EP4598948A2 - Interleukin-21 mimetics - Google Patents

Interleukin-21 mimetics

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Publication number
EP4598948A2
EP4598948A2 EP23875860.1A EP23875860A EP4598948A2 EP 4598948 A2 EP4598948 A2 EP 4598948A2 EP 23875860 A EP23875860 A EP 23875860A EP 4598948 A2 EP4598948 A2 EP 4598948A2
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EP
European Patent Office
Prior art keywords
residue
polypeptide
amino acid
seq
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP23875860.1A
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German (de)
French (fr)
Inventor
Jung Ho CHUN
Daniel Adriano SILVA MANZANO
Umut ULGE
Alfredo QUIJANO RUBIO
Huiyun SUN
Neil P. KING
David Baker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Washington
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University of Washington
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Publication of EP4598948A2 publication Critical patent/EP4598948A2/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • IL-21 Interleukin-21
  • CD4+ T cells IL-21 induces differentiation of various immune cells.
  • IL-21 is involved in the regulation of immune responses to infections, cancer, and autoimmune diseases and has been a target of clinical research in recent years.
  • IL-21 has been investigated as a single agent or in combination with other immunotherapy agents, such as checkpoint inhibitors or cancer vaccines. Harnessing the full potential of native IL-21 for therapeutic purposes has proved challenging due to its poor stability, low cross-reactivity of human IL-21 in mouse models, and lack of engineerability. The restricted efficacy of human IL-21 in murine models further hampers the ⁇ predictive assessment of IL-21-based therapeutic candidates regarding toxicity and activity.
  • the disclosure provides polypeptides comprising domains X2, X3, and optionally X4, wherein: (a) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence ⁇ DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); (b) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of (i) KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934), or (ii) KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937
  • the polypeptide comprises domains X2, X3, and X4, wherein: (a) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); ⁇ (b) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934); and (c) X4 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence ⁇ KEVMERAKSAAQKILGRFL (SEQ ID NO: 9
  • X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, ⁇ 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); and
  • X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937), wherein relative to SEQ ID NO: 937, 1, ⁇ 2, or all 3 of the following are true: residue 4 is Y, residue 6 is F, and/or residue 9 is D.
  • X2 and X3 may be in any order in the polypeptide; wherein amino acid linkers may be present between the domains; and wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21R ⁇ CD360) but is not capable of simultaneously binding to the ⁇ c, CD132 receptor.
  • the polypeptides further comprise an X1 domain, wherein the X1 domain comprises a helical structure, and wherein X1, X2, X3, and X4, when present, may be in any order in the polypeptide.
  • the disclosure provides fusion proteins comprising: (a) the polypeptide of any embodiment herein; and ⁇ (b) one or more functional domains.
  • conditionally active IL-21 receptor binding ⁇ proteins comprising a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not both present in the same fusion protein, wherein in total the first polypeptide component and the second polypeptide component comprise domains X2, X3, and optionally X4, as defined in any one of claims 1-34, wherein: ⁇ (i) the first polypeptide component comprises at least one of X2, X3, and X4 (when present) but does not comprise each of X2, X3, and X4 (when present); and (ii) the second polypeptide component comprises each of X2, X3, and X4 (when present) that is not present in the first polypeptide component; wherein the first polypeptide component and the second polypeptide component are ⁇ not active receptor binding proteins individually, and wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the
  • the first polypeptide component comprises at least one of X2, X3, and X4 but ⁇ does not comprise each of X2, X3, and X4; and (ii) the second polypeptide component comprises each of X2, X3, and X4 that is not present in the first polypeptide component; wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL- ⁇ 21R ⁇ CD360) and is capable of binding to the ⁇ c, CD132 receptor.
  • IL-21 agonistic mimics can bind to both IL-21 receptor (IL- 21R; CD360) and common gamma receptor ( ⁇ c ; CD132) for receptor heterodimerization.
  • A Design, screening, optimization, and characterization schematics of ⁇ IL-21 mimics.
  • B A model of an agonistic mimic of IL-21 in complex with hIL-21R and h ⁇ c.
  • C SDS-PAGE of human IL-21 (lane 2), mouse IL-21 (lane 3), and 21h10 (SEQ ID NO:24) (lane 4) with Precision Plus Protein Kaleidoscope ladder (lane 1).
  • IL-21 agonistic mimics can be fused to antibodies for targeted cytokine delivery.
  • the stoichiometry could also be a 2:1 construct with Knobs/Holes mutations in the heavy chain for specific heterodimeric ⁇ configuration; a heavy chain with knobs mutation with the cytokine fusion can be coupled with another heavy chain with holes mutations without the cytokine fusion.
  • B SDS-PAGE of Atezolizumab-21h10 fusion construct in non-reducing condition, showing a band around the expected size of 179.2kDa.
  • Atezolizumab-21h10 fusion construct can bind to human IL-21R and human PD-L1 independently.
  • Atezolizumab-21h10 fusion construct ⁇ can bind to human IL-21R and human PD-L1 concurrently.
  • Atezolizumab-21h10 fusion construct is at 10 nM, with human PD-L1 immobilized to streptavidin (SA) tips and associated with Atezolizumab-21h10 fusion construct then associated with 50 nM human IL- 21R, or vice versa.
  • SA streptavidin
  • De novo IL-21 antagonistic mimics can bind only to IL-21 receptor (IL- ⁇ 21R; CD360) but not ⁇ c (CD132).
  • X axes either represent binding affinity to hIL-21R or mIL-21R
  • Y axes either represent binding affinity to ⁇ hIL-21R/h ⁇ c or mIL-21R/m ⁇ c , in molar units [M].
  • the constructs with mIL- 21R/m ⁇ c affinity worse than 10 -4 M is populated at 10 -4 M.
  • Numberings 1 to 21 correspond to 21A1 to 21A21 (Table 1, SEQ ID NO:164-184), respectively. Detailed Description ⁇ All references cited are herein incorporated by reference in their entirety.
  • amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; ⁇ P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).
  • any N-terminal methionine residues are optional (i.e.: the N-terminal methionine residue may be present or may be absent, and when absent is not considered in determining percent identity).
  • any N- ⁇ or C-terminal histidine tags are optional and me be present or absent and, when absent, are not considered in determining percent identity. All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.
  • the polypeptide comprises domains X2, X3, and X4, wherein: ⁇ (a) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence DTRILKVSLKKIVRAANVIVRM (SEQ ID NO:933); (b) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence ⁇ KRFEIRMRQLIDIVDHVKRE (SEQ ID NO:934); and (c) X4 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence KEVMERAKSAAQKILGRFL (SEQ ID NO:933)
  • polypeptides of this embodiment are capable of acting as agonists to activate IL-21-like signaling and inducing cell differentiation in both human ⁇ and mouse cells by specifically binding and promoting dimerization of the IL-21 receptor (IL-21R ⁇ CD360) and the common- ⁇ chain-receptor ( ⁇ c, CD132).
  • the designed polypeptides show an anti-tumor effect in mouse MC38 adenocarcinoma models, B16F10 melanoma models, and pancreatic cancer models, and thus can be used, for example, to treat cancer.
  • X2 is a peptide comprising the amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933
  • X3 is a peptide comprising the amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID ⁇ NO: 934
  • X4 is a peptide comprising the amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 935.
  • X2 is a peptide comprising the amino acid sequence at ⁇ least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933;
  • X3 is a peptide comprising the amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 934; and
  • X4 is a peptide comprising the amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 935.
  • X2 is a peptide comprising the amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933;
  • X3 is a peptide comprising the amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 934;
  • X4 is a peptide comprising the amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 935.
  • X2 is a peptide comprising the amino acid sequence at least ⁇ 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933;
  • X3 is a peptide comprising the amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 934; and
  • X4 is a peptide comprising the amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 935.
  • X2 is a peptide comprising the amino acid sequence at least 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933; ⁇ (b) X3 is a peptide comprising the amino acid sequence at least 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 934; and (c) X4 is a peptide comprising the amino acid sequence at least 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 935.
  • the inventors have identified residues 11, 14, 18, and 21 of X2 (SEQ ID NO: 933) as present at the binding interface with IL-21R ⁇ (CD360).
  • the ⁇ polypeptide has 1, 2, 3, or all 4 of the following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 11 is K, E, or R; (b) X2 residue 14 is R, E, or K; (c) X2 residue 18 is V, R, H, or K; and/or (d) X2 residue 21 is R or K.
  • the inventors have identified residues 4, 7, 10, and 15 of X2 (SEQ ID NO: 933) as present at the binding interface with IL-21R ⁇ (CD360).
  • the polypeptide has 1, 2, 3, or all 4 of the following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 4 is I or W; (b) X2 residue 7 is V, D. or E or I; ⁇ (c) X2 residue 10 is K or R; and/or (d) X2 residue 15 is A, I, or L or M.
  • the polypeptide has an amino acid selected from the following residues relative to X3 (SEQ ID NO: 934): Residue 1: P, N, D, E, R, K, and H; ⁇ Residue 2: G, F, Y, E, R, and K; Residue 3: V, I, M, L, F, T, and Q; Residue 4: A, I, L, S, T, E, R, and K; Residue 5: A, V, I, L, and E; Residue 6: A, V, L, E, R, and K; ⁇ Residue 7: M and L; Residue 8: V, L, F, Y, T, N, R, and K; Residue 9: G, A, V, Q, and E; Residue 10: A, I, M, and L; Residue 11: A, V, I and L; ⁇ Residue 12: D, E, and K; Residue 1
  • the polypeptide has 1, 2, 3, 4, or all 5 of the following residues relative to X3 (SEQ ID NO: 934): (a) X3 residue 2 is R or K; ⁇ (b) X3 residue 6 is R or K ; (c) X3 residue 8 is R or K; (d) X3 residue 12 is D or E; and/or (e) X3 residue 16 is H or Y or W.
  • the polypeptide has 1, 2, 3, 4, 5, or all 6 of the following residues relative to X3 (SEQ ID NO: 934): (a) X3 residue 1 is K or R; (b) X3 residue 5 is I or V; ⁇ (c) X3 residue 9 is Q or G; (d) X3 residue 11 is I or L; (e) X3 residue 13 is I or V; and/or (f) X3 residue 19 is R or K.
  • the inventors have identified residues 9 and 16 of X4 (SEQ ID NO: 935) as present at the binding interface with ⁇ c (CD132) receptor.
  • the polypeptide has one or both of the following residues relative to X4 (SEQ ID NO: 935): (a) X4 residue 9 is S or T; and/or ⁇ (b) X4 residue 16 is G or Y.
  • the inventors have identified residues 8, 12, 13, 15, and 19 of X4 (SEQ ID NO: 935) as present at the binding interface with ⁇ c (CD132) receptor.
  • the polypeptide has 1, 2, 3, 4, or all 5 of the following residues relative to X4 (SEQ ID NO: 935): (a) X4 residue 8 is K or R; ⁇ (b) X4 residue 12 is Q; (c) X4 residue 13 is K; (d) X4 residue 15 is L or I; and/or (e) X4 residue 19 is L, F, or Y.
  • the polypeptide comprises: ⁇ (1) the following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 11 is K, E, or R; (b) X2 residue 14 is R, E, or K; (c) X2 residue 18 is V, R, H, or K; (d) X2 residue 21 is R or K; ⁇ (e) X2 residue 4 is I or W; (f) X2 residue 7 is V, D.
  • X2 residue 10 is K or R; and (h) X2 residue 15 is A, I, or L or M; and (2) the following residues relative to X3 (SEQ ID NO: 934): ⁇ (a) X3 residue 2 is R or K; (b) X3 residue 6 is R or K ; (c) X3 residue 8 is R or K; (d) X3 residue 12 is D or E; ⁇ (e) X3 residue 16 is H or Y or W; (f) X3 residue 1 is K or R; (g) X3 residue 5 is I or V; (h) X3 residue 9 is Q or G; (i) X3 residue 11 is I or L; ⁇ (j) X3 residue 13 is I or V; and (k) X3 residue 19 is R or K; and (3) the following residues relative to X4 (SEQ ID NO: 935): (a) X4 residue 9 is S or T; and (b) X4 residue 16
  • the disclosure provides polypeptides comprising domains X2 and X3, wherein: (a) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence ⁇ DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); and (b) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937), wherein relative to SEQ ID NO: 937, 1, 2, or all 3 of the following are true: residue 4 is Y, residue 6 is F, and/or residue 9 is D. ⁇ wherein X2 and X3 may be in any order in the polypeptide; wherein amino acid linkers may
  • the polypeptides are capable of acting as IL-21 antagonists.
  • Antagonistic IL-21 mimics are constructs that can bind to IL-21 receptor (IL-21R ⁇ CD360) but cannot bind to the common- ⁇ chain-receptor ( ⁇ c, CD132) simultaneously, potentially prohibiting heterodimerization of IL-21 receptor (IL-21R ⁇ CD360) and the common- ⁇ chain-receptor ( ⁇ c, CD132) and resultant potential downstream cell signaling. Designs can be tested for antagonist activity by first confirming binding to IL-21R, and then to confirm lack of simultaneous binding to ⁇ c receptor.
  • binding assay for ⁇ c receptor need to be done in the presence of IL-21R. Confirmation of IL-21R binding is critical as lack of binding ⁇ to IL-21R can also cause false positives in the result of the lack of binding against the ⁇ c receptor. Similar to agonist identification, the assay should be performed at the highest concentration possible, 1000 nM for IL-21R and 1000 nM for ⁇ c receptor. The inventors have provided extensive teaching of such polypeptides capable of acting as IL-21 receptor antagonists.
  • polypeptides in ⁇ Table 5 have been identified as having antagonistic capability: SEQ ID NO:627-631, 633, 635-641, 643-644, 648-654, 656, 658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681- 685, 687-695, 698, 700-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746, 749-751, 771, 849-854, 856-862, 864-870, 872-874, 876-898, 900, and 902-907.
  • the polypeptide antagonists may be used, for example, in modulating an immune ⁇ response in a subject in need thereof, such as immunoinhibitory purposes for autoimmune diseases, including but not limited to multiple sclerosis, lupus, and rheumatoid arthritis.
  • the X2 domain of this embodiment is the same as the X2 domain of the agonists detailed above, as the X2 domain mediates interaction with the IL-21 receptor (IL-21R ⁇ CD360) but not the ⁇ c, CD132 receptor.
  • X2 is a peptide comprising the amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933; and (b) X3 is a peptide comprising the amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 937.
  • X2 is a peptide comprising the amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933; and (b) X3 is a peptide comprising the amino acid sequence at least ⁇ 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 937.
  • X2 is a peptide comprising the amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID ⁇ NO: 933; and (b) X3 is a peptide comprising the amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 937.
  • X2 is a peptide comprising the amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933; and (b) X3 is a peptide comprising the amino acid sequence at least 90%, 95%, 98%, or 100% ⁇ identical to the amino acid sequence of SEQ ID NO: 937.
  • X2 is a peptide comprising the amino acid sequence at least 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933; and (b) X3 is a peptide comprising the amino acid sequence at least 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 937.
  • SSM site-saturation mutagenesis
  • BLI biolayer interferometry
  • the polypeptide has an amino acid selected from the following residues relative ⁇ to X2 (SEQ ID NO: 933): Residue 1: G, A, S, T, Q, D, E, R, K, and H; Residue 2: G, A, L, S, T, N, and Q; Residue 3: A, M, Y, W, N, Q, E, R, and K; Residue 4: G, A, V, I, M, L, F, Y, W, N, Q, E, and H; ⁇ Residue 5: M, L, Q, E, and K; Residue 6: V, I, L, Y, R, and K; Residue 7: G, V, I, M, L, F, Y, W, S, T, N, Q, D, E, and R; Residue 8: A, L, F, S, T, E, and H; Residue 9: A, V, I, and L;
  • the inventors have identified residues 11, 14, 18, and 21 of X2 (SEQ ID NO: 933) as ⁇ present at the binding interface with IL-21R ⁇ CD360.
  • the polypeptide has 1, 2, 3, or all 4 of the following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 11 is K, E, or R; (b) X2 residue 14 is R, E, or K; (c) X2 residue 18 is V, R, H, or K; and/or ⁇ (d) X2 residue 21 is R or K.
  • the inventors have identified residues 4, 7, 10, and 15 of X2 (SEQ ID NO: 933) as present at the binding interface with IL-21R ⁇ CD360.
  • the polypeptide has 1, 2, 3, or all 4 of the following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 4 is I or W; ⁇ (b) X2 residue 7 is V, D. or E or I; (c) X2 residue 10 is K or R; and/or (d) X2 residue 15 is A, I, or L or M.
  • the polypeptide has an amino acid selected from the following residues relative to X3 (SEQ ID NO: 937): ⁇ Residue 1: P, N, D, E, R, K, and H; Residue 2: G, F, Y, E, and R, and K; Residue 3: V, I, M, L, F, T, and Q; Residue 4: Y; Residue 5: A, V, I, L, and E; ⁇ Residue 6: F; Residue 7: M and L; Residue 8: Q and E; Residue 9: D; ⁇ Residue10: A, I, M, and L; Residue 11: A, V, I, and L; Residue 12: D, E, and K; Residue 13: A, V, I, L, and T; ⁇ Residue 14: C, A, V, S, T, and E; Residue 15: T, N,
  • the polypeptide has 1, 2, or all 3 of the following residues relative to X3 (SEQ ID NO: 937): (a) X3 residue 2 is R or K; ⁇ (b) X3 residue 12 is D or E; and/or (c) X3 residue 16 is H or Y/W. In other embodiments, the polypeptide has 1, 2, 3, 4, or all 5 of the following residues relative to X3 (SEQ ID NO: 937): (a) X3 residue 1 is K or R; ⁇ (b) X3 residue 5 is I or V; (c) X3 residue 11 is I or L; (d) X3 residue 13 is I or V; and/or (e) X3 residue 19 is R or K.
  • the antagonist polypeptides further comprise an X4 domain, wherein the X4 domain comprises a helical structure, wherein the X4 domain does not permit simultaneous binding of the polypeptide to the IL-21 receptor (IL-21R ⁇ CD360) and the ⁇ c, CD132 receptor, and wherein X2, X3, and X4 may be in any order in the polypeptide.
  • the X4 domain may be any helical domain as suitable for an intended purpose, ⁇ so long as it does not permit simultaneous binding of the polypeptide to the IL-21 receptor (IL-21R ⁇ CD360) and the ⁇ c, CD132 receptor,.
  • X4 comprises a 19-residue peptide.
  • the X4 peptide has an amino acid at each position selected from the following amino acid residues: ⁇ Residue 1: R and K; Residue 2: I, W, S, E, R, and K; Residue 3: V; Residue 4: C, A, and M; Residue 5: A, L, S, E, R, and K; ⁇ Residue 6: M, N, Q, D, E, R, and K; Residue 7: A, L, and F; Residue 8: A, V, and D; Residue 9: G, W, E, and K; Residue 10: A, E, and K; ⁇ Residue 11: C and A; Residue 12: G, I, E, and K; Residue 13: V, L, Q, D, E, and R; Residue 14: C, G, A, V, I, M, L, Y, S, T, Q, D, E, and R; Res
  • the polypeptides may further comprise a helical X1 domain.
  • the helical X1 domain may comprise any amino acid sequence.
  • X1, X2, X3, and X4 may be in any order in the polypeptide.
  • X1 is a peptide comprising the amino acid ⁇ sequence at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence LAEIMKEVAECARKEA (SEQ ID NO: 936).
  • the polypeptide has an amino acid selected from the following residues relative to X1 (SEQ ID NO: 936): Residue 1: V, I, and L; ⁇ Residue 2: A, I, S, N, R, and K; Residue 3: D and E; Residue 4: V and I; Residue 5: M and F; Residue 6: K; ⁇ Residue 7: E; Residue 8: A and V; Residue 9: A and R; Residue 10: A, V, I, L, T, Q, E, and K; Residue 11: C, V, I, M, F, Y, Q, R, and K; ⁇ Residue 12: A; Residue 13: R and K; Residue 14: K; Residue 15: E; and Residue 16: A, I, M, L, F, Y, S, and N.
  • the polypeptide does not have a cysteine residue at position 11 relative to X1 (SEQ ID NO: 936).
  • the X2, X3, X4 (when present), and X1 (when present) domains may be present in any order in the polypeptide of any embodiment herein.
  • the inventors have provided extensive teaching of “shuffled” versions of the polypeptides; see Table 2 and the column ⁇ listing the domain order of X1, X2, X3, and X4 domains.
  • the polypeptide comprises in domain order, X2-X3-X4, or X1-X2-X3, or X2-X3, or X1-X2-X3- X4.
  • the polypeptide comprises in domain order, X4-X3-X2, X3-X2, X1-X3-X2, X2-X1-X3, X3-X2-X1, X3-X1-X2, X2-X4-X3, X3-X2-X4, X3-X4-X2, X4-X2- ⁇ X3, X1-X2-X3-X4, X1-X4-X3-X2, X2-X1-X4-X3, X2-X3-X4-X1, X3-X2-X1-X4, X3-X4- X1-X2, X4-X1-X2-X3, X3-X2, or X4-X3-X2-X1
  • the polypeptides of the disclosure may comprise amino acid linkers between any of the domains.
  • the polypeptides may ⁇ comprise a linker between none of the domains, or may comprise a linker between some but not all of the domains.
  • the linkers may be of any length or amino acid composition.
  • suitable linkers include, but are not limited to GS , GGS , GGGGG (SEQ ID NO: 939), GSGGG (SEQ ID NO: 940), GGGGGG (SEQ ID NO: 941), GGSGGG (SEQ ID NO: 942), GGSGGSGGGSGGSGSG (SEQ ID NO: 943), ⁇ GSGGSGGGSGGSGSG (SEQ ID NO: 944), GSGSGSG GSGGSCKKISGGSGGGGS (SEQ ID NO: 945), and (GGGGX)n (SEQ ID NO: 946), where X is Q, E, or S and n is 2-5.
  • polypeptides may be fused to one or more additional domains as appropriate for an intended use.
  • the disclosure provides fusion proteins comprising a polypeptide of any embodiment or combination of embodiments of the disclosure, and one or ⁇ more functional domains. Any functional domain may be fused to the polypeptide of the disclosure.
  • the one or more functional domains comprises cell targeting domains (including but not limited to antibodies, antibody fragments, domains to extend protein half-life (such as albumin, albumin-binding protein, Fc fragment of antibody), proteins that bind to biological markers, antigens, ligands, peptides, etc.) or ⁇ detectable domains (including but not limited to fluorescent proteins, luminescent proteins, protein tags, etc.)
  • the one or more functional domain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:599-606.
  • the polypeptide and the one or more functional domains may directly abut each other in the ⁇ fusion protein, or may be linked by a polypeptide linker suitable for an intended purpose.
  • the one or more functional domain may be present at the N-terminus, the C-terminus, or position between the X2, X3, X4, or X1 (when present) domains.
  • the polypeptide or fusion protein comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% ⁇ identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-598 627-631, 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746- 747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, ⁇ 856-862, 864-874, 876-898, and 900-931.
  • the polypeptide or fusion protein comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:627-631, 633, 635-641, 643-644, 648-654, 656, 658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698, 700-702, 706-707, 709-711, ⁇ 714-715, 717-735, 738-739, 742-743, 746, 749-751, 771, 849-854, 856-862, 864-870, 872- 874, 876-898, 900, and 902-907.
  • the polypeptides are capable of antagonist activity.
  • the polypeptide or fusion protein comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID ⁇ NO:1-598, 655, 657, 699, 747, 752-770, 772-798, 800-811, 813-821, 823, 825-829, 831-832, 834-835, 843, 845-847, 871, and 908-931.
  • the polypeptides are capable of agonist activity.
  • the disclosure provides conditionally active IL-21 receptor binding proteins, comprising a first polypeptide component and a second polypeptide component, ⁇ wherein the first polypeptide component and the second polypeptide component are not both present in the same fusion protein, wherein in total the first polypeptide component and the second polypeptide component comprise domains X2, X3, and optionally X4 as defined in any embodiment or combination of embodiments of the polypeptides of disclosure, wherein: (i) the first polypeptide component comprises at least one of X2, X3, and X4 ⁇ (when present) but does not comprise each of X2, X3, and X4 (when present); and (ii) the second polypeptide component comprises each of X2, X3, and X4 that is not present in the first polypeptide component; wherein the first polypeptide component and the second polypeptide component are not active receptor binding proteins individually, and wherein the first polypeptide ⁇ component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable
  • the first polypeptide component comprises at least one of X2, X3, and X4 but does not comprise each of X2, X3, and X4; and (ii) the second polypeptide component comprises each of X2, X3, and X4 that is not present in the first polypeptide component; ⁇ wherein the first polypeptide component and the second polypeptide component are not active receptor binding proteins individually, and wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL-21R ⁇ CD360) and is capable of binding to the ⁇ c, CD132 receptor.
  • IL-21R ⁇ CD360 active IL-21 receptor
  • the first polypeptide component comprises at least one of X1, X2, X3, and X4 but does not comprise each of X1, X2, X3, and X4; and (ii) the second polypeptide component comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide component; ⁇ wherein the first polypeptide component and the second polypeptide component are not active receptor binding proteins individually, and wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL-21R ⁇ CD360) and is capable of binding to the ⁇ c, CD132 receptor.
  • IL-21R ⁇ CD360 active IL-21 receptor
  • conditionally active receptor IL-21 agonists comprising the recited separate first and second polypeptides that individually are not receptor agonists, but which can interact non-covalently to form an active agonist of IL- 21, by binding to both the IL-21 receptor (IL-21R ⁇ CD360) and to the ⁇ c, CD132 receptor.
  • conditionally active receptor agonists of the current disclosure can be used for any ⁇ uses that the polypeptide agonists of the disclosure can be used.
  • the first polypeptide component comprises X2 but does not comprise X3; and (ii) the second polypeptide component comprises X3; wherein the first polypeptide component and the second polypeptide component are ⁇ not active receptor binding proteins individually, and wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL-21R ⁇ CD360) but does not bind simultaneously bind to the ⁇ c, CD132 receptor.
  • IL-21R ⁇ CD360 an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL-21R ⁇ CD360) but does not bind simultaneously bind to the ⁇ c, CD132 receptor.
  • This embodiment provides conditionally active receptor antagonists, which can be used for any uses that the polypeptide antagonists of the disclosure can be used.
  • Split IL-21 antagonists are designed to achieve targeted delivery of IL-21 antagonist with minimal off- target activity.
  • the first polypeptide or the second polypeptide may ⁇ comprise an X1 domain and/or an X4 domain as disclosed in any embodiment or combination of embodiments
  • the polypeptides are typically split at sites that won’t interfere with the function of the protein (e.g., linker sections in embodiments with linkers).
  • the split proteins can comprise any combination of the domains.
  • X1 (when present), X2, X3, and X4 (when present) may be in any order in the first and second polypeptide; in non-limiting embodiments: (i) the first polypeptide comprises X1 and the second polypeptide comprises X2, X3, and X4; ⁇ (ii) the first polypeptide comprises X2 and the second polypeptide comprises X1, X3, and X4; (iii) the first polypeptide comprises X3 and the second polypeptide comprises X1, X2, and X4; (iv) the first polypeptide comprises X4 and the second polypeptide comprises X1, ⁇ X2, and X3; (v) the first polypeptide comprises X1 and X2, and the second polypeptide comprises X3 and X4; (vi) the first polypeptide comprises X1 and X3, and the second polypeptide comprises X2 and X4; ⁇ (vii) the first polypeptide comprises X1 and X4, and the second polypeptid
  • the domains may in some embodiments be separated by amino acid linkers of any suitable length or amino acid composition. There is no requirement ⁇ for linkers; in one embodiment there are no linkers present between any of the domains. In other embodiments, an amino acid linker may be present between 0, 1, or 2 junctions between domains X1, X2, X3, and X4 in the first polypeptide and/or the second polypeptide.
  • the amino acid linkers may be of any length as deemed appropriate for an intended use and may, for example, comprise any of the linker embodiments disclosed herein.
  • a linker is at the N terminus or C terminus and is referred to as a linker despite not linking two domains together.
  • X1, when present, X2, X3, and X4, respectively, are as recited in any embodiment or combination of embodiments of the polypeptides of the disclosure.
  • the first and second polypeptides in total comprise an ⁇ amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-598627-631, 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742- 743, 746-747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, ⁇ 849-854, 856-862, 864-874, 876-898, and 900-931; or (b) at least 50%, 55%, 60%, 65%, 70%,
  • the first and second polypeptides comprise: (a) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 608; and (ii) a second polypeptide comprising an amino acid ⁇ sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 609; (b) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 607; and (ii) a second polypeptide comprising an amino acid ⁇ sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
  • the first and second polypeptides comprise: (a) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%, ⁇ 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 608; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 609; (b) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%, ⁇ 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 607; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:
  • the first and second polypeptides comprise: (a) (i) a first polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 608; and (ii) a second polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, ⁇ 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 609; (b) (i) a first polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 607; and (ii) a second polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 612; ⁇ (c) (i) a first polypeptide comprising an amino acid sequence at least 75%, 80%,
  • the first and second polypeptides comprise: ⁇ (a) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 608; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 609; ⁇ (b) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 607; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 612; ⁇ (c) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 610; and (ii) a second polypeptide
  • first polypeptide and the second polypeptide are fused to a targeting domain, permitting targeted delivery.
  • the targeting domain may comprise an antibody or nanobody that can direct the split cytokine mimics to desired cells or tissues of interest (Fig.2C).
  • the first and second polypeptides are fused to anti-HER2-DARPin or anti-EGFR-DARPin (Table 4, SEQ ID NO:613-624) to demonstrate that the first and/or second polypeptides can be fused to ⁇ such targeting domain for use and can be reconstituted (Fig.2D.
  • the disclosure provides polypeptide comprising the amino acid sequence of any first polypeptide or second polypeptide as described in the disclosure.
  • polypeptides may be used, for example, in the conditionally-active receptor agonists of the disclosure.
  • the polypeptides comprise an amino acid sequence at ⁇ least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of selected from the group consisting of SEQ ID NO: 607-624.
  • the disclosure provides non-naturally occurring polypeptides comprising domains X1, X2, X3, and X4, wherein: (a) X1 is a peptide comprising the amino acid sequence at least 25%, 30%, 35%, ⁇ 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence LAEIMKEVAECARKEA (SEQ ID NO:936); (b) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence DTRILKVSLKKIVRA (SEQ ID NO:933); ⁇ (c) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence
  • X1 residues 1-16 are selected from: Residue 1: V, I, and L; Residue 2: A, I, S, N, R, and K; ⁇ Residue 3: D and E; Residue 4: V and I; Residue 5: M and F; Residue 6: K; ⁇ Residue 7: E; Residue 8: A and V; Residue9: A and R; Residue 10: A, V, I, L, T, Q, E, and K; Residue 11: C, V, I, M, F, Y, Q, R, and K; ⁇ Residue 12: A; Residue 13: R and K; Residue 14: K; Residue 15: E; and Residue 16: A, I, M, L, F, Y, S
  • X1 residue 16 is not A.
  • X2 residues 1- 16 are selected from: Residue 1: G, A, T, Q, D, R, K, and H; Residue 2: G, A, S, T, and N; ⁇ Residue 3: A, M, Y, N, Q, E, and R; Residue 4: G, A, V, I, M, L, F, Y, W, N, Q, E, and H; Residue 5: M, L, Q, and K; Residue 6: V, I, L, Y, R, and K; Residue 7: G, V, I, M, L, F, Y, W, S, T, N, Q, D, and E; ⁇ Residue 8: F, S, T, and E; Residue 9: L; Residue 10: K; Residue 11: K; Residue 12: I; ⁇ Residue 13
  • X2 residue 3 is not R; ⁇ (b) X2 residue 4 is not I; (c) X2 residue 7 is not V; and/or (d) X2 residue 8 is not S.
  • X3 residues 1-20 are selected from: ⁇ Residue 1: P, N, R, and K; Residue 2: F, Y, and R; Residue 3: V, I, M, L, F, T, and Q; Residue 4: E; Residue 5: I; ⁇ Residue 6: R; Residue 7: M; Residue 8: V, L, F, Y, T, and R; Residue 9: G, A, and Q; Residue10: I, M, and L; ⁇ Residue 11: I and L; Residue 12: D; Residue 13: I; Residue 14: C, A, V, S, and T; Residue 15: D and E; ⁇ Residue 16: H; Residue 17: V; Residue 18: K; Residue 19: R; and Residue 20: T, N, and E.
  • X3 residue 2 is not R
  • X3 residue 8 is not R
  • X3 residue 9 is not Q
  • X3 residue 10 is not L
  • X3 residue 14 is not V
  • g X3 residue 15 is not D.
  • X4 residues 1-19 are selected from: Residue 1: K; ⁇ Residue 2: E; Residue 3: V; Residue 4: M; Residue 5: E; ⁇ Residue 6: R; Residue 7: A; Residue 8: R and K; Residue 9: V, M, S, and T; Residue 10: A; ⁇ Residue 11: A; Residue 12: Q; Residue 13: K; Residue 14: C, G, A, V, I, M, L, S, T, Q, D, and K; Residue 15: L; ⁇ Residue 16: G; Residue 17: R and K; Residue 18: F, Y, and W; and Residue 19: L, F, and Y.
  • ⁇ (a) X4 residue 8 is not K; (b) X4 residue 9 is not S; and/or (c) X4 residue 19 is not L.
  • the polypeptide is an antagonist of the IL-21 receptor.
  • X1 residue 11 is C. This embodiment helps increase binding affinity to human ⁇ common Gamma chain (CD132), as well as mouse CD360 and mouse CD132.
  • the domains are arranged N-terminal to C-terminal in an arrangement selected from the group consisting of X1-2-3-4, X1-4-3-2, X2-1-4-3, X2-3-4-1, X3-2-1-4, X3-4-1-2, X4-1-2-3, and X4-3-2-1.
  • amino acid linkers are present between the domains.
  • the polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 938.
  • residues at non-helix positions may be selected from the group consisting of: Residue 01: V, I, M, F, Y, and D; Residue 02: A, V, I, M, L, F, Y, W, Q, D, E, R, and H; ⁇ Residue 03: E; Residue 04: D and E; Residue 21: A, T, and E; Residue 22: R and K; Residue 23: I, M, L, F, and Y; ⁇ Residue 24: P, G, A, V, I, S, TN, Q, D, R, and H; Residue 25: G, A, V, N, Q, and K; Residue 01: V, I, M, F, Y, and D; Residue 02: A, V, I, M, L, F, Y, W, Q, D, E, R, and H; ⁇ Residue 03: E; Residue 04:
  • polypeptides of this aspect of the disclosure comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-598627-631, 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, ⁇ 677-679, 681-685, 687-695, 698-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742- 743, 746-747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898, and 900-931.
  • the polypeptides further comprise one or more functional domains.
  • the polypeptides may be fused to any functional domain as suitable for an intended ⁇ use, included but not limited to cell targeting domains and detectable domains (including but not limited to fluorescent proteins, luminescent proteins, etc.)
  • the one or more functional domains is a translational fusion with the polypeptide.
  • the disclosure provides non-naturally occurring conditionally active IL-21 receptor binding proteins, comprising a first polypeptide ⁇ component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not present in a fusion protein, wherein in total the first polypeptide component and the second polypeptide component comprise domains X1, X2, X3, and X4 as defined in any preceding claim; wherein: (i) the first polypeptide component comprises at least one of X1, X2, X3, and X4 ⁇ but does not comprise each of X1, X2, X3, and X4; and (ii) the second polypeptide component comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide component; wherein the first polypeptide component and the second polypeptide component are not active receptor binding proteins individually, and wherein the first polypeptide ⁇ component and the second polypeptide interact to form an active IL-21 receptor binding protein.
  • the polypeptide may be selected from the group consisting of: (i) a polypeptide comprising X1 and excluding X2, X3, and X4; ⁇ (ii) a polypeptide comprising X2 and excluding X1, X3, and X4; (iii) a polypeptide comprising X3 and excluding X1, X2, and X4; (iv) a polypeptide comprising X4 and excluding X1, X2, and X3; (v) a polypeptide comprising X1 and X2, and excluding X3 and X4; (vi) a polypeptide comprising X1 and X3, and excluding X2 and X4; ⁇ (vii) a polypeptide comprising X1 and X4, and excluding X2 and X3; (viii) a polypeptide comprising X2 and X3, and excluding X1 and X4; (ix) a polypeptide comprising
  • X1, X2, X3, and X4, respectively comprise an amino acid ⁇ sequence that are at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NO:1-598 627-631, 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746- 747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, ⁇ 856-862, 864-874, 876-898, and 900-931.
  • the present disclosure provides nucleic acids, including isolated nucleic acids, encoding the polypeptides, fusion proteins, and first and second polypeptides of the present disclosure.
  • the isolated nucleic acid sequence may comprise RNA or DNA.
  • Such isolated nucleic acid sequences may comprise additional sequences useful for ⁇ promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptides of the invention.
  • the present disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable control sequence.
  • “Expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product.
  • “Control sequences” operably linked to the nucleic acid sequences of the invention are nucleic acid ⁇ sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered "operably linked" to the coding sequence.
  • control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites.
  • expression vectors include but are not limited to, plasmid and viral-based expression vectors.
  • the control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be ⁇ constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive).
  • the expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA.
  • the expression vector may comprise a ⁇ plasmid, viral-based vector (including but not limited to a retroviral vector or oncolytic virus), or any other suitable expression vector.
  • the expression vector can be administered in the methods of the disclosure to express the polypeptides in vivo for therapeutic benefit.
  • the expression vectors can be used to transfect or transduce cell therapeutic targets (including but not limited to CAR-T cells or ⁇ tumor cells) to effect the therapeutic methods disclosed herein.
  • the present disclosure provides host cells that comprise the expression vectors, polypeptides, fusion proteins, first and/or second polypeptides, polypeptide components, conditionally active agonists and antagonists, and/or nucleic acids and expression vectors disclosed herein, wherein the host cells can be either prokaryotic or ⁇ eukaryotic.
  • the cells can be transiently or stably engineered to incorporate the expression vector of the invention, using techniques including but not limited to bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection.
  • techniques including but not limited to bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection.
  • a method of producing a polypeptide according to the invention is an additional part of the invention.
  • the method comprises the steps of (a) culturing a host according to this aspect of the invention under conditions conducive to the expression of the polypeptide, and (b) optionally, recovering the expressed ⁇ polypeptide.
  • the expressed polypeptide can be recovered from the cell free extract, but preferably they are recovered from the culture medium.
  • the present disclosure provides pharmaceutical compositions, comprising the polypeptide, fusion protein, first polypeptide, second polypeptide, recombinant nucleic acid, expression vector and/or, recombinant host cell of any embodiment ⁇ or combination of embodiments herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions of the disclosure can be used, for example, in the methods of the disclosure described herein.
  • the active agents of the disclosure may be the sole active agent in the composition or may be combined with one or more other active agents, including ⁇ but not limited to checkpoint inhibitors or cancer vaccines
  • the pharmaceutical composition may further comprise (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and/or (g) a buffer.
  • the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer.
  • the pharmaceutical ⁇ composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose.
  • the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof.
  • the ⁇ pharmaceutical composition includes a bulking agent, like glycine.
  • the pharmaceutical composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof.
  • the ⁇ pharmaceutical composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood.
  • Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride.
  • the pharmaceutical composition additionally includes a stabilizer, e.g., a ⁇ molecule which, when combined with a protein of interest substantially prevents or reduces chemical and/or physical instability of the protein of interest in lyophilized or liquid form.
  • Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.
  • the polypeptide, fusion protein, first polypeptide, second polypeptide, recombinant ⁇ nucleic acid, expression vector and/or, recombinant host cell of any embodiment or combination of embodiments herein may be the sole active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for an intended use.
  • the disclosure provides methods for treating cancer, comprising administering to a subject having cancer the polypeptide agonist or fusion protein, conditionally active receptor agonist, recombinant nucleic acid, expression vector, recombinant host cell, and/or the pharmaceutical composition of any embodiment of IL-21 ⁇ agonists herein, in an amount effective to treat the tumor.
  • the cancer is selected from the group consisting of colon cancer, melanoma, renal cell cancer, head and neck squamous cell cancer, gastric cancer, urothelial carcinoma, Hodgkin lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, pancreatic cancer, Merkel cell carcinoma, colorectal cancer, acute myeloid leukemia, acute ⁇ lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, ovarian cancer, cervical cancer, and any tumor types selected by a diagnostic test, such as microsatellite instability, tumor mutational burden, PD-L1 expression level, or the immunoscore assay (as developed by the Society for Immunotherapy of Cancer).
  • a diagnostic test such as microsatellite instability, tumor mutational burden, PD-L1 expression level, or the immunoscore assay (as developed by the Society for Immunotherapy of Cancer).
  • the disclosure provides methods for modulating an immune ⁇ response in a subject comprising the polypeptide antagonist or fusion protein thereof, conditionally active receptor antagonist, recombinant nucleic acid, expression vector, recombinant host cell, and/or the pharmaceutical composition of embodiment of IL-21 antagonist disclosed herein.
  • the immune response is an anti-cancer immune response.
  • treat or “treating” means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously ⁇ symptomatic for the disorder(s).
  • the subject may be any subject that has a relevant disorder. In one embodiment, the subject is a mammal, including but not limited to humans, dogs, cats, horses, cattle, etc.
  • IL-21 mimetic proteins that recapitulate its interactions with receptors and the biology of native IL-21 in humans and mice, which is ideal for therapeutic use.
  • the IL-21 mimetic proteins designed include agonists, antagonists, immunocytokine, or split agonists/antagonists, as detailed in the examples and listed in ⁇ Tables 1-6. Binding is measured for two receptors by the two setups: (i) hIL-21R only for hIL-21R binding and (ii) h ⁇ c with hIL-21R for h ⁇ c receptor binding.
  • hIL-21R is needed for binding testing because the affinity for h ⁇ c alone is very weak and thus, reasonable h ⁇ c binding measurement can only be done when hIL-21R is present.
  • hIL-21R binding the measurement is done in flow cytometry-based yeast surface display or biolayer interferometry (BLI) as mentioned in Methods section.
  • yeast surface display method 1-1000 nM of hIL-21R labeled with SA-PE is used along with anti- Myc FITC antibody to label the cells that express the IL-21 mimics that binds to the hIL-21R.
  • the proteins that bind to hIL-21R show the PE+FITC+ population equal or more than 0.5% ⁇ of the singlet-gated population, when tested with 1000 nM of hIL-21R.
  • hIL- 21R is loaded to the analysis tip and tested for association with IL-21 mimics provided at 100 pM-1000 nM range.
  • polyhistidine-tagged IL-21 mimics can be loaded to the analysis tip and measured for their association to 100-1000 nM of hIL-21R.
  • the proteins that bind to hIL-21R show at least 0.1nm shift during the association, in either of the ⁇ cases.
  • the provided yeast surface display data shows the results with 100 nM of PE-labeled hIL-21R.
  • the provided BLI data shows hIL-21R-loaded analysis method with the aforementioned range for appropriate Kd calculations, and polyhistidine-tagged protein- loaded analysis with 100nM of hIL-21R for binding testing.
  • mouse cross-reactivity by testing binding to mIL-21R works the same as hIL-21R binding assay and it showed similar results to that of human receptor.
  • h ⁇ c receptor binding the measurement is done in flow cytometry-based yeast surface display or biolayer interferometry (BLI) as mentioned in Methods section.
  • yeast surface display method 1-1000 nM of unlabeled hIL-21R and 1-1000 nM of SA-PE-labeled ⁇ h ⁇ c receptor are used along with anti-Myc FITC antibody to label the cells that express the IL-21 mimics that binds to h ⁇ c receptor in the presence of hIL-21R.
  • the proteins that bind to h ⁇ c receptor in the presence of hIL-21R show the PE+FITC+ population equal or more than 0.5% of the singlet-gated population, when tested with 1000 nM of hIL-21R and 1000 nM of h ⁇ c receptor.
  • h ⁇ c receptor is loaded to the analysis tip and tested for ⁇ association with IL-21 mimics provided at 100 pM-1000 nM range, in the presence of hIL- 21R at 1.5-fold higher concentration compared to that of the IL-21 mimics.
  • the proteins that bind to h ⁇ c receptor and hIL-21R show at least 0.1nm shift during the association.
  • the provided yeast surface display data shows the results with 1000 nM of unlabeled hIL-21R and 200 nM PE-labeled h ⁇ c receptor, or 1000 nM of unlabeled hIL-21R and 1000 nM PE-labeled h ⁇ c receptor.
  • the provided BLI data shows h ⁇ c receptor-loaded analysis method with the aforementioned range for appropriate Kd calculations.
  • mouse cross-reactivity by testing binding to m ⁇ c receptor and mIL- 21R works the same as h ⁇ c receptor binding assay and it showed similar results to that of human receptor.
  • Agonistic mimics of IL-21 are designed to have two interfaces, one for IL-21R and the other for CD360. These designed agonists can bind to both receptors at the same time to ⁇ heterodimerize the two receptor subunits. Furthermore, the designs are cross-reactive to human and murine receptors.
  • Antagonistic mimics of IL-21 are designed only to retain the binding capability to IL-21R but not ⁇ c so that it can occupy the same space as an agonist yet interfere ⁇ c from associating for heterodimerization of the receptor subunits. Similarly, these designs retain human and mouse cross-reactivity.
  • Immunocytokines of IL-21 mimics, ⁇ antibody-fused IL-21 mimics, are examples of molecular fusions for targeted cytokine delivery.
  • Split IL-21 mimics are designed to achieve targeted delivery of IL-21 mimic with minimal off-target cytokine activity.
  • Wild-type hIL-21 consists of four helices, with helix A (the ⁇ first helix from the N-terminus) and helix C (the third helix from the N-terminus) consisting of an interface to hIL-21R, and helix D (the fourth helix from the N-terminus) forming an interface to h ⁇ c .
  • the upper two helices B (the second helix from the N-terminus) and C are not long enough to form ideal intramolecular interactions and intermolecular contacts with the receptor chains.
  • This variant demonstrated binding to not only hIL-21R and hIL- 21R/h ⁇ c , but also mIL-21R and mIL-21R/m ⁇ c , indicating cross-reactivity between human and murine receptors. Furthermore, we employed site-saturation mutagenesis (SSM) to mutate ⁇ interface residues and evaluated their impact on affinity individually (Fig.5; Table 1, SEQ ID NO:9-36).
  • SSM site-saturation mutagenesis
  • 21h10 showed the best cross- reactivity and interestingly exhibited only 44.9% sequence identity to hIL-21 and 23.5% to ⁇ mIL-21, as determined by BLASTP analysis.21h10 was efficiently expressed in Escherichia coli and displayed monodispersity in size-exclusion chromatography (Fig.1D). Additionally, circular dichroism analysis confirmed the presence of helical secondary structures (Fig.1E) in the protein and demonstrated its superior thermal stability (Fig.1F). The agonists were diversified by redesigning non-interface residues and some of the ⁇ interface residues using ProteinMPNN to allow possible substitutions on such positions, with experimental validation (Table 1, SEQ ID NO:37-163).
  • affinity variants are generated based on 21h10 (Table 1, SEQ ID NO:24) by attenuating the affinity of both interfaces using SSM data (Table 1, SEQ ID NO:164-263; Fig.6) and ProteinMPNN (Table ⁇ 1, SEQ ID NO:264 -432). These affinity variants may achieve alterations in orientation and half-life of receptor complex which can affect EC50, Emax, signaling pathway biases, or further downstream differentiating phenotype from the native IL-21 signaling. This confirms that agonistic IL-21 mimics are stable to permit interface engineering to achieve various ⁇ affinities on both interfaces.
  • Native-like, high affinity variants are useful to recapitulate full activity of native IL-21 biology with its full human and mouse cross-reactivity, such as to use these variants for surrogates of native IL-21.
  • Lower affinity variants may have less potency than the native IL-21 or have function distinctive from the native IL-21, however it is useful when antibody or other targeting domain fused IL-21 mimic constructs needs biodistribution ⁇ that is directed by the fused targeting domain’s target-binding affinity rather than the affinity between the fused IL-21 mimic against the IL-21 receptor.
  • the lower affinity variants may have higher EC50 or lower Emax in cell signaling which might be the desired function to use.
  • SEQ ID NO:1-8 in Table 1 are the agonistic mimics derived from 21d26 (Table 5, ⁇ SEQ ID NO:627) with combinations of mutations – W15C, W49R, Q73H, and H99G – that was applied with all four mutations to generate 21JC15 (Table 1, SEQ ID NO:8).
  • SEQ ID NO: 9-24 in Table 1 are the agonistic mimics that are optimized from 21JC15 (Table 1, SEQ ID NO:8) with positive mutations found in site saturation mutagenesis (SSM) data.
  • SEQ ID NO: 25-36 in Table 1 are the agonistic mimics with combinations of mutations that are ⁇ applied to 21h10 (Table 1, SEQ ID NO: 24) from 21JC15 (Table 1, SEQ ID NO:8).
  • SEQ ID NO:37-163 in Table 1 are the agonistic mimics that are derived from 21h10 (Table 1, SEQ ID NO: 24) with mutations applied by ProteinMPNN to the residues that are not part of interface residues contributing to IL-21R (CD360) or ⁇ c (CD132) receptor binding.
  • SEQ ID NO: 164- 432 in Table 1 are the agonistic affinity variant mimics that are derived from 21h10 (Table 1, ⁇ SEQ ID NO: 24) with mutations applied by site saturation mutagenesis (SSM) data or ProteinMPNN to the residues that are part of interface residues contributing to IL-21R (CD360) or ⁇ c (CD132) receptor binding, to acquire attenuation in interface binding affinity.
  • Kd1 is Kd against hIL-21R which indicates the binding affinity against hIL-21R
  • Kd2 is Kd against the IL-21 receptor complex (hIL-21R and h ⁇ c receptor) ⁇ which indicates the binding affinity against h ⁇ c receptor, both measured by BLI.
  • Q3 is percentage of the PE+FITC+ population relative to the singlet-gated population at 1000 nM unlabeled hIL-21R and 1000 nM PE-labeled h ⁇ c receptor, which represents the binding to h ⁇ c receptor in the presence of hIL-21R, measured by flow cytometry.
  • X2-X3-X4- X1, X3-X4-X1-X2, and X4-X1-X2-X3 were demonstrated (Table 2) for their engineerability in maintaining their given molecular structure while retaining both interfaces for IL-21R ⁇ (CD360) and ⁇ c (CD132) receptor for their agonistic function.
  • Helical domain-shuffled designs bind to IL-21 receptor and this implies that the helical domains are hyperstable to be shuffled into any order beyond the suggested three orders and retain its ⁇ interfaces to the IL-21 receptors, with new connectivities between the helices (retopology). Sequences of helical domain shuffled constructs are listed in Table 2. SEQ ID NO:433-445 in Table 2 are the agonistic mimics with shuffled helical domains in the order of X2-X3-X4-X1. Similarly, SEQ ID NO:480-569 in Table 2 are the agonistic mimics with shuffled helical domains in the order of X2-X3-X4-X1 but with mutations on X1.
  • SEQ ID ⁇ NO:446-457 in Table 2 are the agonistic mimics with shuffled helical domains in the order of X3-X4-X1-X2.
  • SEQ ID NO: 458-479 in Table 2 are the agonistic mimics with shuffled helical domains in the order of X4-X1-X2-X3.
  • IL-21 mimics being four helical bundle proteins, there are three different ways to divide the helices into two linear groups (fragment X and fragment Y) – H1/H234, H12/H34, and H123/H4 (Fig.2B; Table 4), and this can even be diversified with shuffle of helical domains (Table 2).
  • the use of these split ⁇ IL-21 mimics for targeted delivery is to fuse each of the split fragments X and Y to targeting domain, such as nanobody that can direct the split cytokine mimics to desired cells or tissues of interest (Fig.2C).
  • SEQ ID NO: 607-612 in Table 4 are the split fragments generated from 21h10 (Table 1, SEQ ID NO: 24), as X1 (Table 4, SEQ ID NO: 607), X1-X2 (Table 4, SEQ ID NO: 608), X3-X4 (Table 4, SEQ ID NO: 609), X4 (Table 4, SEQ ID NO: 610), X1-X2-X3 (Table 4, SEQ ID NO: 611), and X2-X3-X4 (Table 4, SEQ ID NO: 612).
  • SEQ ID NO: 613-618 in Table 4 are the split fragments (Table 4, SEQ ID NO: 607-612) fused to anti-HER2-DARPin ⁇ (Table 4, SEQ ID NO: 625).
  • SEQ ID NO: 619-624 in Table 4 are the split fragments (Table 4, SEQ ID NO: 607-612) fused to anti-EGFR-DARPin (Table 4, SEQ ID NO: 626).
  • Kd1 is Kd against hIL-21R which indicates the binding affinity against ⁇ hIL-21R which is dissected into kon1 and kdis1, all measured by BLI.
  • kon1 is the association rate against hIL-21R and kdis1 is the dissociation rate against hIL-21R.
  • Kd2 is Kd against the IL-21 receptor complex (hIL-21R and h ⁇ c receptor) which indicates the binding affinity against h ⁇ c receptor, measured by BLI.
  • Antibody fusions ⁇ For targeting specific cells or tissues of interest, we demonstrated various antibody- fused IL-21 mimic constructs (Table 3). IL-21 mimics were fused to the heavy chain of antibodies.
  • Atezolizumab anti-PD-L1-targeting antibody
  • TA99 anti-TRP1-targeting antibody
  • 21h10-fused Atezolizumab can be ⁇ either configured as homodimeric form or heterodimeric form using Knobs/Holes mutation in the heavy chain of the antibody (Fig.3A) and shows expected size in SDS-PAGE (Fig.3B).
  • Atezolizumab-21h10 constructs can bind to each of their domains target, human IL-21R for 21h10 and human PD-L1 for Atezolizumab (Fig.3C). Also, this construct can bind to both ⁇ target simultaneously (Fig.3D).
  • Other than targeting antibodies we also have made Fc- fusions for the purpose of half-life extension (Table 3, SEQ ID NO:570-571). This proves that agonistic IL-21 mimics can be fused to any available antibodies or other targeting molecules to redirect their biodistribution.
  • SEQ ID NO:570-571 in Table 3 are Fc fragment fusion constructs in which, an ⁇ agonistic mimic, 21AG-M-p3C1 (Table 1, SEQ ID NO:161), is fused to either human Fc fragment (Table 3, SEQ ID NO:599) or murine Fc fragment (Table 3, SEQ ID NO:600), respectively.
  • SEQ ID NO:572-576, 601 in Table 3 are the components used to make fusion constructs of IL-21 mimic with Pembrolizumab.
  • SEQ ID NO:582-583, 603 in Table 3 are the components used to make fusion constructs of IL-21 mimic with TA99.
  • Two identical copies of the light chain of TA99 (Table 3, SEQ ID NO:582) are paired with two identical copies or two different copies of heavy chain of TA99 (Table 3, SEQ ID NO: 583, 603).
  • SEQ ID NO:584-588, 604 in Table 3 are the components ⁇ used to make fusion constructs of IL-21 mimic with an anti-murine PD-1 monoclonal antibody.
  • SEQ ID NO: 594-598, 606 in Table 3 are the components used to make fusion constructs of IL-21 mimic with an anti-human CD8 monoclonal antibody, RED8.
  • Two ⁇ identical copies of the light chain of RED8 (Table 3, SEQ ID NO:594) are paired with two identical copies or two different copies of heavy chain of RED8 (Table 3, SEQ ID NO: 595- 598, 606).
  • Kd is measured against hIL-21R and the antibody’s target, human PD-L1 ⁇ in the case of Atezolizumab fusions.
  • An Atezolizumab-IL-21 mimic fusion construct is tested for hIL-21R binding and hPD-L1 binding by BLI.
  • Atezolizumab- 21h10 fusion construct (Table 3, SEQ ID NO:577, 578) is tested by immobilizing hIL-21R on SA tip and associated and dissociated in 0.313-20 nM of the fusion construct (Fig.3C).
  • Atezolizumab-21h10 fusion construct (Table 3, SEQ ID NO: 577, ⁇ 578) is tested by immobilizing hPD-L1 on SA tip and associated and dissociated in 0.027-20 nM of the fusion construct (Fig.3C).
  • hPD-L1 is immobilized to the analysis tip and associated it with Atezolizumab-21h10 fusion construct (Table 3, SEQ ID NO: 577, 578) then hIL-21R; in the second experiment, hIL-21R is immobilized to the analysis tip and associated it with ⁇ Atezolizumab-21h10 fusion construct (Table 3, SEQ ID NO: 577, 578) then hPD-L1.
  • Antagonists Antagonistic IL-21 mimics are the designs that only bind to IL-21R, but not to ⁇ c.
  • IL- 21R binding was assed by flow cytometry using 100-1000nM of human or mouse IL-21R labeled with Streptavidin R-Phycoerythrin Conjugate (SAPE), and/or biolayer interferometry ⁇ using human or mouse IL-21R with Avi tag loaded on Streptavidin (SA) tip.
  • SAPE Streptavidin R-Phycoerythrin Conjugate
  • SAPE Streptavidin R-Phycoerythrin Conjugate
  • SAPE Streptavidin R-Phycoerythrin Conjugate
  • Generating antagonists from agonist designs also implies superior molecular stability of the IL-21 mimics, as knocking out the ⁇ c-interface without perturbing overall molecular integrity or the other interface (IL-21R interface) is often not permissible for native IL-21 to withhold.
  • IL-21 mimics are stable to permit interface mutations to achieve antagonistic ⁇ properties.
  • SEQ ID NO:627-631 in Table 5 are the five antagonist hits found during initial screening of IL-21 mimic designs.
  • SEQ ID NO:632-687 (named 21AT-R-p###) in Table 5 are mimics derived from 21h10 (Table 1, SEQ ID NO: 24) that are mutated on ⁇ c-interface residues by Rosetta TM .
  • SEQ ID NO:688- 751 (named 21AT-S-p###) in Table 5 are mimics ⁇ derived from 21h10 (Table 1, SEQ ID NO: 24) that are mutated on ⁇ c-interface residues based on site saturation mutagenesis (SSM) data.
  • SEQ ID NO:752- 907 (named 21AT-M- p###) in Table 5 are mimics derived from 21h10 (Table 1, SEQ ID NO: 24) that are mutated on ⁇ c -interface residues by ProteinMPNN.
  • Q1 is percentage of the PE+FITC+ population relative to the singlet-gated ⁇ population at 100 nM PE-labeled hIL-21R, which represents the binding to hIL-21R, measured by flow cytometry.
  • Q2 is percentage of the PE+FITC+ population relative to the singlet-gated population at 1000 nM unlabeled hIL-21R and 200 nM PE-labeled h ⁇ c receptor, which represents the binding to h ⁇ c receptor in the presence of hIL-21R, measured by flow cytometry.
  • IL-21 mimics.21h10 Table 1, SEQ ID NO:24
  • Cys15 was able to be chemically conjugated to PEG with various sizes to achieve increase in molecular size. This confirms that IL-21 mimics can be fused to other functional domains, along with antibodies or aforementioned targeting ⁇ domains, by genetic fusions or chemical conjugation to achieve their half-life extension or other functional benefits.
  • SEQ ID NO:908-909 in Table 6 are albumin-binding peptide-fusion constructs in which 21AG-M-p3C1 (Table 1, SEQ ID NO:161) fused to an albumin-binding peptide, G148ABD3 (Table 6, SEQ ID NO:932), being designed for the purpose of half-life ⁇ extension.
  • G148ABD3 domain binds albumin in vivo, it increases the size of the overall molecular weight which can increase half-life of the constructs.
  • SEQ ID NO:910-931 in Table 6 are agonistic mimics derived from 21h10 (Table 1, SEQ ID NO:24) or 21AG-M- p3C1 (Table 1, SEQ ID NO:161) which have added or removed Lys (K) or Cys (C) on their non-interface residues (Table 6, SEQ ID NO:910-925) or interface residues (Table 6, SEQ ID ⁇ NO:926-931) to chemically conjugate functional domains, including but not limited to PEG, peptide, and small molecules, for various purposes, such as half-life extension and conditional activation.
  • residues from the human IL-21 are designated to be fixed during scaffold generation and interface residue ⁇ design: R2, I5, R6, R8, Q9, L10, I11, D12, I13, D15, Q16, K18, Y20, R62, I63, V66, S67, K69, K70, R73, K74, P75, P76, S77, K98, E99, E102, R103, K105, S106, Q109, K110, H113, and L116.
  • the non-fixed residues are designed using Rosetta TM FastDesign and relaxed using Rosetta TM FastRelax with ‘beta_nov16’ score function.
  • the vector was linearized by 100-fold overdigestion by NdeI and XhoI (New England Biolabs) and then purified by gel ⁇ extraction (Qiagen).
  • Yeast transformations are validated by colony sequencing; validated colonies are grown in SDCAA media (2.0% Glucose, 0.67% Yeast nitrogen base, 0.5% Casamino acids, 0.54% Di-sodium phosphate, 0.86% MonoSodium phosphate) and induced in SGCAA media (2% Galactose, 0.67% Yeast Nitrogen Base, 0.5% Casamino Acids, 0.54% Na2HPO4, 0.86% NaH2PO4).
  • SGCAA induced the designed proteins to be expressed and ⁇ presented on the yeast surface through Aga2p membrane proteins.
  • the induced cells are resuspended in the running buffer (Phosphate-buffered saline (PBS)+1% w/v Bovine Serum Albumin, pH 7.2-7.4) for all flow cytometry analyses.
  • the induced cells with the designed proteins on their surfaces were labeled with two fluorophores - FITC and PE.
  • Anti-c-myc mAb conjugated to FITC was used to label the myc tag to represent the degree of protein ⁇ expression.
  • IL-21R labeling either human or murine IL-21R with human Fc-tag (R&D Systems 991-R2, R&D Systems 596-MR) were used.
  • IL-21R with His-tag R&D Systems 9249-R2
  • human ⁇ c with Fc-tag Acro Biosystems ILG-H5256
  • murine IL-21R with His-tag Sino Biological 51184-M08H
  • murine ⁇ c with Fc-tag R&D Systems 784-MR
  • SA- PE streptavidin-PE
  • Yeast was analyzed by flow cytometers (BD Accuri C6, Thermo Fisher Attune NxT) or sorted by a Fluorescence-Activated Cell Sorting (FACS) cell sorter (Sony SH800).
  • FACS Fluorescence-Activated Cell Sorting
  • ⁇ Directed evolution and design optimization Using a random mutagenesis library kit (Agilent 200550), the 21d26 DNA was amplified, and the library was partially sequenced using BL21(DE3) (New England Biolabs). The sequencing showed that approximately 2-4 mutations were applied to the design while the DNA was amplified with the error-prone PCR.
  • the library was transformed to yeast ⁇ using electroporation with an approximated diversity of 3.4E7.
  • the yeast library was sorted for direct evolution using FACS with different combinations and concentrations of labeled receptor subunits, with the abovementioned labeling.
  • SSM Site-saturation mutagenesis
  • Sortings with four different receptor conditions (1) labeled human IL-21R, (2) unlabeled human IL-21R with labeled ⁇ human (3) labeled murine IL-21R, and (4) unlabeled murine IL-21R with labeled murine ⁇ c) saturated mutants with mutants with favorable mutations, and depleted mutants with unfavorable mutations.
  • DNA of the sorted SSM library was prepared before MiSeq TM (Illumina) using Zymoprep TM (Zymo Research), qPCR, and gel extraction (Qiagen).
  • the sorted SSM library revealed several mutations that can improve binding affinities to each ⁇ receptor - human IL-21R, human ⁇ c, murine IL-21R, and murine ⁇ c.
  • a combinatorial library that incorporated the mutations by using a PCR-based assembly of pools of 8 sets of primers (Integrated DNA Technologies) incorporating mutations with degenerate codons, which was then sorted against human or murine receptors. It yielded 16 candidates with significantly improved ⁇ affinities to the receptors, 10 human receptor-optimized candidates (21h1–21h10) and 6 murine receptor-optimized candidates (21m1–21m6).
  • the Kd of 16 candidates toward each of the receptors was measured by biolayer interferometry (ForteBio, See Methods). Cytokine mimics and split cytokine mimics protein expression (E. coli expression)
  • the DNA fragments (Integrated DNA Technologies) that encode the designed ⁇ proteins were cloned into pET-29b(+) plasmid with an N-terminal polyhistidine tag.
  • the cloned plasmids were transformed into competent BL21 cells (New England Biolabs), whose cultures were grown in Terrific Broth II and induced using 1 mM isopropyl b-D- thiogalactopyranoside (IPTG).
  • the harvested cultures were either lysed by sonication (Qsonica T M Q500).
  • the lysed cultures were ultracentrifuged at 18,000g for 30 minutes and purified using immobilized metal affinity chromatography (IMAC).
  • IMAC immobilized metal affinity chromatography
  • the elution proteins were separated by HPLC size-exclusion chromatography using Superdex TM 7510/300 GL column (GE Healthcare) on Akta TM (GE Healthcare AKTA TM Pure).
  • Octet binding buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)+1% w/v ⁇ Bovine Serum Albumin, pH 7.2-7.4) was used for all biolayer interferometry analyses.
  • biotinylated hIL-21R R&D Systems AVI9249
  • biotinylated mIL-21R Acro Biosystems ILR-M82E3
  • SAForteBio streptavidin-coated biosensors
  • biotinylated h ⁇ c (Acro Biosystems ILG-H85E8) or biotinylated m ⁇ c (Acro Biosystems ILA-M82E3), respectively, were immobilized on streptavidin coated biosensors (SAForteBio) at 5 ⁇ g/ml in the octet binding buffer and tested for 250-second association and 250-second dissociation in the ⁇ presence of 1.5-fold molar excess of corresponding hIL-21Ra (R&D Systems 9249-R2) or mIL-21Ra (Sino Biological 51184-M08H).
  • SAForteBio streptavidin coated biosensors
  • IL-21 mimics For polyhistidine-tagged IL-21 mimics loading, anti-penta-his (His1K) tips are used for immobilization and loaded to 1.0-1.5nm shift.
  • hIL-21 R&D Systems 8879-IL
  • mIL-21 R&D Systems 594-ML
  • IL-21 mimics See Methods
  • Data was processed using ForteBio Data Analysis Software ⁇ version 9.0.0.10.and the parameters were reported with standard error.
  • Circular dichroism and thermal stability assay Far-ultraviolet circular dichroism measurements were executed with a spectropolarimeter (JASCO J-1500 Spectropolarimeter).
  • the proteins were dissolved in PBS (pH 7.4) within 1 mm path length cuvettes at 0.5 mg/mL.
  • the wavelength scan was ⁇ performed within the 195 nm to 260 nm wavelength range.
  • the thermal melt experiment was performed by heating samples from 25°C to 95°C and cooled back down to 25°C while observing absorption at 222 nm.
  • Reference ⁇ 1. Hashmi, M. H. & Van Veldhuizen, P. J. Interleukin-21: updated review of Phase I and II clinical trials in metastatic renal cell carcinoma, metastatic melanoma and relapsed/refractory indolent non-Hodgkin’s lymphoma. Expert Opin. Biol. Ther.10, 807–817 (2010). 2.

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Abstract

Disclosed are de novo designed polypeptide agonists and antagonists of IL-21, conditionally-active IL-21 agonists and antagonists, and methods for using them to treat cancer or to modulate an immune response.

Description

MBHB 22-1530-US-WO UW 49605.02WO2 Interleukin-21 mimetics ^ Cross Reference This application claims priority to U.S. Provisional Patent Application Serial Number 63/378,797 filed October 7, 2022, incorporated by reference herein in its entirety. ^ Sequence Listing Statement A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on October 6, 2023 having the file name^ “22-1530-WO.xml” and is 892,905 bytes in size. Federal Funding Statement This invention was made with government support under grant number R01CA240339 awarded by the National Cancer Institute, and grant number R01AI160052^ awarded by the National Institute of Allergy and Infectious Disease. The government has certain rights in the invention. Background Interleukin-21 (IL-21) is a pleiotropic cytokine that plays pivotal roles in innate and^ adaptive immune responses. Being primarily produced by CD4+ T cells, IL-21 induces differentiation of various immune cells. IL-21 is involved in the regulation of immune responses to infections, cancer, and autoimmune diseases and has been a target of clinical research in recent years. Clinical trials involving IL-21 have been executed for various types of cancer, including melanoma, renal cell carcinoma, ovarian cancer, and non-Hodgkin's^ lymphoma. In this context, IL-21 has been investigated as a single agent or in combination with other immunotherapy agents, such as checkpoint inhibitors or cancer vaccines. Harnessing the full potential of native IL-21 for therapeutic purposes has proved challenging due to its poor stability, low cross-reactivity of human IL-21 in mouse models, and lack of engineerability. The restricted efficacy of human IL-21 in murine models further hampers the ^ predictive assessment of IL-21-based therapeutic candidates regarding toxicity and activity. Human and murine cross-reactive IL-21 mimetic proteins give a promising solution for cytokine-based immunotherapies using IL-21. ^ Summary In one aspect, the disclosure provides polypeptides comprising domains X2, X3, and optionally X4, wherein: (a) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence^ DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); (b) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of (i) KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934), or (ii) KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937), wherein relative to SEQ ID NO: 937, 1,^ 2, or all 3 of the following are true: residue 4 is Y, residue 6 is F, and/or residue 9 is D; and (c) X4 is optional and, when present, comprises a helical structure; wherein X2, X3, and X4, when present, may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; and wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21R^^^ CD360). In one embodiment, the polypeptide comprises domains X2, X3, and X4, wherein: (a) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); ^ (b) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934); and (c) X4 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence^ KEVMERAKSAAQKILGRFL (SEQ ID NO: 935); wherein X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; and ^ wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21R^^ CD360) and is capable of binding to the ^c, CD132 receptor. In another embodiment, (a) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%,^ 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); and (b) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937), wherein relative to SEQ ID NO: 937, 1,^ 2, or all 3 of the following are true: residue 4 is Y, residue 6 is F, and/or residue 9 is D. wherein X2 and X3 may be in any order in the polypeptide; wherein amino acid linkers may be present between the domains; and wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21R^^ CD360) but is not capable of simultaneously binding to the ^c, CD132 receptor. ^ In one embodiment, the polypeptides further comprise an X1 domain, wherein the X1 domain comprises a helical structure, and wherein X1, X2, X3, and X4, when present, may be in any order in the polypeptide. In another embodiment, the disclosure provides fusion proteins comprising: (a) the polypeptide of any embodiment herein; and ^ (b) one or more functional domains. In further embodiments, the polypeptide or fusion protein comprises an amino acid sequence comprising (a) an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group^ consisting of the polypeptides shown in Tables 1-6; or (b) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-598627-631, 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-^ 743, 746-747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898, and 900-931; or (c) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID ^ NO: SEQ ID NO:627-631, 633, 635-641, 643-644, 648-654, 656, 658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698, 700-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746, 749-751, 771, 849-854, 856-862, 864-870, 872-874, 876- 898, 900, and 902-907; or ^ (d) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO:1-598, 655, 657, 699, 747, 752-770, 772-798, 800-811, 813-821, 823, 825- 829, 831-832, 834-835, 843, 845-847, 871, and 908-931. In another aspect, the disclosure provides conditionally active IL-21 receptor binding^ proteins, comprising a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not both present in the same fusion protein, wherein in total the first polypeptide component and the second polypeptide component comprise domains X2, X3, and optionally X4, as defined in any one of claims 1-34, wherein: ^ (i) the first polypeptide component comprises at least one of X2, X3, and X4 (when present) but does not comprise each of X2, X3, and X4 (when present); and (ii) the second polypeptide component comprises each of X2, X3, and X4 (when present) that is not present in the first polypeptide component; wherein the first polypeptide component and the second polypeptide component are^ not active receptor binding proteins individually, and wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL-21R^^ CD360). In one embodiment, (i) the first polypeptide component comprises at least one of X2, X3, and X4 but^ does not comprise each of X2, X3, and X4; and (ii) the second polypeptide component comprises each of X2, X3, and X4 that is not present in the first polypeptide component; wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL-^ 21R^^ CD360) and is capable of binding to the ^c, CD132 receptor. In another embodiment, the conditionally active IL-21 receptor binding proteins of comprise a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not both present in ^ the same fusion protein, wherein in total the first polypeptide component and the second polypeptide component comprise domains X1, X2, X3, and X4 as defined in any embodiment herein; wherein: (i) the first polypeptide component comprises at least one of X1, X2, X3, and X4^ but does not comprise each of X1, X2, X3, and X4; and (ii) the second polypeptide component comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide component; wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL-^ 21R^^ CD360) and is capable of binding to the ^c, CD132 receptor The disclosure also provides recombinant nucleic acid encoding the polypeptide, first polypeptide, or second polypeptide of any embodiment herein; expression vectors comprising the recombinant nucleic acid of the disclosure operatively linked to a promoter; recombinant host cells comprising the nucleic acid, expression vector, polypeptide, first polypeptide,^ and/or second polypeptide of any embodiment herein; pharmaceutical compositions, comprising the polypeptide, fusion protein, first polypeptide, second polypeptide, recombinant nucleic acid, expression vector and/or recombinant host cell of any embodiment herein, and a pharmaceutically acceptable carrier; and methods treating cancer, or for modulating an immune response in a subject. ^ Figure Legends Figure 1. De novo IL-21 agonistic mimics can bind to both IL-21 receptor (IL- 21R; CD360) and common gamma receptor (^c; CD132) for receptor heterodimerization. (A) Design, screening, optimization, and characterization schematics of^ IL-21 mimics. (B) A model of an agonistic mimic of IL-21 in complex with hIL-21R and h^c. (C) SDS-PAGE of human IL-21 (lane 2), mouse IL-21 (lane 3), and 21h10 (SEQ ID NO:24) (lane 4) with Precision Plus Protein Kaleidoscope ladder (lane 1). (D) Expression of 21h10 shows a monomeric peak at expected retention volume in size exclusion chromatography using the S75 increase column. (E) Circular dichroism shows the presence of helical^ secondary structures in 21h10 (SEQ ID NO:24). (F) 222nm wavelength scan with thermal melt starting from 25°C to 95°C shows superior thermal stability of 21h10 (SEQ ID NO:24). Helical structures start to unfold at 65°C. (G-J) Receptor binding affinity measurement examples of 21h10 (SEQ ID NO:24) by biolayer interferometry as described in the Methods ^ section in the Examples. Measured Kd values of this and related designs are reported in Table 1. Figure 2. Shuffling of helical domains for retopology and de novo split IL-21 agonistic mimics. (A) Monomeric IL-21 mimic can be circularly permuted or shuffled with^ its helical domains (H1-H4) to generate different topologies while maintaining its IL-21R (CD360) and common ^c (CD132) receptor interfaces. (B) Three split pairs - H1/H234, H12/H34, and H123/H4 - can be generated from the given topology of the IL-21 mimic. (C) Split fragments of an IL-21 mimic can be fused to anti-A nanobody and anti-B nanobody, or other targeting domains (A and B are markers and could be identical or different), ^ respectively, to implement conditional activation for reconstituting active IL-21 mimic structure only when the two components colocalize. The reconstituted mimic transduces the IL-21 signal to the target cell. (D) Three split 21h10 pairs – H1/H234, H12/H34, and H123/H4 – can be reconstituted to their original four-helical bundle structure and form an IL- 21 receptor complex by heterodimerizing IL-21R (CD360) and common ^c (CD132)^ receptor. Figure 3. De novo IL-21 agonistic mimics can be fused to antibodies for targeted cytokine delivery. (A) Schematics of immunocytokine in the heavy chain:cytokine=2:2 construct. Example with Atezolizumab fused to IL-21 mimics. The stoichiometry could also be a 2:1 construct with Knobs/Holes mutations in the heavy chain for specific heterodimeric^ configuration; a heavy chain with knobs mutation with the cytokine fusion can be coupled with another heavy chain with holes mutations without the cytokine fusion. (B) SDS-PAGE of Atezolizumab-21h10 fusion construct in non-reducing condition, showing a band around the expected size of 179.2kDa. (C) The Atezolizumab-21h10 fusion construct can bind to human IL-21R and human PD-L1 independently. (D) Atezolizumab-21h10 fusion construct^ can bind to human IL-21R and human PD-L1 concurrently. Atezolizumab-21h10 fusion construct is at 10 nM, with human PD-L1 immobilized to streptavidin (SA) tips and associated with Atezolizumab-21h10 fusion construct then associated with 50 nM human IL- 21R, or vice versa. Figure 4. De novo IL-21 antagonistic mimics can bind only to IL-21 receptor (IL-^ 21R; CD360) but not ^c (CD132). (A) Five antagonistic hits (Table 5, SEQ ID NO:627-631) from the initial screening show human IL-21R binding in flow cytometry. Using yeast surface display, five hits were identified for human IL-21R binding.100nM of human IL-21R was used to label yeast cells that express designs. All of these hits could not bind to ^c, thus ^ categorized as antagonists. (B) Other antagonistic mimics were designed from agonistic mimics of IL-21 using RosettaTM, site saturation mutagenesis (SSM) data, or ProteinMPNN to remove ^c interface. Figure 5. Site saturation mutagenesis study of an agonist, 21JC15. Mutations of^ all positions on 21JC15 (SEQ ID NO:8) revealed positive (black) and negative (white) mutations that increased and decreased binding affinity to IL-21 receptors, respectively, using yeast surface display. Darker the color, more likely the mutation increases the binding affinity to IL-21R (CD360) and common ^c (CD132) receptor. The library was sorted against human IL-21 receptors to populate the data. This study reveals residue-level information of^ how mutations affect binding affinity towards receptors. Figure 6. Agonist designs vary with their interface affinities to IL-21R or IL-21 receptor complex (IL-21R/^c). Two-dimensional plot of Kd from IL-21Ra binding and ternary complex binding (binding ^c in the presence of IL-21Ra). X axes either represent binding affinity to hIL-21R or mIL-21R, whereas Y axes either represent binding affinity to^ hIL-21R/h^c or mIL-21R/m^c, in molar units [M]. In mouse plot, the constructs with mIL- 21R/m^c affinity worse than 10-4M is populated at 10-4M. Numberings 1 to 21 correspond to 21A1 to 21A21 (Table 1, SEQ ID NO:164-184), respectively. Detailed Description ^ All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol.185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA),^ “Guide to Protein Purification” in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R.I. Freshney.1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp.109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.),^ and the Ambion 1998 Catalog (Ambion, Austin, TX). As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. ^ As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro;^ P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). In all embodiments of polypeptides disclosed herein, any N-terminal methionine residues are optional (i.e.: the N-terminal methionine residue may be present or may be absent, and when absent is not considered in determining percent identity). Similarly, any N-^ or C-terminal histidine tags are optional and me be present or absent and, when absent, are not considered in determining percent identity. All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise. Unless the context clearly requires otherwise, throughout the description and the^ claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this^ application as a whole and not to any particular portions of the application. In a first aspect, the disclosure provides polypeptides comprising domains X2, X3, and optionally X4, wherein: (a) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence^ DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); (b) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of (i) KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934), or (ii) KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937), wherein relative to SEQ ID NO: 937, 1,^ 2, or all 3 of the following are true: residue 4 is Y, residue 6 is F, and/or residue 9 is D; and (c) X4 is optional and, when present, comprises a helical structure; wherein X2, X3, and X4, when present, may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; and ^ wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21R^^ CD360). The inventors have shown that polypeptides of this aspect of the disclosure are capable of binding to the IL-21 receptor, and demonstrated that some embodiments are^ capable of acting as agonists, and some embodiments are capable of acting as antagonists. The polypeptides are hyperstable de novo IL-21 mimetic proteins that recapitulate the native IL-21’s interactions with receptors and the biology of native IL-21 in humans and mice, which is ideal for therapeutic use. In one embodiment, the polypeptide comprises domains X2, X3, and X4, wherein:^ (a) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence DTRILKVSLKKIVRAANVIVRM (SEQ ID NO:933); (b) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence^ KRFEIRMRQLIDIVDHVKRE (SEQ ID NO:934); and (c) X4 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence KEVMERAKSAAQKILGRFL (SEQ ID NO:935); wherein X2, X3, and X4 may be in any order in the polypeptide; ^ wherein amino acid linkers may be present between any of the domains; and wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21R^^ CD360) and is capable of binding to the ^c, CD132 receptor. The inventors have shown that polypeptides of this embodiment are capable of acting as agonists to activate IL-21-like signaling and inducing cell differentiation in both human^ and mouse cells by specifically binding and promoting dimerization of the IL-21 receptor (IL-21R^^ CD360) and the common-^ chain-receptor (^c, CD132). The designed polypeptides show an anti-tumor effect in mouse MC38 adenocarcinoma models, B16F10 melanoma models, and pancreatic cancer models, and thus can be used, for example, to treat cancer. ^ Agonistic IL-21 mimics are constructs that can bind to IL-21 receptor (IL-21R^^ CD360) and the common-^ chain-receptor (^c, CD132), not only individually, but also can bind both receptors simultaneously for allowing potential downstream cell signaling through receptor heterodimerization. The way to identify capability to act as an agonist is to confirm ^ simultaneous binding to IL-21R and ^c receptor. As ^c receptor binding is hard to detect by itself due to very weak affinity between IL-21 and receptor, binding assay for ^c receptor in the presence of IL-21R will allow identifying the agonistic variants, at the highest concentration possible, 1000 nM of IL-21R and 1000 nM of ^c receptor. ^ In one embodiment, (a) X2 is a peptide comprising the amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933; (b) X3 is a peptide comprising the amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID^ NO: 934; and (c) X4 is a peptide comprising the amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 935. In another embodiment, (a) X2 is a peptide comprising the amino acid sequence at^ least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933; (b) X3 is a peptide comprising the amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 934; and ^ (c) X4 is a peptide comprising the amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 935. In a further embodiment, (a) X2 is a peptide comprising the amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933; ^ (b) X3 is a peptide comprising the amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 934; and (c) X4 is a peptide comprising the amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 935. In one embodiment, (a) X2 is a peptide comprising the amino acid sequence at least^ 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933; (b) X3 is a peptide comprising the amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 934; and ^ (c) X4 is a peptide comprising the amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 935. In another embodiment, (a) X2 is a peptide comprising the amino acid sequence at least 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933; ^ (b) X3 is a peptide comprising the amino acid sequence at least 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 934; and (c) X4 is a peptide comprising the amino acid sequence at least 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 935. The inventors have conducted extensive site-saturation mutagenesis (SSM), yeast^ surface display assay using flow cytometry, biolayer interferometry (BLI), and other functional studies as described in the Methods section of the Examples, to determine various substitutions that retain the receptor binding, and thus agonist, activity. In one embodiment, the polypeptide has an amino acid selected from the following residues relative to X2 (SEQ ID NO: 933): ^ Residue 1: G, A, S, T, Q, D, E, R, K, and H; Residue 2: G, A, L, S, T, N, and Q; Residue 3: A, M, Y, W, N, Q, E, R, and K; Residue 4: G, A, V, I, M, L, F, Y, W, N, Q, E, and H; Residue 5: M, L, Q, E, and K; ^ Residue 6: V, I, L, Y, R, and K; Residue 7: G, V, I, M, L, F, Y, W, S, T, N, Q, D, E, and R; Residue 8: A, L, F, S, T, E, and H; Residue 9: A, V, I, and L; Residue 10: E, R, and K; ^ Residue 11: V, E, K, R, and H; Residue 12: A, I, and L; Residue 13: V and I; Residue 14: S, E, K, and R; Residue 15: A, I, M, L, and E; ^ Residue 16: C, A, and S; Residue 17: N and R; Residue 18: V, S, N, E, K, R, and H; Residue 19: A, V, I, and L; Residue 20: V and I; ^ Residue 21: W, S, E, K, and R; and Residue 22: M and D. The inventors have identified residues 11, 14, 18, and 21 of X2 (SEQ ID NO: 933) as present at the binding interface with IL-21R^ (CD360). In some embodiments, the ^ polypeptide has 1, 2, 3, or all 4 of the following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 11 is K, E, or R; (b) X2 residue 14 is R, E, or K; (c) X2 residue 18 is V, R, H, or K; and/or (d) X2 residue 21 is R or K. ^ The inventors have identified residues 4, 7, 10, and 15 of X2 (SEQ ID NO: 933) as present at the binding interface with IL-21R^ (CD360). In some embodiments, the polypeptide has 1, 2, 3, or all 4 of the following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 4 is I or W; (b) X2 residue 7 is V, D. or E or I; ^ (c) X2 residue 10 is K or R; and/or (d) X2 residue 15 is A, I, or L or M. In another embodiment, the polypeptide has an amino acid selected from the following residues relative to X3 (SEQ ID NO: 934): Residue 1: P, N, D, E, R, K, and H; ^ Residue 2: G, F, Y, E, R, and K; Residue 3: V, I, M, L, F, T, and Q; Residue 4: A, I, L, S, T, E, R, and K; Residue 5: A, V, I, L, and E; Residue 6: A, V, L, E, R, and K; ^ Residue 7: M and L; Residue 8: V, L, F, Y, T, N, R, and K; Residue 9: G, A, V, Q, and E; Residue 10: A, I, M, and L; Residue 11: A, V, I and L; ^ Residue 12: D, E, and K; Residue 13: A, V, I, L, and T; Residue 14: C, A, V, S, T, and E; Residue 15: A, D, E, and R; Residue 16: A, I, L, Y, W, S, Q, E, R, K, and H; ^ Residue 17: A, V and I; Residue 18: A, L, Y, Q, E, R, and K; Residue 19: T, D, E, R, and K; and Residue 20: Y, T, N, E, and H. ^ The inventors have identified residues 2, 6, 8, 12, and 16 of X3 (SEQ ID NO: 934) as present at the binding interface with IL-21R^ (CD360) or ^c (CD132) receptor. In some embodiments, the polypeptide has 1, 2, 3, 4, or all 5 of the following residues relative to X3 (SEQ ID NO: 934): (a) X3 residue 2 is R or K; ^ (b) X3 residue 6 is R or K ; (c) X3 residue 8 is R or K; (d) X3 residue 12 is D or E; and/or (e) X3 residue 16 is H or Y or W. The inventors have identified residues 1, 5, 9, 11, 13, and 19 of X3 (SEQ ID NO: 934)^ as present at the binding interface with IL-21R^ (CD360) or ^c (CD132) receptor. In some embodiments, the polypeptide has 1, 2, 3, 4, 5, or all 6 of the following residues relative to X3 (SEQ ID NO: 934): (a) X3 residue 1 is K or R; (b) X3 residue 5 is I or V; ^ (c) X3 residue 9 is Q or G; (d) X3 residue 11 is I or L; (e) X3 residue 13 is I or V; and/or (f) X3 residue 19 is R or K. In a further embodiment, the polypeptide has an amino acid selected from the^ following residues relative to X4 (SEQ ID NO: 935): Residue 1: R and K; Residue 2: I, W, S, E, R, and K; Residue 3: A, V, I, and L; Residue 4: C, A, and M; ^ Residue 5: A, L, S, E, R, and K; Residue 6: M, N, Q, D, E, R, and K; Residue 7: A, L, and F; Residue 8: Y, S, E, R, and K; Residue 9: V, M, S, and T; ^ Residue 10: A, E, and K; Residue 11: C and A; Residue 12: Q and R; Residue 13: A and K; ^ Residue 14: C, G, A, V, I, M, L, Y, S, T, Q, D, E, and K; Residue 15: I, L, and F; Residue 16: G, F, and Y; Residue 17: S, R, and K; Residue 18: L, F, Y, W, and E; and ^ Residue 19: L, F, Y, and E. The inventors have identified residues 9 and 16 of X4 (SEQ ID NO: 935) as present at the binding interface with ^c (CD132) receptor. In some embodiments, the polypeptide has one or both of the following residues relative to X4 (SEQ ID NO: 935): (a) X4 residue 9 is S or T; and/or ^ (b) X4 residue 16 is G or Y. The inventors have identified residues 8, 12, 13, 15, and 19 of X4 (SEQ ID NO: 935) as present at the binding interface with ^c (CD132) receptor. In some embodiments, the polypeptide has 1, 2, 3, 4, or all 5 of the following residues relative to X4 (SEQ ID NO: 935): (a) X4 residue 8 is K or R; ^ (b) X4 residue 12 is Q; (c) X4 residue 13 is K; (d) X4 residue 15 is L or I; and/or (e) X4 residue 19 is L, F, or Y. In one embodiment, the polypeptide comprises: ^ (1) the following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 11 is K, E, or R; (b) X2 residue 14 is R, E, or K; (c) X2 residue 18 is V, R, H, or K; (d) X2 residue 21 is R or K; ^ (e) X2 residue 4 is I or W; (f) X2 residue 7 is V, D. or E or I; (g) X2 residue 10 is K or R; and (h) X2 residue 15 is A, I, or L or M; and (2) the following residues relative to X3 (SEQ ID NO: 934): ^ (a) X3 residue 2 is R or K; (b) X3 residue 6 is R or K ; (c) X3 residue 8 is R or K; (d) X3 residue 12 is D or E; ^ (e) X3 residue 16 is H or Y or W; (f) X3 residue 1 is K or R; (g) X3 residue 5 is I or V; (h) X3 residue 9 is Q or G; (i) X3 residue 11 is I or L; ^ (j) X3 residue 13 is I or V; and (k) X3 residue 19 is R or K; and (3) the following residues relative to X4 (SEQ ID NO: 935): (a) X4 residue 9 is S or T; and (b) X4 residue 16 is G or Y; and ^ (c) X4 residue 8 is K or R; (d) X4 residue 12 is Q; (e) X4 residue 13 is K; (f) X4 residue 15 is L or I; and (g) X4 residue 19 is L, F, or Y. ^ In another embodiment, the disclosure provides polypeptides comprising domains X2 and X3, wherein: (a) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence^ DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); and (b) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937), wherein relative to SEQ ID NO: 937, 1, 2, or all 3 of the following are true: residue 4 is Y, residue 6 is F, and/or residue 9 is D.^ wherein X2 and X3 may be in any order in the polypeptide; wherein amino acid linkers may be present between the domains; and wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21R^^ CD360) but is not capable of simultaneously binding to the ^c, CD132 receptor. ^ In this embodiment, the polypeptides are capable of acting as IL-21 antagonists. Antagonistic IL-21 mimics are constructs that can bind to IL-21 receptor (IL-21R^^ CD360) but cannot bind to the common-^ chain-receptor (^c, CD132) simultaneously, potentially prohibiting heterodimerization of IL-21 receptor (IL-21R^^ CD360) and the common-^^ chain-receptor (^c, CD132) and resultant potential downstream cell signaling. Designs can be tested for antagonist activity by first confirming binding to IL-21R, and then to confirm lack of simultaneous binding to ^c receptor. As ^c receptor binding is hard to detect by itself due to very weak affinity between IL-21 and ^c receptor, binding assay for ^c receptor need to be done in the presence of IL-21R. Confirmation of IL-21R binding is critical as lack of binding^ to IL-21R can also cause false positives in the result of the lack of binding against the ^c receptor. Similar to agonist identification, the assay should be performed at the highest concentration possible, 1000 nM for IL-21R and 1000 nM for ^c receptor. The inventors have provided extensive teaching of such polypeptides capable of acting as IL-21 receptor antagonists. For example, at least the following polypeptides in^ Table 5 have been identified as having antagonistic capability: SEQ ID NO:627-631, 633, 635-641, 643-644, 648-654, 656, 658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681- 685, 687-695, 698, 700-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746, 749-751, 771, 849-854, 856-862, 864-870, 872-874, 876-898, 900, and 902-907. The polypeptide antagonists may be used, for example, in modulating an immune^ response in a subject in need thereof, such as immunoinhibitory purposes for autoimmune diseases, including but not limited to multiple sclerosis, lupus, and rheumatoid arthritis. The X2 domain of this embodiment is the same as the X2 domain of the agonists detailed above, as the X2 domain mediates interaction with the IL-21 receptor (IL-21R^^ CD360) but not the ^c, CD132 receptor. ^ In one embodiment, (a) X2 is a peptide comprising the amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933; and (b) X3 is a peptide comprising the amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 937. ^ In another embodiment, (a) X2 is a peptide comprising the amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933; and (b) X3 is a peptide comprising the amino acid sequence at least ^ 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 937. In a further embodiment, (a) X2 is a peptide comprising the amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID^ NO: 933; and (b) X3 is a peptide comprising the amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 937. In one embodiment, (a) X2 is a peptide comprising the amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933; and (b) X3 is a peptide comprising the amino acid sequence at least 90%, 95%, 98%, or 100%^ identical to the amino acid sequence of SEQ ID NO: 937. In another embodiment, (a) X2 is a peptide comprising the amino acid sequence at least 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 933; and (b) X3 is a peptide comprising the amino acid sequence at least 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 937. ^ The inventors have conducted extensive site-saturation mutagenesis (SSM), yeast surface display assay using flow cytometry, biolayer interferometry (BLI), and other functional studies, as described in the Methods section of the Examples, to determine various substitutions that retain the receptor binding, and thus antagonist activity. In one embodiment, the polypeptide has an amino acid selected from the following residues relative^ to X2 (SEQ ID NO: 933): Residue 1: G, A, S, T, Q, D, E, R, K, and H; Residue 2: G, A, L, S, T, N, and Q; Residue 3: A, M, Y, W, N, Q, E, R, and K; Residue 4: G, A, V, I, M, L, F, Y, W, N, Q, E, and H; ^ Residue 5: M, L, Q, E, and K; Residue 6: V, I, L, Y, R, and K; Residue 7: G, V, I, M, L, F, Y, W, S, T, N, Q, D, E, and R; Residue 8: A, L, F, S, T, E, and H; Residue 9: A, V, I, and L; ^ Residue 10: E, R, and K; Residue 11: V, E, K, R, and H; Residue 12: A, I, and L; Residue 13: V and I; Residue 14: S, E, K, and R; ^ Residue 15: A, I, M, L, and E; Residue 16: C, A, and S; Residue 17: N and R; Residue 18: V, S, N, E, K, R, and H; ^ Residue 19: A, V, I, and L; Residue 20: V and I; Residue 21: W, S, E, K, and R; and Residue 22: M and D. The inventors have identified residues 11, 14, 18, and 21 of X2 (SEQ ID NO: 933) as^ present at the binding interface with IL-21R^^ CD360. In some embodiments, the polypeptide has 1, 2, 3, or all 4 of the following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 11 is K, E, or R; (b) X2 residue 14 is R, E, or K; (c) X2 residue 18 is V, R, H, or K; and/or ^ (d) X2 residue 21 is R or K. The inventors have identified residues 4, 7, 10, and 15 of X2 (SEQ ID NO: 933) as present at the binding interface with IL-21R^^ CD360. In some embodiments, the polypeptide has 1, 2, 3, or all 4 of the following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 4 is I or W; ^ (b) X2 residue 7 is V, D. or E or I; (c) X2 residue 10 is K or R; and/or (d) X2 residue 15 is A, I, or L or M. In another embodiment, the polypeptide has an amino acid selected from the following residues relative to X3 (SEQ ID NO: 937): ^ Residue 1: P, N, D, E, R, K, and H; Residue 2: G, F, Y, E, and R, and K; Residue 3: V, I, M, L, F, T, and Q; Residue 4: Y; Residue 5: A, V, I, L, and E; ^ Residue 6: F; Residue 7: M and L; Residue 8: Q and E; Residue 9: D; ^ Residue10: A, I, M, and L; Residue 11: A, V, I, and L; Residue 12: D, E, and K; Residue 13: A, V, I, L, and T; ^ Residue 14: C, A, V, S, T, and E; Residue 15: T, N, and K; Residue 16: A, I, L, Y, W, S, Q, E, R, K, and H; Residue 17: A, V and I; Residue 18: A, L, Y, Q, E, R, and K; ^ Residue 19: T, D, E, R, and K; and Residue 20: Y, T, N, E, and H. In some embodiments, the polypeptide has 1, 2, or all 3 of the following residues relative to X3 (SEQ ID NO: 937): (a) X3 residue 2 is R or K; ^ (b) X3 residue 12 is D or E; and/or (c) X3 residue 16 is H or Y/W. In other embodiments, the polypeptide has 1, 2, 3, 4, or all 5 of the following residues relative to X3 (SEQ ID NO: 937): (a) X3 residue 1 is K or R; ^ (b) X3 residue 5 is I or V; (c) X3 residue 11 is I or L; (d) X3 residue 13 is I or V; and/or (e) X3 residue 19 is R or K. In a further embodiment, (i) relative to X3 (SEQ ID NO: 937): ^ (a) all 3 of the following are true: residue 4 is Y, residue 6 is F, and/or residue 9 is D; (b) X3 residue 2 is R or K; (c) X3 residue 12 is D or E; (d) X3 residue 16 is H, Y, or W. ^ (e) X3 residue 1 is K or R; (f) X3 residue 5 is I or V; (g) X3 residue 11 is I or L; (h) X3 residue 13 is I or V; and (i) X3 residue 19 is R or K; and ^ (ii) relative to X2 (SEQ ID NO: 933): (a) X2 residue 11 is K, E, or R; (b) X2 residue 14 is R, E, or K; (c) X2 residue 18 is V, R, H, or K; and ^ (d) X2 residue 21 is R or K; (e) X2 residue 4 is I or W; (f) X2 residue 7 is V, D. or E or I; (g) X2 residue 10 is K or R; and (h) X2 residue 15 is A, I, or L or M. ^ In another embodiment, the antagonist polypeptides further comprise an X4 domain, wherein the X4 domain comprises a helical structure, wherein the X4 domain does not permit simultaneous binding of the polypeptide to the IL-21 receptor (IL-21R^^ CD360) and the ^c, CD132 receptor, and wherein X2, X3, and X4 may be in any order in the polypeptide. In this embodiment, the X4 domain may be any helical domain as suitable for an intended purpose,^ so long as it does not permit simultaneous binding of the polypeptide to the IL-21 receptor (IL-21R^^ CD360) and the ^c, CD132 receptor,. In a further embodiment, X4 comprises a 19-residue peptide. In one such embodiment, the X4 peptide has an amino acid at each position selected from the following amino acid residues: ^ Residue 1: R and K; Residue 2: I, W, S, E, R, and K; Residue 3: V; Residue 4: C, A, and M; Residue 5: A, L, S, E, R, and K; ^ Residue 6: M, N, Q, D, E, R, and K; Residue 7: A, L, and F; Residue 8: A, V, and D; Residue 9: G, W, E, and K; Residue 10: A, E, and K; ^ Residue 11: C and A; Residue 12: G, I, E, and K; Residue 13: V, L, Q, D, E, and R; Residue 14: C, G, A, V, I, M, L, Y, S, T, Q, D, E, and K; ^ Residue 15: I, L, and F; Residue 16: R, K, and H; Residue 17: S, R, and K; Residue 18: L, F, Y, W, and E; and ^ Residue 19: L, F, Y, and E. In one embodiment of any of the Il-21 binding polypeptides of the disclosure, the polypeptides may further comprise a helical X1 domain. The helical X1 domain may comprise any amino acid sequence. In this embodiment, X1, X2, X3, and X4 may be in any order in the polypeptide. In one embodiment, X1 is a peptide comprising the amino acid^ sequence at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence LAEIMKEVAECARKEA (SEQ ID NO: 936). In another embodiment, the polypeptide has an amino acid selected from the following residues relative to X1 (SEQ ID NO: 936): Residue 1: V, I, and L; ^ Residue 2: A, I, S, N, R, and K; Residue 3: D and E; Residue 4: V and I; Residue 5: M and F; Residue 6: K; ^ Residue 7: E; Residue 8: A and V; Residue 9: A and R; Residue 10: A, V, I, L, T, Q, E, and K; Residue 11: C, V, I, M, F, Y, Q, R, and K; ^ Residue 12: A; Residue 13: R and K; Residue 14: K; Residue 15: E; and Residue 16: A, I, M, L, F, Y, S, and N. ^ In one embodiment, the polypeptide does not have a cysteine residue at position 11 relative to X1 (SEQ ID NO: 936). The X2, X3, X4 (when present), and X1 (when present) domains may be present in any order in the polypeptide of any embodiment herein. The inventors have provided extensive teaching of “shuffled” versions of the polypeptides; see Table 2 and the column ^ listing the domain order of X1, X2, X3, and X4 domains. In one embodiment, the polypeptide comprises in domain order, X2-X3-X4, or X1-X2-X3, or X2-X3, or X1-X2-X3- X4. In other embodiments, the polypeptide comprises in domain order, X4-X3-X2, X3-X2, X1-X3-X2, X2-X1-X3, X3-X2-X1, X3-X1-X2, X2-X4-X3, X3-X2-X4, X3-X4-X2, X4-X2-^ X3, X1-X2-X3-X4, X1-X4-X3-X2, X2-X1-X4-X3, X2-X3-X4-X1, X3-X2-X1-X4, X3-X4- X1-X2, X4-X1-X2-X3, X3-X2, or X4-X3-X2-X1 The polypeptides of the disclosure may comprise amino acid linkers between any of the domains. In some embodiments, there is a linker between each of domains X2, X3, X4 (when present), and X1 (when present). In other embodiments, the polypeptides may^ comprise a linker between none of the domains, or may comprise a linker between some but not all of the domains. The linkers may be of any length or amino acid composition. In various non-limiting embodiments, suitable linkers include, but are not limited to GS , GGS , GGGGG (SEQ ID NO: 939), GSGGG (SEQ ID NO: 940), GGGGGG (SEQ ID NO: 941), GGSGGG (SEQ ID NO: 942), GGSGGSGGGSGGSGSG (SEQ ID NO: 943), ^ GSGGSGGGSGGSGSG (SEQ ID NO: 944), GSGSGSG GSGGSCKKISGGSGGGSGGGGS (SEQ ID NO: 945), and (GGGGX)n (SEQ ID NO: 946), where X is Q, E, or S and n is 2-5. The polypeptides may be fused to one or more additional domains as appropriate for an intended use. In one embodiment, the disclosure provides fusion proteins comprising a polypeptide of any embodiment or combination of embodiments of the disclosure, and one or^ more functional domains. Any functional domain may be fused to the polypeptide of the disclosure. In various non-limiting embodiments, the one or more functional domains comprises cell targeting domains (including but not limited to antibodies, antibody fragments, domains to extend protein half-life (such as albumin, albumin-binding protein, Fc fragment of antibody), proteins that bind to biological markers, antigens, ligands, peptides, etc.) or^ detectable domains (including but not limited to fluorescent proteins, luminescent proteins, protein tags, etc.) In various embodiments, the one or more functional domain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:599-606. The polypeptide and the one or more functional domains may directly abut each other in the^ fusion protein, or may be linked by a polypeptide linker suitable for an intended purpose. The one or more functional domain may be present at the N-terminus, the C-terminus, or position between the X2, X3, X4, or X1 (when present) domains. In other embodiments, the polypeptide or fusion protein comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% ^ identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-598 627-631, 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746- 747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854,^ 856-862, 864-874, 876-898, and 900-931. In one embodiment, the polypeptide or fusion protein comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:627-631, 633, 635-641, 643-644, 648-654, 656, 658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698, 700-702, 706-707, 709-711,^ 714-715, 717-735, 738-739, 742-743, 746, 749-751, 771, 849-854, 856-862, 864-870, 872- 874, 876-898, 900, and 902-907. In this embodiment, the polypeptides are capable of antagonist activity. In another embodiment, the polypeptide or fusion protein comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID^ NO:1-598, 655, 657, 699, 747, 752-770, 772-798, 800-811, 813-821, 823, 825-829, 831-832, 834-835, 843, 845-847, 871, and 908-931. In this embodiment, the polypeptides are capable of agonist activity. In another aspect, the disclosure provides conditionally active IL-21 receptor binding proteins, comprising a first polypeptide component and a second polypeptide component,^ wherein the first polypeptide component and the second polypeptide component are not both present in the same fusion protein, wherein in total the first polypeptide component and the second polypeptide component comprise domains X2, X3, and optionally X4 as defined in any embodiment or combination of embodiments of the polypeptides of disclosure, wherein: (i) the first polypeptide component comprises at least one of X2, X3, and X4^ (when present) but does not comprise each of X2, X3, and X4 (when present); and (ii) the second polypeptide component comprises each of X2, X3, and X4 that is not present in the first polypeptide component; wherein the first polypeptide component and the second polypeptide component are not active receptor binding proteins individually, and wherein the first polypeptide ^ component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL-21R^^ CD360). In one embodiment: ^ (i) the first polypeptide component comprises at least one of X2, X3, and X4 but does not comprise each of X2, X3, and X4; and (ii) the second polypeptide component comprises each of X2, X3, and X4 that is not present in the first polypeptide component; ^ wherein the first polypeptide component and the second polypeptide component are not active receptor binding proteins individually, and wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL-21R^^ CD360) and is capable of binding to the ^c, CD132 receptor. ^ In another embodiment, (i) the first polypeptide component comprises at least one of X1, X2, X3, and X4 but does not comprise each of X1, X2, X3, and X4; and (ii) the second polypeptide component comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide component; ^ wherein the first polypeptide component and the second polypeptide component are not active receptor binding proteins individually, and wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL-21R^^ CD360) and is capable of binding to the ^c, CD132 receptor. ^ The present disclosure surprisingly demonstrates conditionally active receptor IL-21 agonists comprising the recited separate first and second polypeptides that individually are not receptor agonists, but which can interact non-covalently to form an active agonist of IL- 21, by binding to both the IL-21 receptor (IL-21R^^ CD360) and to the ^c, CD132 receptor. Thus, the conditionally active receptor agonists of the current disclosure can be used for any^ uses that the polypeptide agonists of the disclosure can be used. In another embodiment, (i) the first polypeptide component comprises X2 but does not comprise X3; and (ii) the second polypeptide component comprises X3; wherein the first polypeptide component and the second polypeptide component are^ not active receptor binding proteins individually, and wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL-21R^^ CD360) but does not bind simultaneously bind to the ^c, CD132 receptor. ^ This embodiment provides conditionally active receptor antagonists, which can be used for any uses that the polypeptide antagonists of the disclosure can be used. Split IL-21 antagonists are designed to achieve targeted delivery of IL-21 antagonist with minimal off- target activity. In further embodiments, the first polypeptide or the second polypeptide may^ comprise an X1 domain and/or an X4 domain as disclosed in any embodiment or combination of embodiments The polypeptides are typically split at sites that won’t interfere with the function of the protein (e.g., linker sections in embodiments with linkers). In addition, just as the X1 (when present), X2, X3, and X4 (when present) domains in the non-split polypeptides can be^ looped together in any order, the split proteins can comprise any combination of the domains. Thus, X1 (when present), X2, X3, and X4 (when present) may be in any order in the first and second polypeptide; in non-limiting embodiments: (i) the first polypeptide comprises X1 and the second polypeptide comprises X2, X3, and X4; ^ (ii) the first polypeptide comprises X2 and the second polypeptide comprises X1, X3, and X4; (iii) the first polypeptide comprises X3 and the second polypeptide comprises X1, X2, and X4; (iv) the first polypeptide comprises X4 and the second polypeptide comprises X1,^ X2, and X3; (v) the first polypeptide comprises X1 and X2, and the second polypeptide comprises X3 and X4; (vi) the first polypeptide comprises X1 and X3, and the second polypeptide comprises X2 and X4; ^ (vii) the first polypeptide comprises X1 and X4, and the second polypeptide comprises X2 and X3; (viii) the first polypeptide comprises X2 and X3, and the second polypeptide comprises X1 and X4; (ix) the first polypeptide comprises X2 and X4, and the second polypeptide^ comprises X1 and X3; (x) the first polypeptide comprises X3 and X4, and the second polypeptide comprises X1 and X2; (xi) the first polypeptide comprises X1, X2, and X3 and the second polypeptide comprises X4; ^ (xii) the first polypeptide comprises X1, X2, and X4 and the second polypeptide comprises X3; (xiii) the first polypeptide comprises X1, X3, and X4 and the second polypeptide comprises X2; or ^ (xiv) the first polypeptide comprises X2, X3, and X4 and the second polypeptide comprises X1. When the first polypeptide and/or the second polypeptide include more than one domain of X1, X2, X3, and X4, the domains may in some embodiments be separated by amino acid linkers of any suitable length or amino acid composition. There is no requirement^ for linkers; in one embodiment there are no linkers present between any of the domains. In other embodiments, an amino acid linker may be present between 0, 1, or 2 junctions between domains X1, X2, X3, and X4 in the first polypeptide and/or the second polypeptide. The amino acid linkers may be of any length as deemed appropriate for an intended use and may, for example, comprise any of the linker embodiments disclosed herein. In some ^ embodiments, a linker is at the N terminus or C terminus and is referred to as a linker despite not linking two domains together. In one embodiment, X1, when present, X2, X3, and X4, respectively, are as recited in any embodiment or combination of embodiments of the polypeptides of the disclosure. In another embodiment, the first and second polypeptides in total comprise an^ amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-598627-631, 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742- 743, 746-747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847,^ 849-854, 856-862, 864-874, 876-898, and 900-931; or (b) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO:627-631, 633, 635-641, 643- 644, 648-654, 656, 658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698, 700-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746, 749-751, 771,^ 849-854, 856-862, 864-870, 872-874, 876-898, 900, and 902-907; or (c) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO:1-598, 655, 657, 699, 747, 752-770, 772-798, 800-811, 813-821, 823, 825-829, 831-832, 834-835, 843, 845- 847, 871, and 908-931. ^ In another embodiment, the first and second polypeptides comprise: (a) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 608; and (ii) a second polypeptide comprising an amino acid^ sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 609; (b) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 607; and (ii) a second polypeptide comprising an amino acid^ sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 612; (c) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 610; and (ii) a second polypeptide comprising an amino acid^ sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 611; (d) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 613; and (ii) a second polypeptide comprising an amino acid^ sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 614; (e) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 615; and (ii) a second polypeptide comprising an amino acid^ sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 616; (f) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 617; and (ii) a second polypeptide comprising an amino acid^ sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 618; (g) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 619; and (ii) a second polypeptide comprising an amino acid ^ sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 620; (h) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid^ sequence of SEQ ID NO: 621; and (ii) a second polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 622; or (i) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid^ sequence of SEQ ID NO: 623; and (ii) a second polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 624. In a further embodiment, the first and second polypeptides comprise: (a) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%,^ 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 608; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 609; (b) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%,^ 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 607; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 612; (c) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%,^ 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 610; and (ii) a second polypeptide comprising an amino acid sequence at least 50%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 611; (d) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%,^ 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 613; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 614; ^ (e) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 615; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid^ sequence of SEQ ID NO: 616; (f) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 617; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid^ sequence of SEQ ID NO: 618; (g) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 619; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid^ sequence of SEQ ID NO: 620; (h) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 621; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid^ sequence of SEQ ID NO: 622; or (i) (i) a first polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 623; and (ii) a second polypeptide comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid^ sequence of SEQ ID NO: 624. In a still further embodiment, the first and second polypeptides comprise: (a) (i) a first polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 608; and (ii) a second polypeptide comprising an amino acid sequence at least 75%, 80%, 85%,^ 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 609; (b) (i) a first polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 607; and (ii) a second polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 612; ^ (c) (i) a first polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 610; and (ii) a second polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 611; ^ (d) (i) a first polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 613; and (ii) a second polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 614; (e) (i) a first polypeptide comprising an amino acid sequence at least 75%, 80%,^ 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 615; and (ii) a second polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 616; (f) (i) a first polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 617;^ and (ii) a second polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 618; (g) (i) a first polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 619; and (ii) a second polypeptide comprising an amino acid sequence at least 75%, 80%, 85%,^ 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 620; (h) (i) a first polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 621; and (ii) a second polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 622; or^ (i) (i) a first polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 623; and (ii) a second polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 624. In a still further embodiment, the first and second polypeptides comprise:^ (a) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 608; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 609; ^ (b) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 607; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 612; ^ (c) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 610; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 611; (d) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%,^ 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 613; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 614; (e) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 615; and (ii) a second^ polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 616; (f) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 617; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical^ to the amino acid sequence of SEQ ID NO: 618; (g) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 619; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 620; ^ (h) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 621; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 622; or (i) (i) a first polypeptide comprising an amino acid sequence at least 90%, 95%,^ 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 623; and (ii) a second polypeptide comprising an amino acid sequence at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 624. In one embodiment, one or both of the first polypeptide and the second polypeptide are fused to a targeting domain, permitting targeted delivery. In one non-limiting ^ embodiment, the targeting domain may comprise an antibody or nanobody that can direct the split cytokine mimics to desired cells or tissues of interest (Fig.2C). In this example, the first and second polypeptides are fused to anti-HER2-DARPin or anti-EGFR-DARPin (Table 4, SEQ ID NO:613-624) to demonstrate that the first and/or second polypeptides can be fused to^ such targeting domain for use and can be reconstituted (Fig.2D. In another embodiment, the disclosure provides polypeptide comprising the amino acid sequence of any first polypeptide or second polypeptide as described in the disclosure. These polypeptides may be used, for example, in the conditionally-active receptor agonists of the disclosure. In one embodiment, the polypeptides, comprise an amino acid sequence at^ least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of selected from the group consisting of SEQ ID NO: 607-624. In another aspect, the disclosure provides non-naturally occurring polypeptides comprising domains X1, X2, X3, and X4, wherein: (a) X1 is a peptide comprising the amino acid sequence at least 25%, 30%, 35%,^ 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence LAEIMKEVAECARKEA (SEQ ID NO:936); (b) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence DTRILKVSLKKIVRA (SEQ ID NO:933); ^ (c) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence KRFEIRMRQLIDIVDHVKRE (SEQ ID NO:934); and (d) X4 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence^ KEVMERAKSAAQKILGRFL (SEQ ID NO:935); wherein X1, X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; and wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21R^^ CD360). The polypeptides of this aspect of the disclosure can be used, for example, to treat^ cancer and/or to modulate an immune response. In one embodiment, X1 residues 1-16 are selected from: Residue 1: V, I, and L; Residue 2: A, I, S, N, R, and K; ^ Residue 3: D and E; Residue 4: V and I; Residue 5: M and F; Residue 6: K; ^ Residue 7: E; Residue 8: A and V; Residue9: A and R; Residue 10: A, V, I, L, T, Q, E, and K; Residue 11: C, V, I, M, F, Y, Q, R, and K; ^ Residue 12: A; Residue 13: R and K; Residue 14: K; Residue 15: E; and Residue 16: A, I, M, L, F, Y, S, and N. ^ In another embodiment, X1 residue 16 is not A. In one embodiment, X2 residues 1- 16 are selected from: Residue 1: G, A, T, Q, D, R, K, and H; Residue 2: G, A, S, T, and N; ^ Residue 3: A, M, Y, N, Q, E, and R; Residue 4: G, A, V, I, M, L, F, Y, W, N, Q, E, and H; Residue 5: M, L, Q, and K; Residue 6: V, I, L, Y, R, and K; Residue 7: G, V, I, M, L, F, Y, W, S, T, N, Q, D, and E; ^ Residue 8: F, S, T, and E; Residue 9: L; Residue 10: K; Residue 11: K; Residue 12: I; ^ Residue 13: V and I; Residue 14: R; and Residue 15: A, I, M, and L. In various embodiments, 1, 2, 3, or all 4 of the following are true: (a) X2 residue 3 is not R; ^ (b) X2 residue 4 is not I; (c) X2 residue 7 is not V; and/or (d) X2 residue 8 is not S. In a further embodiment, X3 residues 1-20 are selected from:^ Residue 1: P, N, R, and K; Residue 2: F, Y, and R; Residue 3: V, I, M, L, F, T, and Q; Residue 4: E; Residue 5: I; ^ Residue 6: R; Residue 7: M; Residue 8: V, L, F, Y, T, and R; Residue 9: G, A, and Q; Residue10: I, M, and L; ^ Residue 11: I and L; Residue 12: D; Residue 13: I; Residue 14: C, A, V, S, and T; Residue 15: D and E; ^ Residue 16: H; Residue 17: V; Residue 18: K; Residue 19: R; and Residue 20: T, N, and E. ^ In various embodiments, 1, 2, 3, 4, 5, 6, or all 7 of the following are true: (a) X3 residue 2 is not R; (b) X3 residue 8 is not R; (c) X3 residue 9 is not Q; (d) X3 residue 10 is not L; ^ (e) X3 residue 11 is not I; (f) X3 residue 14 is not V; and/or (g) X3 residue 15 is not D. In one embodiment, X4 residues 1-19 are selected from: Residue 1: K; ^ Residue 2: E; Residue 3: V; Residue 4: M; Residue 5: E; ^ Residue 6: R; Residue 7: A; Residue 8: R and K; Residue 9: V, M, S, and T; Residue 10: A; ^ Residue 11: A; Residue 12: Q; Residue 13: K; Residue 14: C, G, A, V, I, M, L, S, T, Q, D, and K; Residue 15: L; ^ Residue 16: G; Residue 17: R and K; Residue 18: F, Y, and W; and Residue 19: L, F, and Y. In another embodiment, 1, 2, or all 3 of the following are true: ^ (a) X4 residue 8 is not K; (b) X4 residue 9 is not S; and/or (c) X4 residue 19 is not L. In one embodiment, the polypeptide is an antagonist of the IL-21 receptor. In another embodiment, X1 residue 11 is C. This embodiment helps increase binding affinity to human^ common Gamma chain (CD132), as well as mouse CD360 and mouse CD132. In various embodiments, the domains are arranged N-terminal to C-terminal in an arrangement selected from the group consisting of X1-2-3-4, X1-4-3-2, X2-1-4-3, X2-3-4-1, X3-2-1-4, X3-4-1-2, X4-1-2-3, and X4-3-2-1. In other embodiments, amino acid linkers are present between the domains. ^ In one embodiment, the polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 938. DEEELAEIMKEVAECARKEAEKIDNTDEDTRILKVSLKKIVRAANVIVRMREREADSKRFEI RMRQLIDIVDHVKREFASEDLKEVMERAKSAAQKILGRFL (SEQ ID NO: 938) ^ In this embodiment, residues at non-helix positions may be selected from the group consisting of: Residue 01: V, I, M, F, Y, and D; Residue 02: A, V, I, M, L, F, Y, W, Q, D, E, R, and H; ^ Residue 03: E; Residue 04: D and E; Residue 21: A, T, and E; Residue 22: R and K; Residue 23: I, M, L, F, and Y; ^ Residue 24: P, G, A, V, I, S, TN, Q, D, R, and H; Residue 25: G, A, V, N, Q, and K; Residue 26: T, N, R, and K; Residue 27: G, A, S, T, N, D, R, K, and H; Residue 28: P, E, and H; ^ Residue 44: A and S; Residue 45: N and K; Residue 46: G, A, V, S, T, N, R, K, and H; Residue 47: V and I; Residue 48: V, I, Y, Q, R, and K; ^ Residue 49: L, R, and K; Residue 50: G, M, Y, S, Q, and H; Residue 51: R; Residue 52: G, M, L, F, Y, W, N, D, E, and H; Residue 53: G, R, and K; ^ Residue 54: G, A, V, I, L, N, E, and K; Residue 55: A, V, I, M, L, F, Y, W, T, Q, and H; Residue 56: D; Residue 57: P, A, V, Y, W, S, D, E, and H; Residue 78: F; ^ Residue 79: A, Y, S, and K; Residue 80: S; Residue 81: E; Residue 82: D; and Residue 83: L and F. ^ In a further embodiment, the polypeptides of this aspect of the disclosure comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-598627-631, 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675,^ 677-679, 681-685, 687-695, 698-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742- 743, 746-747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898, and 900-931. In one embodiment, the polypeptides further comprise one or more functional domains. The polypeptides may be fused to any functional domain as suitable for an intended^ use, included but not limited to cell targeting domains and detectable domains (including but not limited to fluorescent proteins, luminescent proteins, etc.) In one embodiment, the one or more functional domains is a translational fusion with the polypeptide. In another embodiment of this aspect, the disclosure provides non-naturally occurring conditionally active IL-21 receptor binding proteins, comprising a first polypeptide ^ component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not present in a fusion protein, wherein in total the first polypeptide component and the second polypeptide component comprise domains X1, X2, X3, and X4 as defined in any preceding claim; wherein: (i) the first polypeptide component comprises at least one of X1, X2, X3, and X4^ but does not comprise each of X1, X2, X3, and X4; and (ii) the second polypeptide component comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide component; wherein the first polypeptide component and the second polypeptide component are not active receptor binding proteins individually, and wherein the first polypeptide ^ component and the second polypeptide interact to form an active IL-21 receptor binding protein. In various embodiments, the polypeptide may be selected from the group consisting of: (i) a polypeptide comprising X1 and excluding X2, X3, and X4; ^ (ii) a polypeptide comprising X2 and excluding X1, X3, and X4; (iii) a polypeptide comprising X3 and excluding X1, X2, and X4; (iv) a polypeptide comprising X4 and excluding X1, X2, and X3; (v) a polypeptide comprising X1 and X2, and excluding X3 and X4; (vi) a polypeptide comprising X1 and X3, and excluding X2 and X4; ^ (vii) a polypeptide comprising X1 and X4, and excluding X2 and X3; (viii) a polypeptide comprising X2 and X3, and excluding X1 and X4; (ix) a polypeptide comprising X2 and X4, and excluding X1 and X3; (x) a polypeptide comprising X3 and X4, and excluding X1 and X2; ^ (xi) a polypeptide comprising X1, X2, and X3 and excluding X4; (xii) a polypeptide comprising X1, X2, and X4 and excluding X3; (xiii) a polypeptide comprising X1, X3, and X4 and excluding X2; and (xiv) a polypeptide comprising X2, X3, and X4 and excluding X1. In other embodiments, X1, X2, X3, and X4, respectively, comprise an amino acid^ sequence that are at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NO:1-598 627-631, 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746- 747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854,^ 856-862, 864-874, 876-898, and 900-931. In a further aspect, the present disclosure provides nucleic acids, including isolated nucleic acids, encoding the polypeptides, fusion proteins, and first and second polypeptides of the present disclosure. The isolated nucleic acid sequence may comprise RNA or DNA. Such isolated nucleic acid sequences may comprise additional sequences useful for ^ promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptides of the invention. ^ In another aspect, the present disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable control sequence. "Expression vector" includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the invention are nucleic acid^ sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered "operably linked" to the coding sequence. ^ Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors include but are not limited to, plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be ^ constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. In various embodiments, the expression vector may comprise a^ plasmid, viral-based vector (including but not limited to a retroviral vector or oncolytic virus), or any other suitable expression vector. In some embodiments, the expression vector can be administered in the methods of the disclosure to express the polypeptides in vivo for therapeutic benefit. In non-limiting embodiments, the expression vectors can be used to transfect or transduce cell therapeutic targets (including but not limited to CAR-T cells or^ tumor cells) to effect the therapeutic methods disclosed herein. In a further aspect, the present disclosure provides host cells that comprise the expression vectors, polypeptides, fusion proteins, first and/or second polypeptides, polypeptide components, conditionally active agonists and antagonists, and/or nucleic acids and expression vectors disclosed herein, wherein the host cells can be either prokaryotic or^ eukaryotic. The cells can be transiently or stably engineered to incorporate the expression vector of the invention, using techniques including but not limited to bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring^ Harbor Laboratory Press); Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R.I. Freshney.1987. Liss, Inc. New York, NY)). A method of producing a polypeptide according to the invention is an additional part of the invention. The method comprises the steps of (a) culturing a host according to this aspect of the invention under conditions conducive to the expression of the polypeptide, and (b) optionally, recovering the expressed^ polypeptide. The expressed polypeptide can be recovered from the cell free extract, but preferably they are recovered from the culture medium. In another aspect, the present disclosure provides pharmaceutical compositions, comprising the polypeptide, fusion protein, first polypeptide, second polypeptide, recombinant nucleic acid, expression vector and/or, recombinant host cell of any embodiment ^ or combination of embodiments herein and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the disclosure can be used, for example, in the methods of the disclosure described herein. The active agents of the disclosure may be the sole active agent in the composition or may be combined with one or more other active agents, including^ but not limited to checkpoint inhibitors or cancer vaccines The pharmaceutical composition may further comprise (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and/or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The pharmaceutical^ composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the ^ pharmaceutical composition includes a bulking agent, like glycine. In yet other embodiments, the pharmaceutical composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The ^ pharmaceutical composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the pharmaceutical composition additionally includes a stabilizer, e.g., a^ molecule which, when combined with a protein of interest substantially prevents or reduces chemical and/or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride. The polypeptide, fusion protein, first polypeptide, second polypeptide, recombinant^ nucleic acid, expression vector and/or, recombinant host cell of any embodiment or combination of embodiments herein may be the sole active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for an intended use. ^ In another aspect, the disclosure provides methods for treating cancer, comprising administering to a subject having cancer the polypeptide agonist or fusion protein, conditionally active receptor agonist, recombinant nucleic acid, expression vector, recombinant host cell, and/or the pharmaceutical composition of any embodiment of IL-21^ agonists herein, in an amount effective to treat the tumor. In various embodiments, the cancer is selected from the group consisting of colon cancer, melanoma, renal cell cancer, head and neck squamous cell cancer, gastric cancer, urothelial carcinoma, Hodgkin lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, pancreatic cancer, Merkel cell carcinoma, colorectal cancer, acute myeloid leukemia, acute^ lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, ovarian cancer, cervical cancer, and any tumor types selected by a diagnostic test, such as microsatellite instability, tumor mutational burden, PD-L1 expression level, or the immunoscore assay (as developed by the Society for Immunotherapy of Cancer). In another aspect, the disclosure provides methods for modulating an immune^ response in a subject comprising the polypeptide antagonist or fusion protein thereof, conditionally active receptor antagonist, recombinant nucleic acid, expression vector, recombinant host cell, and/or the pharmaceutical composition of embodiment of IL-21 antagonist disclosed herein. In one embodiment, the immune response is an anti-cancer immune response. ^ As used herein, "treat" or "treating" means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously^ symptomatic for the disorder(s). The subject may be any subject that has a relevant disorder. In one embodiment, the subject is a mammal, including but not limited to humans, dogs, cats, horses, cattle, etc. Examples ^ Summary Here, we created hyperstable de novo IL-21 mimetic proteins that recapitulate its interactions with receptors and the biology of native IL-21 in humans and mice, which is ideal for therapeutic use. The IL-21 mimetic proteins designed include agonists, antagonists, immunocytokine, or split agonists/antagonists, as detailed in the examples and listed in ^ Tables 1-6. Binding is measured for two receptors by the two setups: (i) hIL-21R only for hIL-21R binding and (ii) h^c with hIL-21R for h^c receptor binding. For the h^c receptor binding, hIL-21R is needed for binding testing because the affinity for h^c alone is very weak and thus, reasonable h^c binding measurement can only be done when hIL-21R is present.^ For (i) hIL-21R binding, the measurement is done in flow cytometry-based yeast surface display or biolayer interferometry (BLI) as mentioned in Methods section. For yeast surface display method, 1-1000 nM of hIL-21R labeled with SA-PE is used along with anti- Myc FITC antibody to label the cells that express the IL-21 mimics that binds to the hIL-21R. The proteins that bind to hIL-21R show the PE+FITC+ population equal or more than 0.5%^ of the singlet-gated population, when tested with 1000 nM of hIL-21R. For BLI method, hIL- 21R is loaded to the analysis tip and tested for association with IL-21 mimics provided at 100 pM-1000 nM range. In addition, alternatively, polyhistidine-tagged IL-21 mimics can be loaded to the analysis tip and measured for their association to 100-1000 nM of hIL-21R. The proteins that bind to hIL-21R show at least 0.1nm shift during the association, in either of the^ cases. The provided yeast surface display data shows the results with 100 nM of PE-labeled hIL-21R. The provided BLI data shows hIL-21R-loaded analysis method with the aforementioned range for appropriate Kd calculations, and polyhistidine-tagged protein- loaded analysis with 100nM of hIL-21R for binding testing. In both yeast surface display and^ BLI cases, mouse cross-reactivity by testing binding to mIL-21R works the same as hIL-21R binding assay and it showed similar results to that of human receptor. For (ii) h^c receptor binding, the measurement is done in flow cytometry-based yeast surface display or biolayer interferometry (BLI) as mentioned in Methods section. For yeast surface display method, 1-1000 nM of unlabeled hIL-21R and 1-1000 nM of SA-PE-labeled^ h^c receptor are used along with anti-Myc FITC antibody to label the cells that express the IL-21 mimics that binds to h^c receptor in the presence of hIL-21R. The proteins that bind to h^c receptor in the presence of hIL-21R show the PE+FITC+ population equal or more than 0.5% of the singlet-gated population, when tested with 1000 nM of hIL-21R and 1000 nM of h^c receptor. For BLI method, h^c receptor is loaded to the analysis tip and tested for^ association with IL-21 mimics provided at 100 pM-1000 nM range, in the presence of hIL- 21R at 1.5-fold higher concentration compared to that of the IL-21 mimics. The proteins that bind to h^c receptor and hIL-21R show at least 0.1nm shift during the association. ^ The provided yeast surface display data shows the results with 1000 nM of unlabeled hIL-21R and 200 nM PE-labeled h^c receptor, or 1000 nM of unlabeled hIL-21R and 1000 nM PE-labeled h^c receptor. The provided BLI data shows h^c receptor-loaded analysis method with the aforementioned range for appropriate Kd calculations. In both yeast surface^ display and BLI cases, mouse cross-reactivity by testing binding to m^c receptor and mIL- 21R works the same as h^c receptor binding assay and it showed similar results to that of human receptor. Agonistic mimics of IL-21 are designed to have two interfaces, one for IL-21R and the other for CD360. These designed agonists can bind to both receptors at the same time to^ heterodimerize the two receptor subunits. Furthermore, the designs are cross-reactive to human and murine receptors. Antagonistic mimics of IL-21 are designed only to retain the binding capability to IL-21R but not ^c so that it can occupy the same space as an agonist yet interfere ^c from associating for heterodimerization of the receptor subunits. Similarly, these designs retain human and mouse cross-reactivity. Immunocytokines of IL-21 mimics,^ antibody-fused IL-21 mimics, are examples of molecular fusions for targeted cytokine delivery. Split IL-21 mimics are designed to achieve targeted delivery of IL-21 mimic with minimal off-target cytokine activity. Design of initial hits and optimization ^ Based on the human IL-21(hIL-21)/human IL-21R(hIL-21R) complex structure, we generated scaffolds that mimic the helical bundle structure of hIL-21 with improved packing of secondary structures to enhance stability. We optimized the helices by adjusting their lengths in the helical bundle scaffolds, minimizing unstructured regions, and eliminating non- ideal long loops in native hIL-21. Wild-type hIL-21 consists of four helices, with helix A (the^ first helix from the N-terminus) and helix C (the third helix from the N-terminus) consisting of an interface to hIL-21R, and helix D (the fourth helix from the N-terminus) forming an interface to h^c. The upper two helices B (the second helix from the N-terminus) and C are not long enough to form ideal intramolecular interactions and intermolecular contacts with the receptor chains. To optimize the helices B and C, we extended them to substitute most of^ the unstructured regions, including two long unstructured regions between helix pairs – helix A/helix B and helix C/helix D, and adjusted the other two helices, helices A and D, for improved packing. To design the interface of the de novo scaffolds, we grafted the interface residues of hIL-21 to the structurally matching positions of the scaffolds and designed the rest ^ of the residues in the context of hIL-21R using RosettaTM. The designed proteins were evaluated and filtered using RosettaTM score metrics and their folding trajectories using Fast Forward Folding (Fig.1A). Utilizing yeast surface display as a screening platform, we identified five hits that can^ bind hIL-21R; however, none showed binding to h^c. To further develop the binding capabilities of our best hit, 21d26 (Table 5, SEQ ID NO:627), to h^c, we used directed evolution and isolated mutants that displayed h^c binding in an hIL-21R-dependent manner. Among these mutations (Table 1, SEQ ID NO:1-8), we isolated a variant, 21JC15, with four mutations on 21d26, W15C, W49R, Q73H, and H99G (W15C is residue 15 in the X1 domain^ (SEQ ID NO:936), W49R is residue 21 in the X2 domain (SEQ ID NO:933), Q73H is residue 16 in the X3 domain, agonist version (SEQ ID NO:934), and H99G is residue 16 in the X4 domain (SEQ ID NO:935)). This variant demonstrated binding to not only hIL-21R and hIL- 21R/h^c, but also mIL-21R and mIL-21R/m^c, indicating cross-reactivity between human and murine receptors. Furthermore, we employed site-saturation mutagenesis (SSM) to mutate^ interface residues and evaluated their impact on affinity individually (Fig.5; Table 1, SEQ ID NO:9-36). Agonists Through this analysis and combining these mutations, we isolated 21h10 (Table 1,^ SEQ ID NO: 24; Fig.1B, 1C), an optimized mutant, that exhibited significantly higher affinity than the parent, 21JC15 (Table 1, SEQ ID NO:8), while retaining cross-reactivity between human and murine receptors (Fig.1G-1J), along with other variants – 21m1 to 21m6 and 21h1 to 21h9 (Table 1, SEQ ID NO:9-23). Particularly, 21h10 showed the best cross- reactivity and interestingly exhibited only 44.9% sequence identity to hIL-21 and 23.5% to^ mIL-21, as determined by BLASTP analysis.21h10 was efficiently expressed in Escherichia coli and displayed monodispersity in size-exclusion chromatography (Fig.1D). Additionally, circular dichroism analysis confirmed the presence of helical secondary structures (Fig.1E) in the protein and demonstrated its superior thermal stability (Fig.1F). The agonists were diversified by redesigning non-interface residues and some of the^ interface residues using ProteinMPNN to allow possible substitutions on such positions, with experimental validation (Table 1, SEQ ID NO:37-163). Furthermore, affinity variants are generated based on 21h10 (Table 1, SEQ ID NO:24) by attenuating the affinity of both interfaces using SSM data (Table 1, SEQ ID NO:164-263; Fig.6) and ProteinMPNN (Table ^ 1, SEQ ID NO:264 -432). These affinity variants may achieve alterations in orientation and half-life of receptor complex which can affect EC50, Emax, signaling pathway biases, or further downstream differentiating phenotype from the native IL-21 signaling. This confirms that agonistic IL-21 mimics are stable to permit interface engineering to achieve various^ affinities on both interfaces. Native-like, high affinity variants are useful to recapitulate full activity of native IL-21 biology with its full human and mouse cross-reactivity, such as to use these variants for surrogates of native IL-21. Lower affinity variants may have less potency than the native IL-21 or have function distinctive from the native IL-21, however it is useful when antibody or other targeting domain fused IL-21 mimic constructs needs biodistribution^ that is directed by the fused targeting domain’s target-binding affinity rather than the affinity between the fused IL-21 mimic against the IL-21 receptor. In addition, the lower affinity variants may have higher EC50 or lower Emax in cell signaling which might be the desired function to use. SEQ ID NO:1-8 in Table 1 are the agonistic mimics derived from 21d26 (Table 5,^ SEQ ID NO:627) with combinations of mutations – W15C, W49R, Q73H, and H99G – that was applied with all four mutations to generate 21JC15 (Table 1, SEQ ID NO:8). SEQ ID NO: 9-24 in Table 1 are the agonistic mimics that are optimized from 21JC15 (Table 1, SEQ ID NO:8) with positive mutations found in site saturation mutagenesis (SSM) data. SEQ ID NO: 25-36 in Table 1 are the agonistic mimics with combinations of mutations that are^ applied to 21h10 (Table 1, SEQ ID NO: 24) from 21JC15 (Table 1, SEQ ID NO:8). SEQ ID NO:37-163 in Table 1 are the agonistic mimics that are derived from 21h10 (Table 1, SEQ ID NO: 24) with mutations applied by ProteinMPNN to the residues that are not part of interface residues contributing to IL-21R (CD360) or ^c (CD132) receptor binding.SEQ ID NO: 164- 432 in Table 1 are the agonistic affinity variant mimics that are derived from 21h10 (Table 1,^ SEQ ID NO: 24) with mutations applied by site saturation mutagenesis (SSM) data or ProteinMPNN to the residues that are part of interface residues contributing to IL-21R (CD360) or ^c (CD132) receptor binding, to acquire attenuation in interface binding affinity. In Table 1, Kd1 is Kd against hIL-21R which indicates the binding affinity against hIL-21R and Kd2 is Kd against the IL-21 receptor complex (hIL-21R and h^c receptor)^ which indicates the binding affinity against h^c receptor, both measured by BLI. Q3 is percentage of the PE+FITC+ population relative to the singlet-gated population at 1000 nM unlabeled hIL-21R and 1000 nM PE-labeled h^c receptor, which represents the binding to h^c receptor in the presence of hIL-21R, measured by flow cytometry. Only the data with the ^ aforementioned concentrations are presented in the Table 5, rather than all from 1-1000 nM range is because the concentration used for the presented data allows the best measurement of the affinities than lower or higher concentrations within the range. Higher up to 1000 nM concentration is best for identification of binder hits, whereas lower down to 1nM is best for^ finding the most affinity-optimized variants among the mimics. Mouse receptor binding assay works the same as human receptor binding assay and its result was similar to that of human receptor. Retopology/shuffle and split cytokines ^ The designed agonists can be shuffled with their helical domains (X1, X2, X3, and X4) yet maintain their ability to heterodimerize the receptor subunits. They retain most of the binding affinity on both interfaces, which is necessary to be agonists (Fig.2A). X2-X3-X4- X1, X3-X4-X1-X2, and X4-X1-X2-X3 were demonstrated (Table 2) for their engineerability in maintaining their given molecular structure while retaining both interfaces for IL-21R^^ (CD360) and ^c (CD132) receptor for their agonistic function. When the helical domains are shuffled and reconnected, the polypeptide linker is either computationally designed linker using Rosetta and ProteinMPNN, or one of (GS)n or (GGS)n linkers with n=2-10. Helical domain-shuffled designs bind to IL-21 receptor and this implies that the helical domains are hyperstable to be shuffled into any order beyond the suggested three orders and retain its^ interfaces to the IL-21 receptors, with new connectivities between the helices (retopology). Sequences of helical domain shuffled constructs are listed in Table 2. SEQ ID NO:433-445 in Table 2 are the agonistic mimics with shuffled helical domains in the order of X2-X3-X4-X1. Similarly, SEQ ID NO:480-569 in Table 2 are the agonistic mimics with shuffled helical domains in the order of X2-X3-X4-X1 but with mutations on X1. SEQ ID^ NO:446-457 in Table 2 are the agonistic mimics with shuffled helical domains in the order of X3-X4-X1-X2. SEQ ID NO: 458-479 in Table 2 are the agonistic mimics with shuffled helical domains in the order of X4-X1-X2-X3. Linkers in between the helical domains for reconnection are generated using RosettaTM or ProteinMPNN, or one of (GS)n or (GGS)n linkers with n=2-10. ^ This molecular stability correlates to engineerability of the molecule and can be extended to split cytokine mimics for conditional cytokine. IL-21 mimics being four helical bundle proteins, there are three different ways to divide the helices into two linear groups (fragment X and fragment Y) – H1/H234, H12/H34, and H123/H4 (Fig.2B; Table 4), and this can even be diversified with shuffle of helical domains (Table 2). The use of these split ^ IL-21 mimics for targeted delivery is to fuse each of the split fragments X and Y to targeting domain, such as nanobody that can direct the split cytokine mimics to desired cells or tissues of interest (Fig.2C). Here, we demonstrate examples of split cytokines being fused to anti- HER2-DARPin or anti-EGFR-DARPin (Table 4, SEQ ID NO:613-624) to demonstrate that^ these IL-21 mimics can be fused to such targeting domain for use and can be reconstituted in vitro using biolayer interferometry as described in the Methods section of the Examples, (Fig. 2D; Table 4). This confirms that split IL-21 mimic fragments can be fused to any available targeting domain. Sequences of split fragments of IL-21 mimics and their fusion constructs are listed in^ Table 4. SEQ ID NO: 607-612 in Table 4 are the split fragments generated from 21h10 (Table 1, SEQ ID NO: 24), as X1 (Table 4, SEQ ID NO: 607), X1-X2 (Table 4, SEQ ID NO: 608), X3-X4 (Table 4, SEQ ID NO: 609), X4 (Table 4, SEQ ID NO: 610), X1-X2-X3 (Table 4, SEQ ID NO: 611), and X2-X3-X4 (Table 4, SEQ ID NO: 612). SEQ ID NO: 613-618 in Table 4 are the split fragments (Table 4, SEQ ID NO: 607-612) fused to anti-HER2-DARPin^ (Table 4, SEQ ID NO: 625). SEQ ID NO: 619-624 in Table 4 are the split fragments (Table 4, SEQ ID NO: 607-612) fused to anti-EGFR-DARPin (Table 4, SEQ ID NO: 626). Linkers in between the split fragments of IL-21 mimics and targeting domains are one of (GS)n or (GGS)n linkers with n=2-10. In Table 2, Kd1 is Kd against hIL-21R which indicates the binding affinity against^ hIL-21R which is dissected into kon1 and kdis1, all measured by BLI. kon1 is the association rate against hIL-21R and kdis1 is the dissociation rate against hIL-21R. In Table 4, Kd2 is Kd against the IL-21 receptor complex (hIL-21R and h^c receptor) which indicates the binding affinity against h^c receptor, measured by BLI. Antibody fusions ^ For targeting specific cells or tissues of interest, we demonstrated various antibody- fused IL-21 mimic constructs (Table 3). IL-21 mimics were fused to the heavy chain of antibodies. For cis-acting, such as targeting effector cells like CD8+ T cells, there are anti- human-PD-1-targeting antibody (Pembrolizumab) fusions (Table 3, SEQ ID NO:572-576), anti-murine-PD-1-targeting antibody fusions (Table 3, SEQ ID NO:584-588), anti-human-^ CD8-targeting antibody (RED8) fusions (Table 3, SEQ ID NO:594-598), and anti-murine- CD8-targeting antibody (G10.1) fusions (Table 3, SEQ ID NO:589-593). For trans-acting, such as targeting tumor tissue, there are anti-PD-L1-targeting antibody (Atezolizumab) fusions (Table 3, SEQ ID NO:577-581) and anti-TRP1-targeting antibody (TA99) fusions (Table 3, SEQ ID NO:582-583). One of the examples, 21h10-fused Atezolizumab, can be ^ either configured as homodimeric form or heterodimeric form using Knobs/Holes mutation in the heavy chain of the antibody (Fig.3A) and shows expected size in SDS-PAGE (Fig.3B). Atezolizumab-21h10 constructs can bind to each of their domains target, human IL-21R for 21h10 and human PD-L1 for Atezolizumab (Fig.3C). Also, this construct can bind to both^ target simultaneously (Fig.3D). Other than targeting antibodies, we also have made Fc- fusions for the purpose of half-life extension (Table 3, SEQ ID NO:570-571). This proves that agonistic IL-21 mimics can be fused to any available antibodies or other targeting molecules to redirect their biodistribution. SEQ ID NO:570-571 in Table 3 are Fc fragment fusion constructs in which, an^ agonistic mimic, 21AG-M-p3C1 (Table 1, SEQ ID NO:161), is fused to either human Fc fragment (Table 3, SEQ ID NO:599) or murine Fc fragment (Table 3, SEQ ID NO:600), respectively. Linkers in between the IL-21 mimic and Fc fragment are one of (GS)n or (GGS)n linkers with n=2-10. SEQ ID NO:572-576, 601 in Table 3 are the components used to make fusion constructs of IL-21 mimic with Pembrolizumab. Two identical copies of the^ light chain of Pembrolizumab (Table 3, SEQ ID NO:572) are paired with two identical copies or two different copies of heavy chain of Pembrolizumab (Table 3, SEQ ID NO: 573-576, 601). SEQ ID NO:577-581, 602 in Table 3 are the components used to make fusion constructs of IL-21 mimic with Atezolizumab. Two identical copies of the light chain of Atezolizumab (Table 3, SEQ ID NO:577) are paired with two identical copies or two^ different copies of heavy chain of Atezolizumab (Table 3, SEQ ID NO: 578-581, 602). SEQ ID NO:582-583, 603 in Table 3 are the components used to make fusion constructs of IL-21 mimic with TA99. Two identical copies of the light chain of TA99 (Table 3, SEQ ID NO:582) are paired with two identical copies or two different copies of heavy chain of TA99 (Table 3, SEQ ID NO: 583, 603). SEQ ID NO:584-588, 604 in Table 3 are the components^ used to make fusion constructs of IL-21 mimic with an anti-murine PD-1 monoclonal antibody. Two identical copies of the light chain of the anti-murine PD-1 monoclonal antibody (Table 3, SEQ ID NO:584) are paired with two identical copies or two different copies of heavy chain of the anti-murine PD-1 monoclonal antibody (Table 3, SEQ ID NO: 585-588, 604). SEQ ID NO:589-593, 605 in Table 3 are the components used to make fusion^ constructs of IL-21 mimic with an anti-murine CD8 monoclonal antibody, G10.1. Two identical copies of the light chain of G10.1 (Table 3, SEQ ID NO:589) are paired with two identical copies or two different copies of heavy chain of G10.1 (Table 3, SEQ ID NO:590- 593, 605). SEQ ID NO: 594-598, 606 in Table 3 are the components used to make fusion constructs of IL-21 mimic with an anti-human CD8 monoclonal antibody, RED8. Two ^ identical copies of the light chain of RED8 (Table 3, SEQ ID NO:594) are paired with two identical copies or two different copies of heavy chain of RED8 (Table 3, SEQ ID NO: 595- 598, 606). In Table 3, Kd is measured against hIL-21R and the antibody’s target, human PD-L1^ in the case of Atezolizumab fusions. An Atezolizumab-IL-21 mimic fusion construct is tested for hIL-21R binding and hPD-L1 binding by BLI. For hIL-21R binding assay, Atezolizumab- 21h10 fusion construct (Table 3, SEQ ID NO:577, 578) is tested by immobilizing hIL-21R on SA tip and associated and dissociated in 0.313-20 nM of the fusion construct (Fig.3C). For hPD-L1 binding assay, Atezolizumab-21h10 fusion construct (Table 3, SEQ ID NO: 577,^ 578) is tested by immobilizing hPD-L1 on SA tip and associated and dissociated in 0.027-20 nM of the fusion construct (Fig.3C). For its capacity to bind both hIL-21R and hPD-L1 at the same time, in the first experiment, hPD-L1 is immobilized to the analysis tip and associated it with Atezolizumab-21h10 fusion construct (Table 3, SEQ ID NO: 577, 578) then hIL-21R; in the second experiment, hIL-21R is immobilized to the analysis tip and associated it with^ Atezolizumab-21h10 fusion construct (Table 3, SEQ ID NO: 577, 578) then hPD-L1. Antagonists Antagonistic IL-21 mimics are the designs that only bind to IL-21R, but not to ^c. IL- 21R binding was assed by flow cytometry using 100-1000nM of human or mouse IL-21R labeled with Streptavidin R-Phycoerythrin Conjugate (SAPE), and/or biolayer interferometry^ using human or mouse IL-21R with Avi tag loaded on Streptavidin (SA) tip. Gc binding was assessed by flow cytometry using 10-1000nM of human or mouse IL- 21R without label and 10-1000nM of human or mouse Gc labeled with Streptavidin R- Phycoerythrin Conjugate (SAPE), and/or biolayer interferometry using human or mouse Gc with Avi tag loaded on Streptavidin (SA) tip. Soluble non-labeled human or mouse IL-21R is^ provided in IL-21R:IL-21 mimic=1.5:1 for testing. Generating antagonists from agonist designs also implies superior molecular stability of the IL-21 mimics, as knocking out the ^c-interface without perturbing overall molecular integrity or the other interface (IL-21R interface) is often not permissible for native IL-21 to withhold. There are four types of mimics based on their origin or design method that are^ experimentally tested – (i) initial antagonist hits (Table 5, SEQ ID NO:627-631, Fig.4A) and mimics (Fig.4B) originated from agonist 21h10 (Table 1, SEQ ID NO:24) using (ii) RosettaTM (Table 5, SEQ ID NO: 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, ), (iii) SSM data (Table 5, 687688-695, 698-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746-747, 749-751 ), and (iv) ProteinMPNN ^ (Table 5, 798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898, and 900-907). Resulting antagonists can bind to IL-21R and are able to partially or fully not engage with ^c for IL-21 receptor complex formation. This proves that IL-21 mimics are stable to permit interface mutations to achieve antagonistic^ properties. SEQ ID NO:627-631 in Table 5 are the five antagonist hits found during initial screening of IL-21 mimic designs. SEQ ID NO:632-687 (named 21AT-R-p###) in Table 5 are mimics derived from 21h10 (Table 1, SEQ ID NO: 24) that are mutated on ^c-interface residues by RosettaTM. SEQ ID NO:688- 751 (named 21AT-S-p###) in Table 5 are mimics^ derived from 21h10 (Table 1, SEQ ID NO: 24) that are mutated on ^c-interface residues based on site saturation mutagenesis (SSM) data. SEQ ID NO:752- 907 (named 21AT-M- p###) in Table 5 are mimics derived from 21h10 (Table 1, SEQ ID NO: 24) that are mutated on ^c-interface residues by ProteinMPNN. In Table 5, Q1 is percentage of the PE+FITC+ population relative to the singlet-gated^ population at 100 nM PE-labeled hIL-21R, which represents the binding to hIL-21R, measured by flow cytometry. Q2 is percentage of the PE+FITC+ population relative to the singlet-gated population at 1000 nM unlabeled hIL-21R and 200 nM PE-labeled h^c receptor, which represents the binding to h^c receptor in the presence of hIL-21R, measured by flow cytometry. Only the data with the aforementioned concentrations are presented in the Table^ 5, rather than all from 1-1000 nM range is because the concentration used for the presented data allows the best measurement of the affinities than lower or higher concentrations within the range. Higher up to 1000 nM concentration is best for identification of binder hits, whereas lower down to 1nM is best for finding the most affinity-optimized variants among the mimics. Mouse receptor binding assay works the same as human receptor binding assay^ and its result was similar to that of human receptor. Others Genetic fusions or chemical conjugation of functional domains for half-life extension of protein or other applications are often explored with protein-based therapeutics. We designed and tested albumin-binding domain fusion constructs with the purpose of half-life^ extension (Table 6, SEQ ID NO:908-909). We also have designed a number of Cys- and/or Lys-relocated variants (Table 6, SEQ ID NO:910-931) from 21h10 (Table 1, SEQ ID NO:24) or 21AG-M-p3C1 (Table 1, SEQ ID NO:161). These variants can be chemically conjugated to PEG to achieve half-life extension or to other functional domains to acquire conditional ^ activation by having chemical conjugation sites within the interfaces of IL-21 mimics.21h10 (Table 1, SEQ ID NO:24) with Cys15 was able to be chemically conjugated to PEG with various sizes to achieve increase in molecular size. This confirms that IL-21 mimics can be fused to other functional domains, along with antibodies or aforementioned targeting^ domains, by genetic fusions or chemical conjugation to achieve their half-life extension or other functional benefits. SEQ ID NO:908-909 in Table 6 are albumin-binding peptide-fusion constructs in which 21AG-M-p3C1 (Table 1, SEQ ID NO:161) fused to an albumin-binding peptide, G148ABD3 (Table 6, SEQ ID NO:932), being designed for the purpose of half-life ^ extension. As G148ABD3 domain binds albumin in vivo, it increases the size of the overall molecular weight which can increase half-life of the constructs. SEQ ID NO:910-931 in Table 6 are agonistic mimics derived from 21h10 (Table 1, SEQ ID NO:24) or 21AG-M- p3C1 (Table 1, SEQ ID NO:161) which have added or removed Lys (K) or Cys (C) on their non-interface residues (Table 6, SEQ ID NO:910-925) or interface residues (Table 6, SEQ ID^ NO:926-931) to chemically conjugate functional domains, including but not limited to PEG, peptide, and small molecules, for various purposes, such as half-life extension and conditional activation. Methods ^ Computational design of de novo IL-21 mimics The crystal structure of hIL-21 with hIL-21R (PDB: 3TGX) was used to design the mimics of native IL-21. The PyRosettaTM script with PDBInfoLabel metadata implementation was used to generate IL-21-like scaffolds. The following residues from the human IL-21 are designated to be fixed during scaffold generation and interface residue^ design: R2, I5, R6, R8, Q9, L10, I11, D12, I13, D15, Q16, K18, Y20, R62, I63, V66, S67, K69, K70, R73, K74, P75, P76, S77, K98, E99, E102, R103, K105, S106, Q109, K110, H113, and L116. The non-fixed residues are designed using RosettaTM FastDesign and relaxed using RosettaTM FastRelax with ‘beta_nov16’ score function. The designs were filtered with RosettaTM score metrics: packstat>0.6, score_per_residue<-2.3, and sspred>0.8.^ Fast forward folding was used on the designs to filter them based on their probability of folding. Yeast surface display and fluorescent-activated cell sorting The amino acid sequences of the designed proteins were reverse-translated to DNA sequences based on the codon frequency table of Saccharomyces cerevisiae, along with ^ pETcon3TM vector-homologous sequences appended to both 5’ and 3’ termini. Using competent yeast cells, EBY100, the DNA gene blocks of the designs were cloned into linearized pETcon3TM vectors by yeast homologous recombination. The vector was linearized by 100-fold overdigestion by NdeI and XhoI (New England Biolabs) and then purified by gel^ extraction (Qiagen). Yeast transformations are validated by colony sequencing; validated colonies are grown in SDCAA media (2.0% Glucose, 0.67% Yeast nitrogen base, 0.5% Casamino acids, 0.54% Di-sodium phosphate, 0.86% MonoSodium phosphate) and induced in SGCAA media (2% Galactose, 0.67% Yeast Nitrogen Base, 0.5% Casamino Acids, 0.54% Na2HPO4, 0.86% NaH2PO4). SGCAA induced the designed proteins to be expressed and^ presented on the yeast surface through Aga2p membrane proteins. The induced cells are resuspended in the running buffer (Phosphate-buffered saline (PBS)+1% w/v Bovine Serum Albumin, pH 7.2-7.4) for all flow cytometry analyses. The induced cells with the designed proteins on their surfaces were labeled with two fluorophores - FITC and PE. Anti-c-myc mAb conjugated to FITC was used to label the myc tag to represent the degree of protein^ expression. For IL-21R labeling, either human or murine IL-21R with human Fc-tag (R&D Systems 991-R2, R&D Systems 596-MR) were used. For receptor complex labeling, either human IL-21R with His-tag (R&D Systems 9249-R2) and human ^c with Fc-tag (Acro Biosystems ILG-H5256), or murine IL-21R with His-tag (Sino Biological 51184-M08H) and murine ^c with Fc-tag (R&D Systems 784-MR) were used. The target receptor fused with the^ Fc domain is labeled by the biotinylated ZZ domain of protein A and streptavidin-PE (SA- PE), which can bind to the biotin of protein A. PE represents the level of receptor binding. For binding hits, their signal is proportional to the expression level of the protein. Yeast was analyzed by flow cytometers (BD Accuri C6, Thermo Fisher Attune NxT) or sorted by a Fluorescence-Activated Cell Sorting (FACS) cell sorter (Sony SH800). ^ Directed evolution and design optimization Using a random mutagenesis library kit (Agilent 200550), the 21d26 DNA was amplified, and the library was partially sequenced using BL21(DE3) (New England Biolabs). The sequencing showed that approximately 2-4 mutations were applied to the design while the DNA was amplified with the error-prone PCR. Next, the library was transformed to yeast^ using electroporation with an approximated diversity of 3.4E7. The yeast library was sorted for direct evolution using FACS with different combinations and concentrations of labeled receptor subunits, with the abovementioned labeling. To analyze and enhance the interface affinities of the interfaces of the 21JC15, we used Site-saturation mutagenesis (SSM) to analyze the two interfaces of the protein in detail. ^ The protein sequence of interest was split into halves with corresponding single mutations and synthesized as two fragments for assembly (Integrated DNA Technologies). Assembly was done with PCR with pETcon3 TM-specific 5’ and 3’ primers. Sortings with four different receptor conditions ((1) labeled human IL-21R, (2) unlabeled human IL-21R with labeled^ human (3) labeled murine IL-21R, and (4) unlabeled murine IL-21R with labeled murine ^c) saturated mutants with mutants with favorable mutations, and depleted mutants with unfavorable mutations. DNA of the sorted SSM library was prepared before MiSeq TM (Illumina) using Zymoprep TM (Zymo Research), qPCR, and gel extraction (Qiagen). The sorted SSM library revealed several mutations that can improve binding affinities to each^ receptor - human IL-21R, human ^c, murine IL-21R, and murine ^c. By merging the positive mutations, we built a combinatorial library that incorporated the mutations by using a PCR-based assembly of pools of 8 sets of primers (Integrated DNA Technologies) incorporating mutations with degenerate codons, which was then sorted against human or murine receptors. It yielded 16 candidates with significantly improved^ affinities to the receptors, 10 human receptor-optimized candidates (21h1–21h10) and 6 murine receptor-optimized candidates (21m1–21m6). The Kd of 16 candidates toward each of the receptors was measured by biolayer interferometry (ForteBio, See Methods). Cytokine mimics and split cytokine mimics protein expression (E. coli expression) The DNA fragments (Integrated DNA Technologies) that encode the designed^ proteins were cloned into pET-29b(+) plasmid with an N-terminal polyhistidine tag. The cloned plasmids were transformed into competent BL21 cells (New England Biolabs), whose cultures were grown in Terrific Broth II and induced using 1 mM isopropyl b-D- thiogalactopyranoside (IPTG). The cultures were grown in baffled flasks at 37C within 225 rpm shakers until they reached approximately ODA600=0.8 for induction. The cultures were^ induced for 14 hours at 18C. The harvested cultures were either lysed by sonication (Qsonica TM Q500). The lysed cultures were ultracentrifuged at 18,000g for 30 minutes and purified using immobilized metal affinity chromatography (IMAC). The elution proteins were separated by HPLC size-exclusion chromatography using Superdex TM 7510/300 GL column (GE Healthcare) on Akta TM (GE Healthcare AKTA TM Pure). ^ Immunocytokine (antibody-cytokine fusion) protein expression (Mammalian expression) The antibody-cytokine fusion constructs are synthesized in CMVR plasmid (Genscript). Cytokine mimics are fused to C-terminal end of heavy chain of antibodies with linkers. Using HEK293 cells, the fusion constructs are expressed and purified by Protein A ^ capture (Genscript). SDS-PAGE and SEC-HPLC was utilized for purification. Biolayer interferometry Binding data were collected with an OctetRED96 and OctetR8 (Sartorius). Octet binding buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)+1% w/v^ Bovine Serum Albumin, pH 7.2-7.4) was used for all biolayer interferometry analyses. To measure the binding affinity to hIL-21R or mIL-21R, biotinylated hIL-21R (R&D Systems AVI9249) or biotinylated mIL-21R (Acro Biosystems ILR-M82E3), respectively, were immobilized on streptavidin-coated biosensors (SAForteBio) at 5 ^g/ml in the octet binding buffer to 0.8-1.0nm shift for loading, with 2-fold serial dilutions of the ligand starting at 1^ uM, with 300 seconds for association and another 300 seconds for dissociation. Similarly, to measure the binding affinity to hIL-21Ra/h^c or mIL-21Ra/m^c, biotinylated h^c (Acro Biosystems ILG-H85E8) or biotinylated m^c (Acro Biosystems ILA-M82E3), respectively, were immobilized on streptavidin coated biosensors (SAForteBio) at 5 ^g/ml in the octet binding buffer and tested for 250-second association and 250-second dissociation in the^ presence of 1.5-fold molar excess of corresponding hIL-21Ra (R&D Systems 9249-R2) or mIL-21Ra (Sino Biological 51184-M08H). For polyhistidine-tagged IL-21 mimics loading, anti-penta-his (His1K) tips are used for immobilization and loaded to 1.0-1.5nm shift. For ligands, hIL-21 (R&D Systems 8879-IL), mIL-21 (R&D Systems 594-ML), or IL-21 mimics (See Methods) were used. Data was processed using ForteBio Data Analysis Software^ version 9.0.0.10.and the parameters were reported with standard error. Circular dichroism and thermal stability assay Far-ultraviolet circular dichroism measurements were executed with a spectropolarimeter (JASCO J-1500 Spectropolarimeter). The proteins were dissolved in PBS (pH 7.4) within 1 mm path length cuvettes at 0.5 mg/mL. The wavelength scan was ^ performed within the 195 nm to 260 nm wavelength range. The thermal melt experiment was performed by heating samples from 25°C to 95°C and cooled back down to 25°C while observing absorption at 222 nm. Reference ^ 1. Hashmi, M. H. & Van Veldhuizen, P. J. Interleukin-21: updated review of Phase I and II clinical trials in metastatic renal cell carcinoma, metastatic melanoma and relapsed/refractory indolent non-Hodgkin’s lymphoma. Expert Opin. Biol. Ther.10, 807–817 (2010). 2. Vasu, S. et al. A Phase I Clinical Trial Testing the Safety of IL-21-Expanded, Off-the- ^ Shelf, Third-Party Natural Killer Cells for Relapsed/Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome. Blood 136, 44–44 (2020). 3. Petrella, T. M. et al. Interleukin-21 has activity in patients with metastatic melanoma: a phase II study. J. Clin. Oncol.30, 3396–3401 (2012). ^ 4. Petrella, T. M. et al. Final efficacy results of NCIC CTG IND.202: A randomized phase II study of recombinant interleukin-21 (rIL21) in patients with recurrent or metastatic melanoma (MM). J. Clin. Oncol. (2013) doi:10.1200/jco.2013.31.15_suppl.9032. 5. Coquet, J. M., Skak, K., Davis, I. D., Smyth, M. J. & Godfrey, D. I. IL-21 Modulates Activation of NKT Cells in Patients with Stage IV Malignant Melanoma. Clin Transl^ Immunology 2, e6 (2013). 6. Bhatia, S. et al. Recombinant interleukin-21 plus sorafenib for metastatic renal cell carcinoma: a phase 1/2 study. J Immunother Cancer 2, 2 (2014). 7. Hamming, O. J. et al. Crystal structure of interleukin-21 receptor (IL-21R) bound to IL- 21 reveals that sugar chain interacting with WSXWS motif is integral part of IL-21R. J.^ Biol. Chem.287, 9454–9460 (2012). 8. Silva, D.-A. et al. De novo design of potent and selective mimics of IL-2 and IL-15. Nature 565, 186–191 (2019). 9. Stauber, D. J., Debler, E. W., Horton, P. A., Smith, K. A. & Wilson, I. A. Crystal structure of the IL-2 signaling complex: paradigm for a heterotrimeric cytokine receptor.^ Proc. Natl. Acad. Sci. U. S. A.103, 2788–2793 (2006). 10. Abhiraman, G. C. et al. A structural blueprint for interleukin-21 signal modulation. Cell Rep.42, 112657 (2023). 11. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol.215, 403–410 (1990). ^ 12. Dauparas, J. et al. Robust deep learning-based protein sequence design using ProteinMPNN. Science 378, 49–56 (2022). 13. Quijano-Rubio, A. et al. A split, conditionally active mimetic of IL-2 reduces the toxicity of systemic cytokine therapy. Nat. Biotechnol.41, 532–540 (2023). ^ ^ ^ ^ ^ ^ ^ Table^1.^Agonists^ *Kd1=Kd(hIL21R) *Kd2=Kd(hIL21R/hGc) *Q3=Q2(1uM_hIL21R/1uM_hGc) ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ Table^2.^^Retopology/shuffle^ * kon1=kon(hIL21R) * kdis1=kdis(hIL21R) * Kd1=Kd(hIL21R) ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ Table^3.^Antibody^fusion^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ Table^4.^^Split^ *Kd2=Kd(hIL21R/hGc) ^ ^ ^ ^ ^ ^ ^ ^ Table^5.^^Antagonists^and^other^designs^ *Q1=100nM_hIL21R *Q2=1uM_hIL21R/200nM_hGc^ 109^ ^ ^ 111^ ^ ^ 113^ ^ ^ 115^ ^ 116^ ^ ^ ^ ^ ^ ^ Table^6.^Others^ ^ ^ ^ ^ ^ ^ ^

Claims

We claim: 1. A polypeptide comprising domains X2, X3, and optionally X4, wherein: (a) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%,^ 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); (b) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of (i) KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934), or (ii) ^ KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937), wherein relative to SEQ ID NO: 937, 1, 2, or all 3 of the following are true: residue 4 is Y, residue 6 is F, and/or residue 9 is D; and (c) X4 is optional and, when present, comprises a helical structure; wherein X2, X3, and X4, when present, may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; and^ wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21R^^ CD360). 2. The polypeptide of claim 1, wherein the polypeptide comprises domains X2, X3, and X4, wherein: ^ (a) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); (b) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence^ KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934); and (c) X4 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence KEVMERAKSAAQKILGRFL (SEQ ID NO: 935); wherein X2, X3, and X4 may be in any order in the polypeptide; ^ wherein amino acid linkers may be present between any of the domains; and wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21R^^ CD360) and is capable of binding to the ^c, CD132 receptor. ^
3. The polypeptide of claim 1 or 2, wherein the polypeptide has an amino acid selected from the following residues relative to X2 (SEQ ID NO: 933): Residue 1: G, A, S, T, Q, D, E, R, K, and H; Residue 2: G, A, L, S, T, N, and Q; ^ Residue 3: A, M, Y, W, N, Q, E, R, and K; Residue 4: G, A, V, I, M, L, F, Y, W, N, Q, E, and H; Residue 5: M, L, Q, E, and K; Residue 6: V, I, L, Y, R, and K; Residue 7: G, V, I, M, L, F, Y, W, S, T, N, Q, D, E, and R; ^ Residue 8: A, L, F, S, T, E, and H; Residue 9: A, V, I, and L; Residue 10: E, R, and K; Residue 11: V, E, K, R, and H; Residue 12: A, I, and L; ^ Residue 13: V and I; Residue 14: S, E, K, and R; Residue 15: A, I, M, L, and E; Residue 16: C, A, and S; Residue 17: N and R; ^ Residue 18: V, S, N, E, K, R, and H; Residue 19: A, V, I, and L; Residue 20: V and I; Residue 21: W, S, E, K, and R; and Residue 22: M and D. ^ 4. The polypeptide of any one of claims 1-3, wherein the polypeptide has 1, 2, 3, or all 4 of the following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 11 is K, E, or R; (b) X2 residue 14 is R, E, or K; ^ (c) X2 residue 18 is V, R, H, or K; and/or (d) X2 residue 21 is R or K. 5. The polypeptide of any one of clams 1-4, wherein the polypeptide has 1, 2, 3, or all 4 of the following residues relative to X2 (SEQ ID NO:933): ^ (a) X2 residue 4 is I or W; (b) X2 residue 7 is V, D, or E or I; (c) X2 residue 10 is K or R; and/or (d) X2 residue 15 is A, I, or L or M. ^ 6. The polypeptide of any one of claims 2-5, wherein the polypeptide has an amino acid selected from the following residues relative to X3 (SEQ ID NO: 934): Residue 1: P, N, D, E, R, K, and H; Residue 2: G, F, Y, E, R, and K; ^ Residue 3: V, I, M, L, F, T, and Q; Residue 4: A, I, L, S, T, E, R, and K; Residue 5: A, V, I, L, and E; Residue 6: A, V, L, E, R, and K; Residue 7: M and L; ^ Residue 8: V, L, F, Y, T, N, R, and K; Residue 9: G, A, V, Q, and E; Residue 10: A, I, M, and L; Residue 11: A, V, I and L; Residue 12: D, E, and K; ^ Residue 13: A, V, I, L, and T; Residue 14: C, A, V, S, T, and E; Residue 15: A, D, E, and R; Residue 16: A, I, L, Y, W, S, Q, E, R, K, and H; Residue 17: A, V and I; ^ Residue 18: A, L, Y, Q, E, R, and K; Residue 19: T, D, E, R, and K; and Residue 20: Y, T, N, E, and H. 7. The polypeptide of any one of claims 2-6, wherein the polypeptide comprises 1, 2, 3,^ or all 4 of the following residues relative to X3 (SEQ ID NO: 934): (a) X3 residue 2 is R or K; (b) X3 residue 6 is R or K ; (c) X3 residue 8 is R or K; (d) X3 residue 12 is D or E; and/or ^ (e) X3 residue 16 is H or Y or W. 8. The polypeptide of any one of claims 2-7, wherein the polypeptide comprises 1, 2, 3, 4, 5, or all 6 of the following residues relative to X3 (SEQ ID NO: 934): ^ (a) X3 residue 1 is K or R; (b) X3 residue 5 is I or V; (c) X3 residue 9 is Q or G; (d) X3 residue 11 is I or L; (e) X3 residue 13 is I or V; and/or ^ (f) X3 residue 19 is R or K. 9. The polypeptide of any one of claims 2-8, wherein the polypeptide has an amino acid selected from the following residues relative to X4 (SEQ ID NO: 935): Residue 1: R and K; ^ Residue 2: I, W, S, E, R, and K; Residue 3: A, V, I, and L; Residue 4: C, A, and M; Residue 5: A, L, S, E, R, and K; Residue 6: M, N, Q, D, E, R, and K; ^ Residue 7: A, L, and F; Residue 8: Y, S, E, R, and K; Residue 9: V, M, S, and T; Residue 10: A, E, and K; Residue 11: C and A; ^ Residue 12: Q and R; Residue 13: A and K; Residue 14: C, G, A, V, I, M, L, Y, S, T, Q, D, E, and K; Residue 15: I, L, and F; Residue 16: G, F, and Y; ^ Residue 17: S, R, and K; Residue 18: L, F, Y, W, and E; and Residue 19: L, F, Y, and E. ^
10. The polypeptide of any one of claims 2-9, wherein the polypeptide comprises one or both of the following residues relative to X4 (SEQ ID NO: 935): (a) X4 residue 9 is S or T; and/or (b) X4 residue 16 is G or Y. ^ 11. The polypeptide of any one of claims 2-10, wherein the polypeptide comprises 1, 2, 3, 4, or all 5 of the following residues relative to X4 (SEQ ID NO: 935): (a) X4 residue 8 is K or R; (b) X4 residue 12 is Q; ^ (c) X4 residue 13 is K; (d) X4 residue 15 is L or I; and/or (e) X4 residue 19 is L or F/Y. 12. The polypeptide of any one of claims 2-11, wherein the polypeptide comprises: ^ (i) the following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 11 is K, E, or R; (b) X2 residue 14 is R, E, or K; (c) X2 residue 18 is V, R, H, or K; and (d) X2 residue 21 is R or K; ^ (e) X2 residue 4 is I or W; (f) X2 residue 7 is V, D. or E or I; (g) X2 residue 10 is K or R; and (h) X2 residue 15 is A, I, or L or M; (ii) the following residues relative to X3 (SEQ ID NO: 934): ^ (a) X3 residue 2 is R or K; (b) X3 residue 6 is R or K ; (c) X3 residue 8 is R or K; (d) X3 residue 12 is D or E; and (e) X3 residue 16 is H or Y or W; ^ (f) X3 residue 1 is K or R; (g) X3 residue 5 is I or V; (h) X3 residue 9 is Q or G; (i) X3 residue 11 is I or L; (j) X3 residue 13 is I or V; and ^ (k) X3 residue 19 is R or K; and (iii) the following residues relative to X4 (SEQ ID NO: 935): (a) X4 residue 9 is S or T; and (b) X4 residue 16 is G or Y; and ^ (c) X4 residue 8 is K or R; (d) X4 residue 12 is Q; (e) X4 residue 13 is K; (f) X4 residue 15 is L or I; and (g) X4 residue 19 is L, F, or Y. ^ 13. The polypeptide of claim 1, wherein: (a) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); and ^ (b) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937), wherein relative to SEQ ID NO: 937, 1, 2, or all 3 of the following are true: residue 4 is Y, residue 6 is F, and/or residue 9 is D. wherein X2 and X3 may be in any order in the polypeptide; ^ wherein amino acid linkers may be present between the domains; and wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21R^^ CD360) but is not capable of simultaneously binding to the ^c, CD132 receptor. 14. The polypeptide of claim 13, wherein relative to SEQ ID NO: 937, 2 or all 3 of the^ following are true: residue 4 is Y, residue 6 is F, and/or residue 9 is D. 15. The polypeptide of claim 13, wherein relative to SEQ ID NO: 937, all 3 of the following are true: residue 4 is Y, residue 6 is F, and/or residue 9 is D. ^ 16. The polypeptide of any one of claims 13-15, wherein the polypeptide has an amino acid selected from the following residues relative to X2 (SEQ ID NO: 933): Residue 1: G, A, S, T, Q, D, E, R, K, and H; Residue 2: G, A, L, S, T, N, and Q; ^ Residue 3: A, M, Y, W, N, Q, E, R, and K; Residue 4: G, A, V, I, M, L, F, Y, W, N, Q, E, and H; Residue 5: M, L, Q, E, and K; Residue 6: V, I, L, Y, R, and K; ^ Residue 7: G, V, I, M, L, F, Y, W, S, T, N, Q, D, E, and R; Residue 8: A, L, F, S, T, E, and H; Residue 9: A, V, I, and L; Residue 10: E, R, and K; Residue 11: V, E, K, R, and H; ^ Residue 12: A, I, and L; Residue 13: V and I; Residue 14: S, E, K, and R; Residue 15: A, I, M, L, and E; Residue 16: C, A, and S; ^ Residue 17: N and R; Residue 18: V, S, N, E, K, R, and H; Residue 19: A, V, I, and L; Residue 20: V and I; Residue 21: W, S, E, K, and R; and ^ Residue 22: M and D. 17. The polypeptide of any one of claims 13-16, wherein the polypeptide has 1, 2, 3, or all 4 of the following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 11 is K, E, or R; ^ (b) X2 residue 14 is R, E, or K; (c) X2 residue 18 is V, R, H, or K; and/or (d) X2 residue 21 is R or K. 18. The polypeptide of any one of claims 13-17, the polypeptide has 1, 2, 3, or all 4 of the^ following residues relative to X2 (SEQ ID NO: 933): (a) X2 residue 4 is I or W; (b) X2 residue 7 is V, D, or E or I; (c) X2 residue 10 is K or R; and/or (d) X2 residue 15 is A, I, or L or M. ^
19. The polypeptide of any one of claims 13-18, wherein the polypeptide has an amino acid selected from the following residues relative to X3 (SEQ ID NO:937): Residue 1: P, N, D, E, R, K, and H; ^ Residue 2: G, F, Y, E, and R, and K; Residue 3: V, I, M, L, F, T, and Q; Residue 4: Y; Residue 5: A, V, I, L, and E; Residue 6: F; ^ Residue 7: M and L; Residue 8: Q and E; Residue 9: D; Residue10: A, I, M, and L; Residue 11: A, V, I, and L; ^ Residue 12: D, E, and K; Residue 13: A, V, I, L, and T; Residue 14: C, A, V, S, T, and E; Residue 15: T, N, and K; Residue 16: A, I, L, Y, W, S, Q, E, R, K, and H; ^ Residue 17: A, V and I; Residue 18: A, L, Y, Q, E, R, and K; Residue 19: T, D, E, R, and K; and Residue 20: Y, T, N, E, and H. ^ 20. The polypeptide of any one of claims 13-19, wherein the polypeptide has 1, 2, or all 3 of the following residues relative to X3 (SEQ ID NO: 937): (a) X3 residue 2 is R or K; (b) X3 residue 12 is D or E; and/or (c) X3 residue 16 is H, Y, or W. ^ 21. The polypeptide of any one of claims 13-20, wherein the polypeptide has 1, 2, 3, 4, or all 5 of the following residues relative to X3 (SEQ ID NO: 937): (a) X3 residue 1 is K or R; (b) X3 residue 5 is I or V; ^ (c) X3 residue 11 is I or L; (d) X3 residue 13 is I or V; and/or (e) X3 residue 19 is R or K. ^ 22. The polypeptide of any one of claims 13-21, wherein (i) relative to X3 (SEQ ID NO: 937): (a) all 3 of the following are true: residue 4 is Y, residue 6 is F, and/or residue 9 is D; (b) X3 residue 2 is R or K; ^ (c) X3 residue 12 is D or E; (d) X3 residue 16 is H, Y, or W. (e) X3 residue 1 is K or R; (f) X3 residue 5 is I or V; (g) X3 residue 11 is I or L; ^ (h) X3 residue 13 is I or V; and (i) X3 residue 19 is R or K; and (ii) relative to X2 (SEQ ID NO: 933): (a) X2 residue 11 is K, E, or R; (b) X2 residue 14 is R, E, or K; ^ (c) X2 residue 18 is V, R, H, or K; and (d) X2 residue 21 is R or K; (e) X2 residue 4 is I or W; (f) X2 residue 7 is V, D. or E or I; (g) X2 residue 10 is K or R; and ^ (h) X2 residue 15 is A, I, or L or M. 23. The polypeptide of any one of claims 13-22, further comprising the X4 domain, wherein the X4 domain does not permit simultaneous binding of the polypeptide to the IL-21 receptor (IL-21R^^ CD360) and the ^c, CD132 receptor, and wherein X2, X3, and X4 may^ be in any order in the polypeptide. ^
24. The polypeptide of claim 23, wherein X4 comprises a 19 residue peptide, and wherein the peptide has an amino acid at each position selected from the following amino acid residues: Residue 1: R and K; ^ Residue 2: I, W, S, E, R, and K; Residue 3: V; Residue 4: C, A, and M; Residue 5: A, L, S, E, R, and K; Residue 6: M, N, Q, D, E, R, and K; ^ Residue 7: A, L, and F; Residue 8: A, V, and D; Residue 9: G, W, E, and K; Residue 10: A, E, and K; Residue 11: C and A; ^ Residue 12: G, I, E, and K; Residue 13: V, L, Q, D, E, and R; Residue 14: C, G, A, V, I, M, L, Y, S, T, Q, D, E, and K; Residue 15: I, L and F; Residue 16: R, K, and H; ^ Residue 17: S, R, and K; Residue 18: L, F, Y, W, and E; and Residue 19: L, F, Y, and E. 25. The polypeptide of any one of claims 1-24, further comprising an X1 domain,^ wherein the X1 domain comprises a helical structure, and wherein X1, X2, X3, and X4, when present, may be in any order in the polypeptide. 26. The polypeptide of claim 25, wherein X1 is a peptide comprising an amino acid sequence at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,^ 90%, 95%, 98%, or 100% identical to the amino acid sequence LAEIMKEVAECARKEA (SEQ ID NO: 936). 27. The polypeptide of claim 26, wherein the polypeptide has an amino acid selected from the following residues relative to X1 (SEQ ID NO: 936): ^ Residue 1: V, I, and L; Residue 2: A, I, S, N, R, and K; Residue 3: D and E; Residue 4: V and I; ^ Residue 5: M and F; Residue 6: K; Residue 7: E; Residue 8: A and V; Residue 9: A and R; ^ Residue 10: A, V, I, L, T, Q, E, and K; Residue 11: C, V, I, M, F, Y, Q, R, and K; Residue 12: A; Residue 13: R and K; Residue 14: K; ^ Residue 15: E; and Residue 16: A, I, M, L, F, Y, S, and N. 28. The polypeptide of claim 26 or 27, wherein the polypeptide does not have a cysteine residue at position 11 relative to X1 (SEQ ID NO: 936). ^ 29. The polypeptide of any one of claims 1-28, wherein the polypeptide comprises in domain order, X2-X3-X4, or X1-X2-X3, or X2-X3, or X1-X2-X3-X4. 30. The polypeptide of any one of claims 1-15, wherein the polypeptide comprises in^ domain order, X4-X3-X2, X3-X2, X1-X3-X2, X2-X1-X3, X3-X2-X1, X3-X1-X2, X2-X4- X3, X3-X2-X4, X3-X4-X2, X4-X2-X3, X1-X2-X3-X4, X1-X4-X3-X2, X2-X1-X4-X3, X2- X3-X4-X1, X3-X2-X1-X4, X3-X4-X1-X2, X4-X1-X2-X3, X3-X2, or X4-X3-X2-X1. 31. A fusion protein, comprising: ^ (a) the polypeptide of any one of claims 1-30; and (b) one or more functional domains. 32. The fusion protein of claim 18, wherein the one or more functional domains comprises cell targeting domains or detectable domains. ^
33. The fusion protein of claim 31 or 32, wherein the one or more functional domain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:599-^ 606. 34. The polypeptide or fusion protein of any one of claims 1-33, comprising an amino acid sequence (a) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or^ 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-598627-631, 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742- 743, 746-747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898, and 900-931; or ^ (b) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO:627-631, 633, 635-641, 643-644, 648-654, 656, 658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698, 700-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746, 749-751, 771, 849-854, 856-862, 864-870, 872-874, 876-^ 898, 900, and 902-907; or (c) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO:1-598, 655, 657, 699, 747, 752-770, 772-798, 800-811, 813-821, 823, 825- 829, 831-832, 834-835, 843, 845-847, 871, and 908-931. ^ 35. A conditionally active IL-21 receptor binding protein, comprising a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not both present in the same fusion protein, wherein in total the first polypeptide component and the second polypeptide component^ comprise domains X2, X3, and optionally X4, as defined in any one of claims 1-34, wherein: (i) the first polypeptide component comprises at least one of X2, X3, and X4 (when present) but does not comprise each of X2, X3, and X4 (when present); and (ii) the second polypeptide component comprises each of X2, X3, and X4 (when present) that is not present in the first polypeptide component; ^ wherein the first polypeptide component and the second polypeptide component are not active receptor binding proteins individually, and wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL-21R^^ CD360). ^ 36. The conditionally active IL-21 receptor binding protein of claim 35, wherein: (i) the first polypeptide component comprises at least one of X2, X3, and X4 but does not comprise each of X2, X3, and X4; and (ii) the second polypeptide component comprises each of X2, X3, and X4 that is^ not present in the first polypeptide component; wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL- 21R^^ CD360) and is capable of binding to the ^c, CD132 receptor. ^ 37. The conditionally active IL-21 receptor binding protein of claim 35, comprising a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not both present in the same fusion protein, wherein in total the first polypeptide component and the second polypeptide component comprise domains X1, X2, X3, and X4 as defined in any one of claims 1-34;^ wherein: (i) the first polypeptide component comprises at least one of X1, X2, X3, and X4 but does not comprise each of X1, X2, X3, and X4; and (ii) the second polypeptide component comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide component; ^ wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL- 21R^^ CD360) and is capable of binding to the ^c, CD132 receptor. 38. The conditionally active IL-21 receptor binding protein of any one of claims 35-37,^ wherein the first and second polypeptides in total comprise (a) an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-598627-631, 633, 635-641, 643-644, 648-658, 660, 662, 664- ^ 666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746-747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898, and 900-931; or (b) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or^ 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO:627-631, 633, 635-641, 643-644, 648-654, 656, 658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687-695, 698, 700-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746, 749-751, 771, 849-854, 856-862, 864-870, 872-874, 876- 898, 900, and 902-907; or ^ (c) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO:1-598, 655, 657, 699, 747, 752-770, 772-798, 800-811, 813-821, 823, 825- 829, 831-832, 834-835, 843, 845-847, 871, and 908-931. ^ 39. The conditionally active IL-21 receptor binding protein of any one of claims 35-38, wherein the first and second polypeptides comprise: (a) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 608; and (ii) a second polypeptide comprising an amino acid^ sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 609; (b) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 607; and (ii) a second polypeptide comprising an amino acid^ sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 612; (c) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 610; and (ii) a second polypeptide comprising an amino acid^ sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 611; (d) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 613; and (ii) a second polypeptide comprising an amino acid ^ sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 614; (e) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid^ sequence of SEQ ID NO: 615; and (ii) a second polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 616; (f) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid^ sequence of SEQ ID NO: 617; and (ii) a second polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 618; (g) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid^ sequence of SEQ ID NO: 619; and (ii) a second polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 620; (h) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid^ sequence of SEQ ID NO: 621; and (ii) a second polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 622; or (i) (i) a first polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid^ sequence of SEQ ID NO: 623; and (ii) a second polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 624. 40. The conditionally active IL-21 receptor binding protein of any one of claims 35-39,^ wherein one or both of the first polypeptide and the second polypeptide are fused to a targeting domain, including but not limited an antibody or nanobody. 41. A non-naturally occurring polypeptide comprising domains X1, X2, X3, and X4, wherein: ^ (a) X1 is a peptide comprising the amino acid sequence at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence LAEIMKEVAECARKEA (SEQ ID NO:936); (b) X2 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%,^ 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence DTRILKVSLKKIVRA (SEQ ID NO:933); (c) X3 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence KRFEIRMRQLIDIVDHVKRE (SEQ ID NO:934); and ^ (d) X4 is a peptide comprising the amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence KEVMERAKSAAQKILGRFL (SEQ ID NO:935); wherein X1, X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; and^ wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21R^^ CD360). 42. The polypeptide of claim 41, wherein X1 residues 1-16 are selected from: Residue 1: V, I, and L; Residue 2: A, I, S, N, R, and K; ^ Residue 3: D and E; Residue 4: V and I; Residue 5: M and F; Residue 6: K; Residue 7: E; ^ Residue 8: A and V; Residue 9: A and R; Residue 10: A, V, I, L, T, Q, E, and K; Residue 11: C, V, I, M, F, Y, Q, R, and K; Residue 12: A; ^ Residue 13: R and K; Residue 14: K; Residue 15: E; and Residue 16: A, I, M, L, F, Y, S, and N. ^
43. The polypeptide of claim 41 or 42, wherein X1 residue 16 is not A. 44. The polypeptide of any one of claims 41-43, wherein X2 residues 1-15 are selected^ from: Residue 1: G, A, T, Q, D, R, K, and H; Residue 2: G, A, S, T, and N; Residue 3: A, M, Y, N, Q, E, and R; Residue 4: G, A, V, I, M, L, F, Y, W, N, Q, E, and H; ^ Residue 5: M, L, Q, and K; Residue 6: V, I, L, Y, R, and K; Residue 7: G, V, I, M, L, F, Y, W, S, T, N, Q, D, and E; Residue 8: F, S, T, and E; Residue 9: L; ^ Residue 10: K; Residue 11: K; Residue 12: I; Residue 13: V and I; Residue 14: R; and ^ Residue 15: A, I, M, and L. 45. The polypeptide of any one of claims 41-44, wherein 1, 2, 3, or all 4 of the following are true: (a) X2 residue 3 is not R; ^ (b) X2 residue 4 is not I; (c) X2 residue 7 is not V; and/or (d) X2 residue 8 is not S. 46. The polypeptide of any one of claims 41-45, wherein X3 residues 1-20 are selected^ from: Residue 1: P, N, R, and K; Residue 2: F, Y, and R; Residue 3: V, I, M, L, F, T, and Q; Residue 4: E; ^ Residue 5: I; Residue 6: R; Residue 7: M; Residue 8: V, L, F, Y, T, and R; ^ Residue 9: G, A, and Q; Residue10: I, M, and L; Residue 11: I and L; Residue 12: D; Residue 13: I; ^ Residue 14: C, A, V, S, and T; Residue 15: D and E; Residue 16: H; Residue 17: V; Residue 18: K; ^ Residue 19: R; and Residue 20: T, N, and E. 47. The polypeptide of any one of claims 41-46, wherein 1, 2, 3, 4, 5, 6, or all 7 of the following are true: ^ (a) X3 residue 2 is not R; (b) X3 residue 8 is not R; (c) X3 residue 9 is not Q; (d) X3 residue 10 is not L; (e) X3 residue 11 is not I; ^ (f) X3 residue 14 is not V; and/or (g) X3 residue 15 is not D. 48. The polypeptide of any one of claims 41-47, wherein X4 residues 1-19 are selected from: ^ Residue 81: K; Residue 2: E; Residue 3: V; Residue 4: M; Residue 5: E; ^ Residue 6: R; Residue 7: A; Residue 8: R and K; Residue 9: V, M, S, and T; ^ Residue 10: A; Residue 11: A; Residue 12: Q; Residue 13: K; Residue 14: C, G, A, V, I, M, L, S, T, Q, D, and K; ^ Residue 15: L; Residue 16: G; Residue 17: R and K; Residue 18: F, Y, and W; and Residue 19: L, F, and Y. ^ 49. The polypeptide of any one of claims 41-48, wherein 1, 2, 3, 4, 5, 6, or all 7 of the following are true: (a) X4 residue 8 is not K; (b) X4 residue 9 is not S; and/or ^ (c) X4 residue 19 is not L. 50. The polypeptide of any one of claims 41-49, wherein the polypeptide is an antagonist of the IL-21 receptor. ^ 51. The polypeptide of any one of claims 41-50, wherein X4 residue 16 is G. 52. The polypeptide of claim 51, wherein X1 residue 11 is C. 53. The polypeptide of any one of claims 41-52, wherein the domains are arranged N-^ terminal to C-terminal in an arrangement selected from the group consisting of X1-2-3-4, X1- 4-3-2, X2-1-4-3, X2-3-4-1, X3-2-1-4, X3-4-1-2, X4-1-2-3, and X4-3-2-1. 54. The polypeptide of any one of claims 41-53, wherein amino acid linkers are present between the domains. ^
55. The polypeptide of any one of claims 41-54, wherein the polypeptide comprises an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 938. ^ 56. The polypeptide of claim 55, wherein residues at non-helix positions may be selected from the group consisting of: Residue 01: V, I, M, F, Y, and D; Residue 02: A, V, I, M, L, F, Y, W, Q, D, E, R, and H; ^ Residue 03: E; Residue 04: D and E; Residue 21: A, T, and E; Residue 22: R and K; Residue 23: I, M, L, F, and Y; ^ Residue 24: P, G, A, V, I, S, TN, Q, D, R, and H; Residue 25: G, A, V, N, Q, and K; Residue 26: T, N, R, and K; Residue 27: G, A, S, T, N, D, R, K, and H; Residue 28: P, E, and H; ^ Residue 44: A and S; Residue 45: N and K; Residue 46: G, A, V, S, T, N, R, K, and H; Residue 47: V and I; Residue 48: V, I, Y, Q, R, and K; ^ Residue 49: L, R, and K; Residue 50: G, M, Y, S, Q, and H: Residue 51: R; Residue 52: G, M, L, F, Y, W, N, D, E, and H; Residue 53: G, R, and K; ^ Residue 54: G, A, V, I, L, N, E, and K; Residue 55: A, V, I, M, L, F, Y, W, T, Q, and H; Residue 56: D; Residue 57: P, A, V, Y, W, S, D, E, and H; Residue 78: F; ^ Residue 79: A, Y, S, and K; Residue 80: S; Residue 81: E; Residue 82: D; and ^ Residue 83: L and F. 57. The polypeptide of any one of claims 41-56, comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:1-598627-631, 633,^ 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685, 687- 695, 698-702, 706-707, 709-711, 714-715, 717-735, 738-739, 742-743, 746-747, 749-798, 800-811, 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864- 874, 876-898, and 900-931. ^ 58. The polypeptide of any one of claims 41-57, further comprising one or more functional domains. 59. The polypeptide of claim 58, wherein the one or more functional domains is a translational fusion with the polypeptide. ^ 60. A non-naturally occurring conditionally active IL-21 receptor binding protein, comprising a first polypeptide component and a second polypeptide component, wherein the first polypeptide component and the second polypeptide component are not present in a fusion protein, wherein in total the first polypeptide component and the second polypeptide^ component comprise domains X1, X2, X3, and X4 as defined in any of claims 41-59; wherein: (i) the first polypeptide component comprises at least one of X1, X2, X3, and X4 but does not comprise each of X1, X2, X3, and X4; and (ii) the second polypeptide component comprises each of X1, X2, X3, and X4 that^ is not present in the first polypeptide component; wherein the first polypeptide component and the second polypeptide component are not active receptor binding proteins individually, and wherein the first polypeptide component and the second polypeptide interact to form an active IL-21 receptor binding protein. ^
61. The conditionally active IL-21 receptor binding protein of claim 60, wherein X1, X2, X3, and X4, respectively, comprise an amino acid sequence as recited in one of claims 52-70. ^ 62. A polypeptide, comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of selected from the group consisting of SEQ ID NO: 607-624. 63. A recombinant nucleic acid encoding the polypeptide, first polypeptide, or second^ polypeptide of any preceding claim. 64. An expression vector comprising the recombinant nucleic acid of claim 63 operatively linked to a promoter. ^ 65. A recombinant host cell comprising the nucleic acid, expression vector, polypeptide, first polypeptide, and/or second polypeptide of any preceding claim. 66 A pharmaceutical composition, comprising the polypeptide, fusion protein, first polypeptide, second polypeptide, recombinant nucleic acid, expression vector, and/or^ recombinant host cell of any preceding claim, and a pharmaceutically acceptable carrier. 67. A method for treating cancer, comprising administering to a subject having cancer the polypeptide, conditionally-active IL-21 receptor agonist, first polypeptide, second polypeptide, recombinant nucleic acid, expression vector, recombinant host cell, and/or the^ pharmaceutical composition of any preceding claim in an amount effective to treat the tumor. 68. The method of claim 67, where the cancer is selected from the group consisting of colon cancer, melanoma, renal cell cancer, head and neck squamous cell cancer, gastric cancer, urothelial carcinoma, Hodgkin lymphoma, non-small cell lung cancer, small cell lung^ cancer, hepatocellular carcinoma, pancreatic cancer, Merkel cell carcinoma, colorectal cancer, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, ovarian cancer, cervical cancer, and any tumor types selected by a diagnostic test, such as microsatellite instability, tumor ^ mutational burden, PD-L1 expression level, or the immunoscore assay (as developed by the Society for Immunotherapy of Cancer). 69. A method for modulating an immune response in a subject comprising the ^ polypeptide antagonist, conditionally-active IL-21 receptor antagonist, first polypeptide, second polypeptide, recombinant nucleic acid, expression vector, recombinant host cell, and/or the pharmaceutical composition of any preceding claim. 70. The method of claim 70 wherein the immune response is an anti-cancer immune^ response, multiple sclerosis, lupus, or rheumatoid arthritis. ^
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