments, X1=G, X2=G, X3=G, X4=G, X5=E, X6=G, X7=G, X8=G, X9=G, X 10 =G, X 11 =G, X 12 =G. In several embodiments, X1=G, X2=G, X3=G, X4=S, X5=G, X6=G, X7=S, X8=G, X9=G, X 10 =E, X 11 =G, X 12=G. In several embodiments, X1=G, X2=G, X3=G, X4=G, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E, X 11 =G, X 12 =G. In some embodiments, X3=G, X4=G, X5=S, X6=G, X7=G, X8=G, X9=S, X 10 =E, X 11 =G, X 12 =S. In some embodiments, X3=G, X4=G, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E, X 11 =G, X 12 =G. In some embodiments, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E, X 11 =G. In several embodiments, X3=G, X4=G, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E, X 11 =G. In some embodiments, X3=G, X4=G, X5=G, X6=E, X7=G, X8=G, X9=G, X 10 =E, X 11 =G. In some embodiments, X3=G, X4=G, X5=E, X6=G, X7=G, X8=G, X9=G, X 10 =E, X 11 =G. In several embodiments, X1=G, X2=G, X3=G, X4=E, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E, X 11 =G, X 12 =G. In several embodiments, X1=G, X2=G, X3=E, X4=G, X5=G, X6=G, X7=G, X8=G, X9=G, X 10 =E, X 11 =G, X 12 =G. 【0144】 In some embodiments, the IL-21 polypeptide comprises an amino acid sequence that begins at position 78 as defined by SEQ ID NO: 1, and the amino acid sequence is PPX1X2X3X4GX5X6X7X8X9X 10 X 11 X12 CP (SEQ ID NO: 386) (wherein, X1=G, S;X2=G, T;X3=G, E, N;X4=G, S, E, A; 10 =A, E, S, G, R;X 11 =S, G, L; and X 12 =G, S, T, where X 5、 X 6、 X 8、 X9, and X 10 At least one of them is not an amino acid residue at the same position as shown in Sequence ID No. 1. 【0145】 In some embodiments, the IL-21 polypeptide comprises an amino acid sequence that begins at position 80 as defined by SEQ ID NO: 1, and the amino acid sequence is X1X2X3X4GX5X6X7X8X9X 10 X 11 X 12 (In the formula, X1=G, S;X2=G, T;X3=G, E, N;X4=G, S, E, A; 10 =A, E, S, G, R;X 11 =S, G, L; and X 12 =G, S, T, where X 5、 X 6、 X 8、 X9, and X 10 At least one of them is not an amino acid residue at the same position as shown in Sequence ID No. 1. 【0146】 Reduced IL-21R binding In some embodiments, the disclosure provides an IL-21 polypeptide or a functional fragment or variant thereof having a reduced binding affinity to IL-21R. In some embodiments, the IL-21 polypeptide or a functional fragment or variant thereof comprises at least one mutation that reduces its binding affinity to IL-21R. Such mutations are, in some embodiments, at one or more positions selected from the group consisting of R5, I8, R9, R11, Q12, I14, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117, where the position number is a number corresponding to the amino acid sequence of SEQ ID NO: 1. In some embodiments, such mutations are at one or more positions selected from the group consisting of R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, and K117, where the position number is a number corresponding to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a mutation at position R5 includes 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, a mutation at position I8 includes an amino acid substitution selected from the group consisting of Q, H, and E. In some embodiments, a mutation at position R9 includes 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, a mutation at position R11 includes an amino acid substitution selected from the group consisting of D or E. In some embodiments, the mutation at position Q12 includes an amino acid substitution selected from the group consisting of L, I, or Y. In some embodiments, the mutation at position L13 includes an amino acid substitution selected from the group consisting of F or R. In some embodiments, the mutation at position I14 includes an amino acid substitution selected from the group consisting of D or E. In some embodiments, the mutation at position D15 includes an amino acid substitution selected from the group consisting of R, K, H, L, Y, or F. In some embodiments, the mutation at position I16 includes an amino acid substitution selected from the group consisting of A, S, or R.In some embodiments, the mutation at position V17 includes an amino acid substitution selected from the group consisting of I or A. In some embodiments, the mutation at position D18 includes an amino acid substitution selected from the group consisting of A, K, or R. In some embodiments, the mutation at position Q19 includes an amino acid substitution selected from the group consisting of L or Y. In some embodiments, the mutation at position Y23 includes an amino acid substitution of E. In some embodiments, the mutation at position R65 includes an amino acid substitution selected from the group consisting of G, S, E, D, or A. In some embodiments, the mutation at position S70 includes an amino acid substitution selected from the group consisting of H, Y, L, V, or F. In some embodiments, the mutation at position K72 includes an amino acid substitution selected from the group consisting of G, S, E, D, or A. In some embodiments, the mutation at position K73 includes 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 includes an amino acid substitution selected from the group consisting of I, F, V, or M. In some embodiments, the mutation at position K75 includes an amino acid substitution selected from the group consisting of G, S, E, D, or A. In some embodiments, the mutation at position R76 includes 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 includes an amino acid substitution selected from the group consisting of G, S, E, D, or A. In some embodiments, the mutation at position S80 includes an amino acid substitution of H, A, G, E, or D. In some embodiments, the mutation at position Q116 includes an amino acid substitution of Y. In some embodiments, the mutation at position K117 includes an amino acid substitution selected from the group consisting of A, D, or E. 【0147】 In some cases, IL-21 polypeptide or its functional fragments or variants are R5F, R5A, R5E, R5S, R5T, R5N, R5Q, R5V, R5I, R5L, R5Y, I8E, R9A, R9D, R9E, R9H, R9S, R9T, R9N, R9G, R9V, R9I, R9L, R9Y, R11D, R11E, L13F, L13R, I14D, I16A, I16S, I1 It contains at least one amino acid substitution selected from 6R, V17I, V17A, D18A, K72A, K72E, K73A, K73E, K75A, K75E, L74I, L74F, L74M, L74V, R76E, R76F, R76A, R76N, R76D, R76S, R76T, R76Q, R76V, R76I, R76L, R76Y, R76M, K77A, K77E, and K117A. In some cases, the IL-21 polypeptide or its functional fragment or variant contains an amino acid substitution and is R76E or R76Q. 【0148】 Exemplary sequences for the IL-21 polypeptide or functional fragments or variants of the IL-21 polypeptide of this disclosure are provided as follows: QGQDX1HMX2X3MX4X5LX6X7IVX8X9LKNX 10 VNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNEX 11 IINVX 12 IX 13 X 14 LX 15 X 16 X 17 PPX 18 TNAGRRQKHRLTTCPSCDSYEKPPPKEFLERFKSLLX 19 X 20MIHQHLSSRTHGSEDS (array number 22). In some embodiments, X1 = R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X2 = I, Q, H, E. In some embodiments, X3 = R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X4 = R, D, or E. In some embodiments, X5 = Q, L, I, or Y. In some embodiments, X6 = I, D, or E. In some embodiments, X7 = D, R, K, H, L, Y, or F. In some embodiments, X8 = D, A, K, or R. In some embodiments, X9 = Q, L, or Y. In some embodiments, X 10 =Y or E. In some embodiments, X 11 = R, G, S, E, D, or A. In some embodiments, X 12 =S, H, Y, L, V, or F. In some embodiments, X 13 =K, G, S, E, D, or A. In some embodiments, X 14 =K, A, Y, L, F, G, S, T, E, A, or D. In some embodiments, X 15 =K, G, S, E, D, or A. In some embodiments, X 16 =R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X 17 =K, G, S, E, D, or A. In some embodiments, X 18 =S, H, A, G, E, or D. In some embodiments, X 19 =Q or Y. In some embodiments, X 20 =K, A, D, or E. 【0149】 In some embodiments, the IL-21 polypeptide or a functional fragment or variant having reduced binding to IL-21R is given the sequence: QGQDX1HMX2X3MX4QX5X6DX7X8X9QLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIX 10 X 11 X 12 X 13 X14 X 15 PPSTNAGRRQKHRLTTCPSCDSYEKPPPKEFLERFKSLLQX 16 MIHQHLSSRTHGSEDS (SEQ ID NO: 384) (wherein, X1=F, A, E, S, T, N, Q, V, I, L, Y, R; X2=E, I; X3=A, D, E, H, S, T, N, G, V, I, L, Y, R;X4=D, E, R;X5=F, R, L;X6=D, I;X7=A, S, R, I;X8=I, A, V;X9=A, D;X 10 =A, E, K;X 11 =A, E, K;X 12 =I, F, M, L;X 13 =A, K, E;X 14 =E, F, A, N, D, S, T, Q, V, I, L, Y, M, R;X 15 =A, E, K;X 16 =A, K; however, X1~X 16 At least one of these includes an amino acid residue that is not at the same position as shown in Sequence ID No. 1. 【0150】 In some embodiments, the Disclosure provides IL-21 polypeptides comprising an amino acid sequence that is 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. 【0151】 combination of mutations Functional fragments of the IL-21 polypeptide or its variants described herein include, in some embodiments, a combination of (a) a mutation that alters its isoelectric point compared to wild-type IL-21 (SEQ ID NO: 1) and (b) a mutation that attenuates its binding to IL-21R compared to wild-type IL-21. In some embodiments, the mutations in group (a) are at positions selected from positions K56, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92; the mutations in group (b) are at positions selected from the group consisting of R5, I8, R9, R11, Q12, I14, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117, with the position numbering following the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutations in group (a) are at positions selected from S80, T81, N82, A83, G84, R85, R86, Q87, K88, H89, R90, L91, and T92; and the mutations in group (b) are at positions selected from the group consisting of R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, and K117, with the position numbering following the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutations in group (a) are at positions selected from S80, T81, N82, A83, R85, R86, Q87, K88, H89, R90, L91, and T92; and the mutations in group (b) are at positions selected from the group consisting of R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, and K117, with the position numbering following the amino acid sequence of SEQ ID NO: 1. In some embodiments, the 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; the mutation in group (b) is at position R76, and the position numbering follows the amino acid sequence of SEQ ID NO: 1. 【0152】 In some embodiments, the IL-21 polypeptide contains the following consensus sequence: QGQDX1HMX2X3MX4X5LX6X7IVX8X9LKNX 10 VNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLX 11 SANTGNNEX 12 IINVX 13 IX 14 X 15 LX 16 X 17 X 18 PPX 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 CPSCDSYEKKPPKEFLERFKSLLX 32 X 33 MIHQHLSSRTHGSEDS(array number 3). In some embodiments, X1 = R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X2 = I, Q, H, E. In some embodiments, X3 = R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X4 = R, D, or E. In some embodiments, X5 = Q, L, I, or Y. In some embodiments, X6 = I, D, or E. In some embodiments, X7 = D, R, K, H, L, Y, or F. In some embodiments, X8 = D, A, K, or R. In some embodiments, X9 = Q, L, or Y. In some embodiments, X 10 =Y or E. X 11 =G, S, E, D, or A. In some embodiments, X 12 = R, G, S, E, D, or A. In some embodiments, X 13 =S, H, Y, L, V, or F. In some embodiments, X 14 =K, G, S, E, D, or A. In some embodiments, X 15=K, A, Y, L, F, G, S, T, E, A, or D. In some embodiments, X 16 =K, G, S, E, D, or A. In some embodiments, X 17 =R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X 18 =K, G, S, E, D, or A. In some embodiments, X 19 =S, H, A, G, E, or D. In some embodiments, X 20 =G, S, E, D, or A. In some embodiments, X 21 =G, S, E, D, or A. In some embodiments, X 22 =G, S, E, or D. In some embodiments, X 23 =A, S, E, or D. In some embodiments, X 24 =G, S, E, D, or A. In some embodiments, X 25 =G, S, E, D, or A. In some embodiments, X 26 =G, S, E, D, or A. In some embodiments, X 27 =G, S, E, D, or A. In some embodiments, X 28 =G, S, E, D, or A. In some embodiments, X 29 =G, S, E, D, or A. In some embodiments, X 30 =G, S, E, D, or A. In some embodiments, X 31 =G, S, E, D, or A. In some embodiments, X 32 =Y. In some embodiments, X 33 = A, D, or E. 【0153】 In some embodiments, the IL-21 polypeptide contains the following consensus sequence: QGQDX1HMX2X3MX4QX5X6DX7X8X9QLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLX 10 SANTGNNERIINVSIX 11 X 12 X 13 X 14 X 15 X16 PPX 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 CPSCDSYEKKPPKEFLERFKSLLQX 30 MIHQHLSSRTHGSEDS (SEQ ID NO: 385) (wherein, X1=F, A, E, S, T, N, Q, V, I, L, Y, R; X2=E, I; X3=A, D, E, H, S, T, N, G, V, I, L, Y, R;X4=D, E, R;X5=F, R, L;X6=D, I;X7=A, S, R, I;X8=I, A, V;X9=A, D;X 10 =A, E, K;X 11 =A, E, K;X 12 =A, E, K;X 13 =I, F, M, L;X 14 =A, K, E;X 15 =E, F, A, N, D, S, T, Q, V, I, L, Y, M, R;X 16 =A, E, K;X 17 =G, S;X 18 =G, T;X 19 =G, E, N;X 20 =G, S, E, A;X 21 =E, G;X 22 =G, E, S, R;X 23 =G, E, R;X 24 =S, G, E, Q;X 25 =G, K;X 26 =G, S, H;X 27 =A, E, S, G, R;X 28 =S, G, L;X 29 =G, S, T;X 30 =A, K, where X1~X9X 11 ~X 16 and X 30 At least one of these reduces binding to IL-21R, rather than the amino acid residue at the same position shown in SEQ ID NO: 1, however X 10 and X 17 ~X 29At least one of these is an amino acid residue at the same position as shown in SEQ ID NO: 1, and reduces the isoelectric point compared to human IL-21 (SEQ ID NO: 1). 【0154】 In some embodiments, an IL-21 polypeptide or a functional fragment or variant containing a combination of mutations contains at least one mutation at the position of SEQ ID NO: 2. In some embodiments, the IL-21 polypeptide or its functional fragment or variant, comprising a combination of mutations, includes at least one mutation at a position selected from the group consisting of positions R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, and K117, the position numbering according to Sequence ID No. 2. 【0155】 In some embodiments, an IL-21 polypeptide or a functional fragment or variant containing a combination of mutations contains at least one mutation at the position of SEQ ID NO: 40. In some embodiments, an IL-21 polypeptide or a functional fragment or variant containing a combination of mutations contains at least one mutation at a position selected from the group consisting of positions: R5, I8, R9, R11, Q12, I14, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117, the position numbering following SEQ ID NO: 40. In some embodiments, the IL-21 polypeptide or its functional fragment or variant, comprising a combination of mutations, includes at least one mutation at a position selected from the group consisting of positions R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, and K117, the position numbering according to Sequence ID No. 40. 【0156】 In some embodiments, the IL-21 polypeptide or its functional fragment or variant is (i) four or more amino acid substitutions that provide a reduced isoelectric point compared to the human IL-21 polypeptide comprising SEQ ID NO: 1, wherein the four or more amino acid substitutions are located within S80-T92 of SEQ ID NO: 1, and at least four of the amino acid substitutions are in residues R85, R86, K88, H89, R90, or a combination thereof, and (ii) (a) R11D; (b) R11E; (c) I14D , D18A and K117A;(d)R76E;(e)R5F;(f)R76F;(g)I8E;(h)R5A;(i)R5E;(j)R5S;(k)R5T;(l)R5N;(m)R5Q;(n)R5V;(o)R5I;(p)R5L;(q)R5Y;(r) R76A;(s)R76N;(t)R76D;(u)R76S;(v)R76T;(w)R76Q;(x)R76V;(y)R76I;(z)R76L;(aa)R76Y;(bb)K77A;(cc)K77E;(dd)K72A;(ee)K72E;(ff )K75A;(gg)K75E;(hh)K73A;(ii)K73E;(jj)R5F and K77A;(kk)R5F and K77E;(ll)R5F and K72A;(mm)R5F and K72E;(nn)R5F and K76A;(oo)R5F and K76E;(pp)K73A and K76F;(qq)K73E and K76F;(rr)R9A;(ss)R9D;(tt)R9E;(uu)R9H;(vv)R9S;(ww)R9T;(xx)R9N;(zz)R9G;(aaa)R9V;(bbb)R9I;(cc c) comprising at least one amino acid substitution selected from R9L;(ddd)R9Y;(eee)K72A and R76F;(fff)K75A and R76F;(ggg)R76F and K77A;(hhh)K75E and R76F;(iii)V17I and L74I;(jjj)I16A and L74F;(kkk)I16S, V17I and L74V;(lll)I16R, V17I and L74I;(mmm)L13F, I16A, V17A and L74M; and (nnn)L13R, I16A, V17I and L74I. 【0157】 In some embodiments, the IL-21 polypeptide or its functional fragment or variant is (i) an amino acid sequence selected from SEQ ID NOs: 2, 4-15 and 23-40, and (ii) (a) R11D; (b) R11E; (c) I14D, D18A and K117A; (d) R76E; (e) R5F; (f) R76F; (g) I8E; (h) R5A; (i) R5E; (j) R5S; (k) R5T; (l) R5N; (m) R5Q; (n) R5V; (o )R5I;(p)R5L;(q)R5Y;(r)R76A;(s)R76N;(t)R76D;(u)R76S;(v)R76T;(w)R76Q;(x)R76V;(y)R76I;(z)R76L;(aa)R76Y; (bb)K77A;(cc)K77E;(dd)K72A;(ee)K72E;(ff)K75A;(gg)K75E;(hh)K73A;(ii)K73E;(jj)R5F and K77A;(kk)R5F and K77E;( ll)R5F and K72A; (mm)R5F and K72E; (nn)R5F and K76A; (oo)R5F and K76E; (pp)K73A and K76F; (qq)K73E and K76F; (rr)R9A; (ss)R9D; (tt)R9E; (uu)R9H; (vv)R9S; (ww)R9T; (xx)R9N; (zz)R9G; (aaa)R9V; (bbb)R9I; (ccc)R9L; (ddd)R9Y; (eee)K72A and R76F; (fff)K75A and R76F; (ggg)R76F and K77A; (hhh)K75E and R76F; (iii)V17I and L74I; (jjj)I16A and L74F; (kkk)I16S, V17I, and L74V; (lll)I16R, V17I, and L74I; (mmm)L13F, I16A, V17A, and L74M; and (nnn)L13R, I16A, V17I, and L74I, comprising at least one amino acid substitution selected from these. 【0158】 In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains the R11D mutation and exhibits approximately 75% to approximately 100% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains the R11D mutation and exhibits at least approximately 75% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains the R11D mutation and exhibits up to approximately 100% sequence identity with SEQ ID NO: 16. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains the R11D mutation and exhibits approximately 75% to 80% sequence identity with SEQ ID NO: 16, approximately 75% to 85% sequence identity, approximately 75% to 90% sequence identity, approximately 75% to 95% sequence identity, approximately 75% to 96% sequence identity, and approximately 75% to 97% sequence identity. Sequence identity of approximately 75% to 98% sequence identity, approximately 75% to 99% sequence identity, approximately 75% to 100% sequence identity, approximately 80% to 85% sequence identity, approximately 80% to 90% sequence identity, approximately 80% to 95% sequence identity, approximately 80% to 96% sequence identity, approximately 80% to 97% sequence identity, approximately 80 % sequence identity ~ approximately 98% sequence identity, approximately 80% sequence identity ~ approximately 99% sequence identity, approximately 80% sequence identity ~ approximately 100% sequence identity, approximately 85% sequence identity ~ approximately 90% sequence identity, approximately 85% sequence identity ~ approximately 95% sequence identity, approximately 85% sequence identity ~ approximately 96% sequence identity, approximately 85% sequence identity ~ approximately 97% sequence identity, approximately 85% sequence identity ~ approximately 98% sequence identity, approximately 85% sequence identity ~ approximately 9 9% sequence identity, approximately 85% to approximately 100% sequence identity, approximately 90% to approximately 95% sequence identity, approximately 90% to approximately 96% sequence identity, approximately 90% to approximately 97% sequence identity, approximately 90% to approximately 98% sequence identity, approximately 90% to approximately 99% sequence identity, approximately 90% to approximately 100% sequence identity, approximately 95% to approximately 96% sequence identity,Approximately 95% sequence identity to approximately 97% sequence identity, approximately 95% sequence identity to approximately 98% sequence identity, approximately 95% sequence identity to approximately 99% sequence identity, approximately 95% sequence identity to approximately 100% sequence identity, approximately 96% sequence identity to approximately 97% sequence identity, approximately 96% sequence identity to approximately 98% sequence identity, approximately 96% sequence identity to approximately 99% sequence identity, approximately 96% Includes sequence identity of ~ approximately 100% sequence identity, approximately 97% to approximately 98% sequence identity, approximately 97% to approximately 99% sequence identity, approximately 97% to approximately 100% sequence identity, approximately 98% to approximately 99% sequence identity, approximately 98% to approximately 100% sequence identity, or approximately 99% to approximately 100% sequence identity. In some embodiments, the IL-21 polypeptide or its functional fragment or variant includes the R11D mutation and comprises approximately 75% sequence identity with SEQ ID NO: 16, approximately 80% sequence identity, approximately 85% sequence identity, approximately 90% sequence identity, approximately 95% sequence identity, approximately 96% sequence identity, approximately 97% sequence identity, approximately 98% sequence identity, approximately 99% sequence identity, or approximately 100% sequence identity. 【0159】 In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains the R11E mutation and exhibits approximately 75% to approximately 100% sequence identity with SEQ ID NO: 17. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains the R11E mutation and exhibits at least approximately 75% sequence identity with SEQ ID NO: 17. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains the R11E mutation and exhibits up to approximately 100% sequence identity with SEQ ID NO: 17. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains the R11E mutation and exhibits approximately 75% to 80% sequence identity with SEQ ID NO: 17, approximately 75% to 85% sequence identity, approximately 75% to 90% sequence identity, approximately 75% to 95% sequence identity, approximately 75% to 96% sequence identity, and approximately 75% to 97% sequence identity. Sequence identity of approximately 75% to 98% sequence identity, approximately 75% to 99% sequence identity, approximately 75% to 100% sequence identity, approximately 80% to 85% sequence identity, approximately 80% to 90% sequence identity, approximately 80% to 95% sequence identity, approximately 80% to 96% sequence identity, approximately 80% to 97% sequence identity, approximately 80 % sequence identity ~ approximately 98% sequence identity, approximately 80% sequence identity ~ approximately 99% sequence identity, approximately 80% sequence identity ~ approximately 100% sequence identity, approximately 85% sequence identity ~ approximately 90% sequence identity, approximately 85% sequence identity ~ approximately 95% sequence identity, approximately 85% sequence identity ~ approximately 96% sequence identity, approximately 85% sequence identity ~ approximately 97% sequence identity, approximately 85% sequence identity ~ approximately 98% sequence identity, approximately 85% sequence identity ~ approximately 9 9% sequence identity, approximately 85% to approximately 100% sequence identity, approximately 90% to approximately 95% sequence identity, approximately 90% to approximately 96% sequence identity, approximately 90% to approximately 97% sequence identity, approximately 90% to approximately 98% sequence identity, approximately 90% to approximately 99% sequence identity, approximately 90% to approximately 100% sequence identity, approximately 95% to approximately 96% sequence identity,Approximately 95% sequence identity to approximately 97% sequence identity, approximately 95% sequence identity to approximately 98% sequence identity, approximately 95% sequence identity to approximately 99% sequence identity, approximately 95% sequence identity to approximately 100% sequence identity, approximately 96% sequence identity to approximately 97% sequence identity, approximately 96% sequence identity to approximately 98% sequence identity, approximately 96% sequence identity to approximately 99% sequence identity, approximately 96% Includes sequence identity of ~ approximately 100% sequence identity, approximately 97% to approximately 98% sequence identity, approximately 97% to approximately 99% sequence identity, approximately 97% to approximately 100% sequence identity, approximately 98% to approximately 99% sequence identity, approximately 98% to approximately 100% sequence identity, or approximately 99% to approximately 100% sequence identity. In some embodiments, the IL-21 polypeptide or its functional fragment or variant includes the R11E mutation and comprises approximately 75% sequence identity with SEQ ID NO: 17, approximately 80% sequence identity, approximately 85% sequence identity, approximately 90% sequence identity, approximately 95% sequence identity, approximately 96% sequence identity, approximately 97% sequence identity, approximately 98% sequence identity, approximately 99% sequence identity, or approximately 100% sequence identity. 【0160】 In some embodiments, the IL-21 polypeptide or its functional fragment or variant, or its functional fragment or variant, includes the I14D, D18A, and K117A mutations and exhibits approximately 75% to approximately 100% sequence identity with SEQ ID NO: 18. In some embodiments, the IL-21 polypeptide or its functional fragment or variant, or its functional fragment or variant, includes the I14D, D18A, and K117A mutations and exhibits at least approximately 75% sequence identity with SEQ ID NO: 18. In some embodiments, the IL-21 polypeptide or its functional fragment or variant, or its functional fragment or variant, includes the I14D, D18A, and K117A mutations and exhibits up to approximately 100% sequence identity with SEQ ID NO: 18. In some embodiments, the IL-21 polypeptide or its functional fragment or variant or its functional fragment or variant includes the I14D, D18A and K117A mutations, and exhibits approximately 75% to 80% sequence identity with SEQ ID NO: 18, approximately 75% to 85%, approximately 75% to 90%, approximately 75% to 95%, approximately 75% to 96%, approximately 75% to 97%, approximately 75% to 98%, approximately 75% to 99%, approximately 75% to 100%, approximately 80% to 85%, and approximately 80% sequence identity. Sex ~ approximately 90% sequence identity, approximately 80% sequence identity ~ approximately 95% sequence identity, approximately 80% sequence identity ~ approximately 96% sequence identity, approximately 80% sequence identity ~ approximately 97% sequence identity, approximately 80% sequence identity ~ approximately 98% sequence identity, approximately 80% sequence identity ~ approximately 99% sequence identity, approximately 80% sequence identity ~ approximately 100% sequence identity, approximately 85% sequence identity ~ approximately 90% Sequence identity of approximately 85% to 95% sequence identity, approximately 85% to 96% sequence identity, approximately 85% to 97% sequence identity, approximately 85% to 98% sequence identity, approximately 85% to 99% sequence identity, approximately 85% to 100% sequence identity, approximately 90% to 95% sequence identity,Approximately 90% sequence identity to approximately 96% sequence identity, approximately 90% sequence identity to approximately 97% sequence identity, approximately 90% sequence identity to approximately 98% sequence identity, approximately 90% sequence identity to approximately 99% sequence identity, approximately 90% sequence identity to approximately 100% sequence identity, approximately 95% sequence identity to approximately 96% sequence identity, approximately 95% sequence identity to approximately 97% sequence identity, approximately 95% sequence identity to approximately 98% sequence identity, approximately 95% sequence identity to approximately 99% sequence identity, approximately 95% sequence identity to approximately 100% sequence identity, approximately 96% Includes sequence identity of approximately 97% to 97% sequence identity, approximately 96% to 98% sequence identity, approximately 96% to 99% sequence identity, approximately 96% to 100% sequence identity, approximately 97% to 98% sequence identity, approximately 97% to 99% sequence identity, approximately 97% to 100% sequence identity, approximately 98% to 99% sequence identity, approximately 98% to 100% sequence identity, or approximately 99% to 100% sequence identity. In some embodiments, the IL-21 polypeptide or its functional fragment or variant, or its functional fragment or variant, includes the I14D, D18A, and K117A mutations and exhibits approximately 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 18. 【0161】 In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains the R76E mutation and exhibits approximately 75% to approximately 100% sequence identity with SEQ ID NO: 19. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains the R76E mutation and exhibits at least approximately 75% sequence identity with SEQ ID NO: 19. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains the R76E mutation and exhibits up to approximately 100% sequence identity with SEQ ID NO: 19. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has approximately 75% to 80% sequence identity with SEQ ID NO: 19, approximately 75% to 85% sequence identity, approximately 75% to 90% sequence identity, approximately 75% to 95% sequence identity, approximately 75% to 96% sequence identity, approximately 75% to 97% sequence identity, and approximately 75% sequence identity to approximately 98% sequence identity, approximately 75% sequence identity to approximately 99% sequence identity, approximately 75% sequence identity to approximately 100% sequence identity, approximately 80% sequence identity to approximately 85% sequence identity, approximately 80% sequence identity to approximately 90% sequence identity, approximately 80% sequence identity to approximately 95% sequence identity, approximately 80% sequence identity to approximately 96% sequence identity, approximately 80% sequence identity to approximately 97% sequence identity, approximately 80% sequence identity Uniformity to approximately 98% sequence identity, approximately 80% to approximately 99% sequence identity, approximately 80% to approximately 100% sequence identity, approximately 85% to approximately 90% sequence identity, approximately 85% to approximately 95% sequence identity, approximately 85% to approximately 96% sequence identity, approximately 85% to approximately 97% sequence identity, approximately 85% to approximately 98% sequence identity, approximately 85% to approximately 99% sequence identity Sequence identity of approximately 85% to 100% sequence identity, approximately 90% to 95% sequence identity, approximately 90% to 96% sequence identity, approximately 90% to 97% sequence identity, approximately 90% to 98% sequence identity, approximately 90% to 99% sequence identity, approximately 90% to 100% sequence identity, approximately 95% to 96% sequence identity,Approximately 95% sequence identity to approximately 97% sequence identity, approximately 95% sequence identity to approximately 98% sequence identity, approximately 95% sequence identity to approximately 99% sequence identity, approximately 95% sequence identity to approximately 100% sequence identity, approximately 96% sequence identity to approximately 97% sequence identity, approximately 96% sequence identity to approximately 98% sequence identity, approximately 96% sequence identity to approximately 99% sequence identity, approximately 96% Includes sequence identity of ~ approximately 100% sequence identity, approximately 97% to approximately 98% sequence identity, approximately 97% to approximately 99% sequence identity, approximately 97% to approximately 100% sequence identity, approximately 98% to approximately 99% sequence identity, approximately 98% to approximately 100% sequence identity, or approximately 99% to approximately 100% sequence identity. In some embodiments, the IL-21 polypeptide or its functional fragment or variant includes the R76E mutation and comprises approximately 75% sequence identity with SEQ ID NO: 19, approximately 80% sequence identity, approximately 85% sequence identity, approximately 90% sequence identity, approximately 95% sequence identity, approximately 96% sequence identity, approximately 97% sequence identity, approximately 98% sequence identity, approximately 99% sequence identity, or approximately 100% sequence identity. 【0162】 In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains the R5F mutation and has approximately 75% to approximately 100% sequence identity with SEQ ID NO: 20. In some embodiments, the IL-21 polypeptide or its functional fragment or variant contains the R5F mutation and has at least approximately 75% sequence identity with SEQ ID NO: 20. In some embodiments, the IL-21 polypeptide or its functional fragment or variant has up to approximately 100% sequence identity with SEQ ID NO: 20. In some embodiments, the IL-21 polypeptide or its functional fragment or variant includes the R5F mutation and exhibits approximately 75% to 80% sequence identity with SEQ ID NO: 29, approximately 75% to 85% sequence identity, approximately 75% to 90% sequence identity, approximately 75% to 95% sequence identity, approximately 75% to 96% sequence identity, and approximately 75% to 97% sequence identity. Sequence identity, approximately 75% sequence identity to approximately 98% sequence identity, approximately 75% sequence identity to approximately 99% sequence identity, approximately 75% sequence identity to approximately 100% sequence identity, approximately 80% sequence identity to approximately 85% sequence identity, approximately 80% sequence identity to approximately 90% sequence identity, approximately 80% sequence identity to approximately 95% sequence identity, approximately 80% sequence identity to approximately 96% sequence identity, approximately 80% sequence identity to approximately 97% sequence identity, approximately 80% Sequence identity ~ approximately 98% sequence identity, approximately 80% sequence identity ~ approximately 99% sequence identity, approximately 80% sequence identity ~ approximately 100% sequence identity, approximately 85% sequence identity ~ approximately 90% sequence identity, approximately 85% sequence identity ~ approximately 95% sequence identity, approximately 85% sequence identity ~ approximately 96% sequence identity, approximately 85% sequence identity ~ approximately 97% sequence identity, approximately 85% sequence identity ~ approx...

Claims

[Claim 1] It is a fusion protein, (a) An IL-21 polypeptide comprising an amino acid sequence that is at least 90% identical to that of a human IL-21 polypeptide comprising the sequence of SEQ ID NO: 1, wherein the IL-21 polypeptide comprises one or more amino acid substitutions in the region of positions S80 to T92 numbered according to the sequence of SEQ ID NO: 1, and the one or more amino acid substitutions are at least 0.6 to 5 units lower in isoelectricity compared to those of the human IL-21 polypeptide comprising the sequence of SEQ ID NO:

1. The points provided, and the one or more amino acid substitutions are selected from the group consisting of S80G, T81G, N82G, N82E, A83G, A83E, A83S, R85G, R85E, R85S, R86G, R86E, Q87G, Q87E, Q87S, K88G, H89G, H89S, R90G, R90S, R90E, R90A, L91G, L91S, T92G, and T92S, which are numbered according to the sequence of SEQ ID NO: 1, and IL-21 polypeptide, and (b) An antibody or antigen-binding fragment that specifically binds to at least one of CD8α, CD8αα, or CD8αβ. Includes, The fusion protein containing the IL-21 polypeptide provides improved systemic exposure compared to the fusion protein containing the human IL-21 polypeptide containing the sequence of SEQ ID NO: 1, as measured by the area under the curve (AUC) of the fusion protein containing the IL-21 polypeptide being at least about 1.5 times greater when administered to a subject at equivalent concentrations. Fusion protein. [Claim 2] The fusion protein according to claim 1, wherein the IL-21 polypeptide has an isoelectric point of 7.12 to 8.

72. [Claim 3] The fusion protein according to claim 1, wherein the IL-21 polypeptide is G84, numbered according to the sequence of SEQ ID NO: 1, and does not contain any amino acid substitutions. [Claim 4] The fusion protein according to claim 1, wherein the IL-21 polypeptide comprises 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions selected from the group consisting of S80G, T81G, N82G, N82E, A83G, A83E, A83S, R85G, R85E, R85S, R86G, R86E, Q87G, Q87E, Q87S, K88G, H89G, H89S, R90G, R90S, R90E, R90A, L91G, L91S, T92G, and T92S, compared to the sequence of SEQ ID NO:

1. [Claim 5] The fusion protein according to claim 1, wherein the IL-21 polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 2, 4-15, and 23-40. [Claim 6] The fusion protein according to claim 1, wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 4-15, and 23-40. [Claim 7] The fusion protein according to claim 1, wherein the IL-21 polypeptide further comprises at least one amino acid substitution that reduces binding to the IL-21 receptor compared to binding by the human IL-21 polypeptide. [Claim 8] The fusion protein according to claim 7, wherein the at least one amino acid substitution that reduces binding to the IL-21 receptor is at one or more amino acid residues at a position selected from the group consisting of R5, I8, R9, R11, L13, I14, I16, V17, D18, K72, K73, L74, K75, R76, K77, and K117, numbered according to the sequence of SEQ ID NO:

1. [Claim 9] The at least one amino acid substitution that reduces binding to the IL-21 receptor is, compared to the sequence of SEQ ID NO: 1, R5F, R5A, R5E, R5S, R5T, R5N, R5Q, R5V, R5I, R5L, R5Y, I8E, R9A, R9D, R9E, R9H, R9S, R9T, R9N, R9G, R9V, R9I, R9L, R9Y, R11D, R11E, L13F, L13R, I14D, I16A, I16S, A fusion protein according to claim 7, selected from the group consisting of I16R, V17I, V17A, D18A, K72A, K72E, K73A, K73E, K75A, K75E, L74I, L74F, L74M, L74V, R76E, R76F, R76A, R76N, R76D, R76S, R76T, R76Q, R76V, R76I, R76L, R76Y, R76M, K77A, K77E, and K117A. [Claim 10] The fusion protein according to claim 7, wherein the at least one amino acid substitution that reduces binding to the IL-21 receptor is R76E or R76Q. [Claim 11] The fusion protein according to claim 1, wherein the IL-21 polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 16-21, 41-98, and 374-379. [Claim 12] The fusion protein according to claim 1, wherein the IL-21 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 16-21, 41-98, and 374-379. [Claim 13] It is a fusion protein, (a) an IL-21 polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 94, and (b) An antibody or antigen-binding fragment that specifically binds to at least one of CD8α, CD8αα, or CD8αβ. Includes, The fusion protein containing the IL-21 polypeptide provides improved systemic exposure compared to the fusion protein containing the human IL-21 polypeptide containing the sequence of SEQ ID NO: 1, as measured by the area under the curve (AUC) of the fusion protein containing the IL-21 polypeptide being at least about 1.5 times greater when administered to a subject at equivalent concentrations. Fusion protein. [Claim 14] The fusion protein according to claim 1, wherein the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO:

94. [Claim 15] The fusion protein according to claim 1, wherein the IL-21 polypeptide and the antibody or its antigen-binding fragment are linked to each other via a linker, and optionally the IL-21 polypeptide is linked to the C-terminus of the antibody or its antigen-binding fragment. [Claim 16] The antibody or its antigen-binding fragment is i) A first polypeptide comprising a light chain variable region and a light chain constant region, ii) A second polypeptide comprising a heavy chain variable region and a heavy chain constant region, and iii) A third polypeptide containing a heavy chain constant region The fusion protein according to claim 1, comprising the heavy chain constant regions of the second polypeptide and the third polypeptide, wherein the heavy chain constant regions of the second polypeptide and the third polypeptide form an Fc domain. [Claim 17] The fusion protein according to claim 16, wherein the third polypeptide comprises a heavy chain variable region, and optionally the antibody or its antigen-binding fragment further comprises a fourth polypeptide comprising a light chain variable region and a light chain constant region. [Claim 18] The fusion protein according to claim 16, wherein the IL-21 polypeptide is linked to the C-terminus of the heavy chain constant region of the second polypeptide or the third polypeptide. [Claim 19] The fusion protein according to claim 16, wherein the Fc domain includes one or more modifications that promote heterodimerization. [Claim 20] The fusion protein according to claim 16, wherein the second polypeptide comprises a knob modification in the heavy chain constant region, and the third polypeptide comprises a hole modification in the heavy chain constant region, optionally the knob modification comprising amino acid substitutions Y349C and T366W according to EU numbering, and the hole modification comprising amino acid substitutions S354C, T366S, L368A and Y407V according to EU numbering. [Claim 21] The fusion protein according to claim 16, wherein the Fc domain comprises an amino acid mutation that reduces the binding of the Fc domain to the Fc gamma receptor, and optionally the Fc domain comprises amino acid substitutions L234A, L235A, and G237A according to EU numbering. [Claim 22] The antibody or its antigen-binding fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain. (a) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 226, CDR-H2 containing the amino acid sequence of SEQ ID NO: 227, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 151; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 152, CDR-L2 containing the amino acid sequence of SEQ ID NO: 153, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

154. (b) The VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 228, CDR-H2 containing the amino acid sequence of SEQ ID NO: 227, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 157, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 158, CDR-L2 containing the amino acid sequence of SEQ ID NO: 159, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

160. (c) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 223, CDR-H2 containing the amino acid sequence of SEQ ID NO: 227, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 163; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 164, CDR-L2 containing the amino acid sequence of SEQ ID NO: 165, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

166. (d) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 227, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 169, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 170, CDR-L2 containing the amino acid sequence of SEQ ID NO: 171, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

172. (e) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 230, CDR-H2 containing the amino acid sequence of SEQ ID NO: 231, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 175, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 176, CDR-L2 containing the amino acid sequence of SEQ ID NO: 177, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

178. (f) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 230, CDR-H2 containing the amino acid sequence of SEQ ID NO: 232, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 181, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 182, CDR-L2 containing the amino acid sequence of SEQ ID NO: 183, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

184. (g) The VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 223, CDR-H2 containing the amino acid sequence of SEQ ID NO: 224, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 225, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 140, CDR-L2 containing the amino acid sequence of SEQ ID NO: 141, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

142. (h) The VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 233, CDR-H2 containing the amino acid sequence of SEQ ID NO: 234, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 145, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 146, CDR-L2 containing the amino acid sequence of SEQ ID NO: 147, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

148. (i) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 235, CDR-H2 containing the amino acid sequence of SEQ ID NO: 236, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 237, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 188, CDR-L2 containing the amino acid sequence of SEQ ID NO: 189, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

190. (j) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 242, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 152, CDR-L2 containing the amino acid sequence of SEQ ID NO: 153, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

202. (k) The VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

207. (l) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 152, CDR-L2 containing the amino acid sequence of SEQ ID NO: 153, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

202. (m) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 244, CDR-H2 containing the amino acid sequence of SEQ ID NO: 245, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 246, and the VL domain includes CDR-L1 containing the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 176), CDR-L2 containing the amino acid sequence of GASSRAT (SEQ ID NO: 177), and CDR-L3 containing the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 178). (n) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 250, CDR-H2 containing the amino acid sequence of SEQ ID NO: 251, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 288, and the VL domain includes CDR-L1 containing the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 176), CDR-L2 containing the amino acid sequence of GASSRAT (SEQ ID NO: 177), and CDR-L3 containing the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 178). (o) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 250, CDR-H2 containing the amino acid sequence of SEQ ID NO: 261, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 288, and the VL domain includes CDR-L1 containing the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 176), CDR-L2 containing the amino acid sequence of GASSRAT (SEQ ID NO: 177), and CDR-L3 containing the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 178), or (p) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 223, CDR-H2 containing the amino acid sequence of SEQ ID NO: 224, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 284, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 285, CDR-L2 containing the amino acid sequence of SEQ ID NO: 286, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

287. The fusion protein according to claim 1. [Claim 23] The fusion protein according to claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 241, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 204, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 205, CDR-L2 containing the amino acid sequence of SEQ ID NO: 206, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

207. [Claim 24] The antibody or its antigen-binding fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain. (a) The VH domain comprises the amino acid sequence of SEQ ID NO: 109, and the VL domain comprises the amino acid sequence of SEQ ID NO:

110. (b) The VH domain comprises the amino acid sequence of SEQ ID NO: 111, and the VL domain comprises the amino acid sequence of SEQ ID NO:

112. (c) The VH domain comprises the amino acid sequence of SEQ ID NO: 113, and the VL domain comprises the amino acid sequence of SEQ ID NO:

114. (d) The VH domain comprises the amino acid sequence of SEQ ID NO: 115, and the VL domain comprises the amino acid sequence of SEQ ID NO:

116. (e) The VH domain comprises the amino acid sequence of SEQ ID NO: 117, and the VL domain comprises the amino acid sequence of SEQ ID NO:

118. (f) The VH domain comprises the amino acid sequence of SEQ ID NO: 119, and the VL domain comprises the amino acid sequence of SEQ ID NO:

120. (g) The VH domain contains the amino acid sequence of SEQ ID NO: 123, and the VL domain contains the amino acid sequence of SEQ ID NO:

124. (h) The VH domain comprises the amino acid sequence of SEQ ID NO: 129, and the VL domain comprises the amino acid sequence of SEQ ID NO:

130. (i) The VH domain comprises the amino acid sequence of SEQ ID NO: 131, and the VL domain comprises the amino acid sequence of SEQ ID NO:

132. (j) The VH domain comprises the amino acid sequence of SEQ ID NO: 125, and the VL domain comprises the amino acid sequence of SEQ ID NO:

126. (k) The VH domain comprises the amino acid sequence of SEQ ID NO: 127, and the VL domain comprises the amino acid sequence of SEQ ID NO:

128. (l) The VH domain contains the amino acid sequence of SEQ ID NO: 133, and the VL domain contains the amino acid sequence of SEQ ID NO:

134. (m) The VH domain contains the amino acid sequence of SEQ ID NO: 135, and the VL domain contains the amino acid sequence of SEQ ID NO:

136. (n) The VH domain comprises the amino acid sequence of SEQ ID NO: 107, and the VL domain comprises the amino acid sequence of SEQ ID NO:

108. (o) The VH domain contains the amino acid sequence of SEQ ID NO: 121, and the VL domain contains the amino acid sequence of SEQ ID NO: 122, or (p) The VH domain contains the amino acid sequence of SEQ ID NO: 258, and the VL domain contains the amino acid sequence of SEQ ID NO:

259. The fusion protein according to claim 1. [Claim 25] The fusion protein according to claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, the VH domain comprising the amino acid sequence of SEQ ID NO: 129, and the VL domain comprising the amino acid sequence of SEQ ID NO:

130. [Claim 26] It comprises a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, (a) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 262, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 263, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 264, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

262. (b) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 266, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 267, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 268, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

266. (c) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 270, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 271, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 272, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

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

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

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

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

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

270. (i) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 274, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 275, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 277, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

274. (j) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 278, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 279, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 281, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

278. (k) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 297, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 298, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 299, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

297. (l) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 301, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 302, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 303, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

301. (m) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 305, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 306, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 307, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

305. (n) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 309, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 310, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 311, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

309. (o) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 297, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 298, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 300, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

297. (p) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 301, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 302, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 304, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

301. (q) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 305, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 306, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 308, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 305, or (r) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 309, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 310, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 312, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

309. The fusion protein according to claim 1. [Claim 27] The fusion protein according to claim 1, comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 262, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 263, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 264, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

262. [Claim 28] The fusion protein according to claim 1, comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 262, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 263, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 265, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

262. [Claim 29] A polynucleotide encoding a fusion protein according to any one of claims 1 to 28, wherein the polynucleotide optionally comprises a nucleotide sequence encoding the IL-21 polypeptide and at least one nucleotide sequence encoding the antibody or its antigen-binding fragment. [Claim 30] A vector comprising polynucleotides as described in claim 29. [Claim 31] A host cell comprising the vector according to claim 30. [Claim 32] A pharmaceutical composition comprising the fusion protein described in any one of claims 1 to 28. [Claim 33] A pharmaceutical composition according to claim 32 for use in activating a population of CD8+ T cells, wherein the activation comprises contacting the population of CD8+ T cells with the pharmaceutical composition. [Claim 34] A pharmaceutical composition according to claim 32 for use in the selective activation of a population of CD8+ T cells, wherein the selective activation comprises contacting a population of cells including CD8+ T cells, CD4+ T cells, and NK cells with the pharmaceutical composition, and optionally the CD8+ T cells are activated with at least 10 to 100,000 times higher potency compared to the activation of the NK cells or CD4+ T cells in the population of cells by the pharmaceutical composition. [Claim 35] A fusion protein according to any one of claims 1 to 28, for use as a pharmaceutical agent. [Claim 36] A fusion protein according to any one of claims 1 to 28, for use in the treatment of cancer or chronic infection in a subject, wherein the treatment comprises administering the fusion protein to the subject. [Claim 37] A fusion protein according to any one of claims 1 to 28 for use in the treatment of cancer in a subject, wherein the treatment comprises administering the fusion protein to the subject, optionally further comprising administering a T-cell therapy, a cancer vaccine, a chemotherapeutic agent, or an immune checkpoint inhibitor (ICI) to the subject, optionally the ICI being an inhibitor of PD-1, PD-L1, or CTLA-4, and optionally the T-cell therapy comprising a chimeric antigen receptor (CAR)-based T-cell therapy, a tumor-infiltrating lymphocyte (TIL)-based therapy, or a therapy using T cells having a transduced TCR. [Claim 38] A kit comprising a container containing a fusion protein according to any one of claims 1 to 28, wherein the kit optionally further comprises a pharmaceutically acceptable carrier and instructions for administering the fusion protein to a subject having a disease, wherein the disease is cancer or a chronic infection. [Claim 39] A method for producing a fusion protein according to any one of claims 1 to 28, wherein the method comprises culturing the host cells according to claim 31, and optionally further comprising collecting the fusion protein from the host cells.

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