Interleukin-21 mimetics

Abstract

De novo designed polypeptide agonists and antagonists of IL-21, conditionally active IL-21 agonists and antagonists, and methods of using them to treat cancer or modulate immune responses are disclosed.

Description

[Technical Field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application No. 63 / 378,797, filed October 7, 2022, the entire contents of which are incorporated herein by reference.

[0002] Explanation of the sequence listing A sequence listing is submitted electronically in computer-readable form accompanying this application, the entire contents of which are incorporated herein by reference. This sequence listing is contained in the file entitled "22-1530-WO.xml," created on October 6, 2023, and having a size of 892,905 bytes.

[0003] Explanation of Federal Research Support This invention was made with government support under Grant No. R01CA240339 awarded by the National Cancer Institute and Grant No. R01AI160052 awarded by the National Institute of Allergy and Infectious Diseases. The federal government has certain rights in this invention. [Background technology]

[0004] Interleukin-21 (IL-21) is a multifunctional cytokine that plays a key role in innate and adaptive immune responses. IL-21 is primarily expressed by CD4 +Produced by T cells, IL-21 induces the differentiation of various immune cells. IL-21 is involved in regulating immune responses to infection, cancer, and autoimmune diseases and has recently become a target of clinical research. Clinical trials involving IL-21 are being conducted 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 immunotherapeutic agents, such as checkpoint inhibitors or cancer vaccines. Harnessing the full efficacy of natural IL-21 for therapeutic purposes has proven challenging due to its poor stability, low cross-reactivity of human IL-21 in mouse models, and lack of engineering potential. The limited efficacy of human IL-21 in mouse models further hinders predictive evaluation of the toxicity and activity of IL-21-based therapeutic candidates. IL-21 mimetic proteins that are cross-reactive between humans and mice offer a promising solution for cytokine-based immunotherapy using IL-21. Summary of the Invention

[0005] In one aspect, the disclosure provides a polypeptide comprising domains X2, X3, and optionally X4; where (a) X2 is a peptide 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); (b) X3 is: (i) KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934), or (ii) KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937), a peptide comprising an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of wherein one, two, or all three of the following are true in comparison with SEQ ID NO:937: the residue at position 4 is Y; the residue at position 6 is F; and / or The residue at position 9 is D; and (c) X4 is optional and, if present, includes a helix; wherein X2, X3, and X4, if present, can be in any order within the polypeptide; wherein there may be an amino acid linker anywhere between the domains; and wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21Rα, CD360).

[0006] In one embodiment, the polypeptide comprises domains X2, X3, and X4; where (a) X2 is a peptide 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); (b) X3 is a peptide 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 KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934); and (c) X4 is a peptide 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 KEVMERAKSAAQKILGRFL (SEQ ID NO: 935); wherein X2, X3, and X4 may be in any order within the polypeptide; wherein there may be an amino acid linker anywhere between the domains; and Here, the polypeptide is capable of binding to the IL-21 receptor (IL-21Rα, CD360) and also to the γc, CD132 receptor.

[0007] In another embodiment, (a) X2 is a peptide 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); and (b) X3 is a peptide 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 KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937); wherein one, two, or all three of the following are true in comparison with SEQ ID NO:937: the residue at position 4 is Y; the residue at position 6 is F; and / or The residue at position 9 is D; wherein X2 and X3 may be in any order within the polypeptide; wherein there may be an amino acid linker between the domains; and Here, the polypeptide is capable of binding to the IL-21 receptor (IL-21Rα, CD360), but is not capable of simultaneously binding to the γc, CD132 receptor.

[0008] In one embodiment, the polypeptide further comprises an X1 domain; wherein the X1 domain comprises a helical structure; and wherein X1, X2, X3, and X4, if present, can be in any order within the polypeptide.

[0009] In another embodiment, the present disclosure provides: (a) a polypeptide of any embodiment described herein; and (b) one or more functional domains; The present invention provides a fusion protein comprising:

[0010] In a further embodiment, the polypeptide or fusion protein comprises: (a) an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of the polypeptides set forth in Tables 1-6; or (b) SEQ ID NOs: 1 to 598, 627 to 631, 633, 635 to 641, 643 to 644, 648 to 658, 660, 662, 664 to 666, 668, 670, 672 to 675, 677 to 679, 681 to 685, 687 to 695, 698 to 702, 706 to 707, 709 to 711, 714 to 715, 717 to 735, 738 to 739, 742 to 743, 746 to 747, 749 to 798, 800 to 802 an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of 11, 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) SEQ ID NOs: 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- an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of 743, 746, 749-751, 771, 849-854, 856-862, 864-870, 872-874, 876-898, 900, and 902-907; or (d) an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 amino acid sequence includes:

[0011] In another aspect, the disclosure provides a conditionally active IL-21 receptor binding protein, wherein the IL-21 receptor binding protein comprises a first polypeptide element and a second polypeptide element; wherein both the first polypeptide element and the second polypeptide element are not both present in the same fusion protein; wherein the first polypeptide element and the second polypeptide element together comprise domains X2, X3, and optionally X4, as defined in any one of claims 1 to 34; where (i) the first polypeptide element comprises at least one of X2, X3, and X4 (if present), but not each of X2, X3, and X4 (if present); and (ii) the second polypeptide element comprises each of X2, X3, and X4 (if present) that is not present in the first polypeptide element; wherein said first polypeptide element and said second polypeptide element are not individually active receptor-binding proteins; and The first polypeptide element and the second polypeptide then interact to form an active IL-21 receptor binding protein capable of binding to the IL-21 receptor (IL-21Rα, CD360).

[0012] In one embodiment, (i) the first polypeptide element comprises at least one of X2, X3, and X4, but not each of X2, X3, and X4; and (ii) the second polypeptide element comprises each of X2, X3, and X4 that is not present in the first polypeptide element; wherein the first polypeptide element and the second polypeptide element interact to form It is capable of binding to the IL-21 receptor (IL-21Rα, CD360), and γc, capable of binding to the CD132 receptor; Forms active IL-21 receptor binding protein.

[0013] In another embodiment, the conditionally active IL-21 receptor binding protein described above comprises a first polypeptide element and a second polypeptide element; wherein both the first polypeptide element and the second polypeptide element are not both present in the same fusion protein; wherein the first polypeptide element and the second polypeptide element together comprise domains X1, X2, X3, and X4, as defined in any embodiment described herein; where (i) the first polypeptide element comprises at least one of X1, X2, X3, and X4, but not each of X1, X2, X3, and X4; and (ii) the second polypeptide element comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide element; wherein the first polypeptide element and the second polypeptide element interact to form It is capable of binding to the IL-21 receptor (IL-21Rα, CD360), and γc, capable of binding to the CD132 receptor; Forms active IL-21 receptor binding protein.

[0014] The present disclosure also provides a recombinant nucleic acid encoding the polypeptide, first polypeptide, or second polypeptide of any of the embodiments described herein; an expression vector comprising a recombinant nucleic acid of the disclosure operably linked to the promoter; a recombinant host cell comprising the nucleic acid, expression vector, polypeptide, first polypeptide, and / or second polypeptide of any of the embodiments described herein; a pharmaceutical composition comprising the polypeptide, fusion protein, first polypeptide, second polypeptide, recombinant nucleic acid, expression vector, and / or recombinant host cell of any of the embodiments described herein and a pharmaceutically acceptable carrier; and a method of treating cancer in a subject or a method of modulating an immune response in a subject. [Brief explanation of the drawings]

[0015] [Figure 1]The de novo IL-21 agonistic mimetic is capable of binding to both the IL-21 receptor (IL-21R; CD360) and the common gamma receptor (γc; CD132) in the context of receptor heterodimerization. (A) Schematic of the design, screening, optimization, and characterization of IL-21 mimetics. (B) Model of an agonistic mimetic of IL-21 in complex with hIL-21R and hγc. (C) SDS-PAGE of human IL-21 (lane 2), murine IL-21 (lane 3), and 21h10 (SEQ ID NO: 24) (lane 4) and the Precision Plus Protein Kaleidoscope Ladder (lane 1). (D) In ​​size-exclusion chromatography using an S75increase column, expression of 21h10 shows a monomer peak at the expected retention volume. (E) Circular dichroism indicates the presence of a helical secondary structure in 21h10 (SEQ ID NO: 24). (F) Thermal melting scans (starting at 25°C and going up to 95°C) at 222 nm show that 21h10 (SEQ ID NO: 24) exhibits excellent thermal stability. The helical structure begins to denature at 65°C. (G-J) Example measurements of receptor binding affinity of 21h10 (SEQ ID NO: 24) in biolayer interferometry as described in the Methods section of the Examples. The measured Kd values ​​of this and related designs are shown in Table 1. [Figure 2]Helix domain shuffling for retopology and de novo split IL-21 agonistic mimics. (A) Monomeric IL-21 mimics can be cyclically swapped or shuffled to form different topologies while maintaining their interfaces with the IL-21R (CD360) and common γc (CD132) receptors. (B) Three split pairs, namely, H1 / H234, H12 / H34, and H123 / H4, can be generated from a given topology of the IL-21 mimic. (C) Split fragments of the IL-21 mimic can be fused to anti-A and anti-B nanobodies (A and B are markers and can be the same or different) or other targeting domains; only when the two elements colocalize can the active IL-21 mimic structure be reconstituted, resulting in conditional activation. This reconstituted mimic delivers IL-21 signals to target cells. (D) Three types of 21h10 split pairs, i.e., H1 / H234, H12 / H34, and H123 / H4, can be reconstituted into the original helical bundle structure consisting of their four helices, and can form the IL-21 receptor complex by heterodimerizing IL-21R (CD360) and the common γc (CD132) receptor. [Figure 3]De novo IL-21 agonistic mimetics can be fused to antibodies for targeted cytokine delivery. (A) Schematic of immunocytokine in a 2:2 heavy chain:cytokine construct. An example is shown using atezolizumab fused to an IL-21 mimetic. For certain heterodimeric structures, constructs with a 2:1 stoichiometry containing knob / hole mutations in the heavy chain are also possible; a heavy chain with a knob mutation and a cytokine fusion can be complexed with another heavy chain with a hole mutation and no cytokine fusion. (B) SDS-PAGE of the atezolizumab-21h10 fusion construct under non-reducing conditions shows a band at the expected size of approximately 179.2 kDa. (C) The atezolizumab-21h10 fusion construct can bind independently to human IL-21R and human PD-L1. (D) The atezolizumab-21h10 fusion construct can simultaneously bind to human IL-21R and human PD-L1. The atezolizumab-21h10 fusion construct is 10 nM; human PD-L1 is immobilized on a streptavidin (SA) assay tip and the atezolizumab-21h10 fusion construct is allowed to bind; then, 50 nM of human IL-21R is allowed to bind; or vice versa. [Figure 4] De novo IL-21 antagonistic mimetics can only bind to the IL-21 receptor (IL-21R; CD360) and not to γc (CD132). (A) Five antagonistic hits (Table 5, SEQ ID NOS: 627-631) from the initial screen demonstrate human IL-21R binding by flow cytometry. Five hits were identified for human IL-21R binding using yeast surface display. 100 nM human IL-21R was used to label yeast cells expressing the designs. None of these hits were able to bind to γc and were therefore classified as antagonists. (B) Other antagonistic mimetics were designed from the agonistic IL-21 mimetics using Rosetta™, site-saturation mutagenesis (SSM) data, or ProteinMPNN to eliminate the γc interface. [Figure 5]Site-saturation mutagenesis study of agonist 21JC15. Using yeast surface display, mutations at every position in 21JC15 (SEQ ID NO: 8) yielded positive (black) and negative (white) mutations that enhanced and attenuated binding affinity to the IL-21 receptor, respectively. The darker the color, the more likely the mutation is to enhance binding affinity to the IL-21R (CD360) and common γc (CD132) receptors. To obtain the data, the library was sorted against the human IL-21 receptor. This study provides residue-level information on the impact of mutations on binding affinity to the receptor. [Figure 6] Agonist designs differ according to their interfacial affinity for IL-21R or the IL-21 receptor complex (IL-21R / γc). Two-dimensional plots of Kd for IL-21Ra binding and ternary complex binding (binding to γc in the presence of IL-21Ra). The X-axis represents binding affinity to either hIL-21R or mIL-21R; while the Y-axis represents binding affinity in molar units [M] to either hIL-21R / hγc or mIL-21R / mγc. In the mouse plot, constructs with mIL-21R / mγc affinity worse than 10-4M are plotted at the 10-4M position. Numbering 1-21 corresponds to 21A1-21A21 (Table 1, SEQ ID NOs: 164-184), respectively. DETAILED DESCRIPTION OF THE INVENTION

[0016] All cited references are incorporated herein by reference in their entirety. In this application, unless otherwise specifically stated, the techniques utilized may be described 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, USA); "Guide to Protein Purification" in Methods in Enzymology (edited by M.P. Deutscher, (1990) Academic Press, Inc.); "PCR Protocols: A Guide to Methods and Applications" (Innis et al., 1990, Academic Press, San Diego, CA, USA); "Culture of Animal Cells: A Manual of Basic Technique, 2nd Edition" (RI Freshney, 1987, Liss, Inc., New York, NY, USA); "Gene Transfer and Expression Protocols" (Innis et al., 1990, Academic Press, San Diego, CA, USA). "Protocols," pp. 109-128, edited by E.J. Murray, The Humana Press Inc., Clifton, NJ; and Ambion 1998 Catalogue (Ambion, Austin, TX, USA).

[0017] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0018] As used herein, 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).

[0019] In all embodiments of the polypeptides disclosed herein, any N-terminal methionine residue is optional (i.e., the N-terminal methionine residue may or may not be present, and if absent, will not be considered when determining percent identity). Similarly, a C-terminal histidine tag is also optional, may or may not be present, and if absent, will not be considered when determining percent identity.

[0020] In this disclosure, all embodiments of any aspect can be used in combination unless the context clearly dictates otherwise.

[0021] Unless the context clearly requires otherwise, throughout the description and claims, the words "comprise," "comprising," and the like are to be construed as inclusive, as opposed to exclusive or exhaustive; that is, as meaning "including, but not limited to." Words using the singular or plural also include the plural and singular, respectively. Furthermore, when used herein, the terms "herein," "above," and "below," and words of similar import, refer to this specification as a whole and not to any particular portion of this specification.

[0022] In one aspect, the disclosure provides a polypeptide comprising domains X2, X3, and optionally X4; where (a) X2 is a peptide 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); (b) X3 is: (i) KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934), or (ii) KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937), a peptide comprising an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of wherein one, two, or all three of the following are true in comparison with SEQ ID NO:937: the residue at position 4 is Y; the residue at position 6 is F; and / or The residue at position 9 is D; and (c) X4 is optional and, when present, includes a helix structure, but is not limited to: wherein X2, X3, and X4, if present, can be in any order within the polypeptide; wherein there may be an amino acid linker anywhere between the domains; and wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21Rα, CD360).

[0023] The inventors have shown that the polypeptides of this aspect of the disclosure can bind to the IL-21 receptor and demonstrate that in some embodiments they can act as agonists and in some embodiments as antagonists. The polypeptides are highly stable de novo IL-21 mimetic proteins that are ideal for therapeutic use, mimicking the interaction of native IL-21 with its receptor and the biology of native IL-21 in humans and mice.

[0024] In one embodiment, the polypeptide comprises domains X2, X3, and X4; where (a) X2 is a peptide 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); (b) X3 is a peptide 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 KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934); and (c) X4 is a peptide 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 KEVMERAKSAAQKILGRFL (SEQ ID NO: 935); wherein X2, X3, and X4 may be in any order within the polypeptide; wherein there may be an amino acid linker anywhere between the domains; and Here, the polypeptide is capable of binding to the IL-21 receptor (IL-21Rα, CD360) and also to the γc, CD132 receptor.

[0025] The present inventors have demonstrated that the polypeptide of this embodiment can act as an agent that activates IL-21-like signaling, induce cell differentiation in both human and mouse cells by specific binding, and promote dimerization of the IL-21 receptor (IL-21Rα, CD360) and the common gamma chain receptor (γc, CD132). The designed polypeptide exhibits antitumor effects in mouse MC38 adenocarcinoma, B16F10 melanoma, and pancreatic cancer models, and can therefore be used, for example, in cancer therapy.

[0026] Agonistic IL-21 mimetics are constructs that can bind to the IL-21 receptor (IL-21Rα, CD360) and the common gamma chain receptor (γc, CD132), not only individually but also simultaneously, allowing for potential downstream cell signaling via receptor heterodimerization. Methods to determine their ability to act as agonists include the binding of IL-21R and γc receptors. c The goal is to confirm simultaneous binding to the receptors. c The affinity between the receptor and γ c Because it is difficult to detect receptor binding by itself, the highest possible concentration in the presence of IL-21R (1000 nM IL-21R and γ c receptor at 1000 nM) c Receptor binding assays would allow identification of agonistic variants.

[0027] In one embodiment: (a) X2 is a peptide 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: 933; (b) X3 is a peptide 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: 934; and (c) X4 is a peptide comprising an amino acid sequence that is 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 an 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 an 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 an amino acid sequence that is 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 an 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 an 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 an amino acid sequence that is 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 an 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 an 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 an amino acid sequence that is 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 an 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 an 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 an amino acid sequence that is at least 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:935.

[0028] In order to identify various substitutions that retain receptor binding and therefore agonist activity, the inventors performed large-scale site-saturation mutagenesis (SSM), yeast surface display assays using flow cytometry, biolayer interferometry (BLI), and other functional studies as described in the Methods section of the Examples. 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.

[0029] The inventors have identified residues 11, 14, 18, and 21 of X2 (SEQ ID NO: 933) as being in the binding interface with IL-21Rα (CD360). In some embodiments, the polypeptide has one, two, three, or all four of the following residues compared to X2 (SEQ ID NO: 933): (a) residue 11 of X2 is K, E, or R; (b) residue 14 of X2 is R, E, or K; (c) residue 18 of X2 is V, R, H, or K; and / or (d) Residue 21 of X2 is R or K.

[0030] The inventors have identified residues 4, 7, 10, and 15 of X2 (SEQ ID NO: 933) as being in the binding interface with IL-21Rα (CD360). In some embodiments, the polypeptide has one, two, three, or all four of the following residues compared to X2 (SEQ ID NO: 933): (a) residue 4 of X2 is I or W; (b) residue 7 of X2 is V, D, E, or I; (c) residue 10 of X2 is K or R; and / or (d) Residue 15 of X2 is A, I, or L or M.

[0031] In another embodiment, the polypeptide has an amino acid sequence 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.

[0032] The inventors have identified residues 2, 6, 8, 12, and 16 of X3 (SEQ ID NO: 934) as being in the binding interface with the IL-21Rα (CD360) or γc (CD132) receptor. In some embodiments, the polypeptide has one, two, three, four, or all five of the following residues relative to X3 (SEQ ID NO: 934): (a) residue 2 of X3 is R or K; (b) residue 6 of X3 is R or K; (c) residue 8 of X3 is R or K; (d) residue 12 of X3 is D or E; and / or (e) Residue 16 of X3 is H or Y or W.

[0033] The inventors have identified residues 1, 5, 9, 11, 13, and 19 of X3 (SEQ ID NO: 934) as being in the binding interface with the IL-21Rα (CD360) or γc (CD132) receptor. In some embodiments, the polypeptide has one, two, three, four, five, or all six of the following residues relative to X3 (SEQ ID NO: 934): (a) residue 1 of X3 is K or R; (b) residue 5 of X3 is I or V; (c) residue 9 of X3 is Q or G; (d) residue 11 of X3 is I or L; (e) residue 13 of X3 is I or V; and / or (f) Residue 19 of X3 is R or K.

[0034] In a further embodiment, the polypeptide has an amino acid sequence 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.

[0035] The inventors have identified residues 9 and 16 of X4 (SEQ ID NO: 935) as being in the binding interface with the γc (CD132) receptor. In some embodiments, the polypeptide has one or both of the following residues relative to X4 (SEQ ID NO: 935): (a) residue 9 of X4 is S or T; and / or (b) Residue 16 of X4 is G or Y.

[0036] The inventors have identified residues 8, 12, 13, 15, and 19 of X4 (SEQ ID NO: 935) as being in the binding interface with the γc (CD132) receptor. In some embodiments, the polypeptide has one, two, three, four, or all five of the following residues compared to X4 (SEQ ID NO: 935): (a) residue 8 of X4 is K or R; (b) residue 12 of X4 is Q; (c) residue 13 of X4 is K; (d) residue 15 of X4 is L or I; and / or (e) Residue 19 of X4 is L, F, or Y. In one embodiment, the polypeptide is: (1) Compared to X2 (SEQ ID NO: 933), (a) Residue 11 of X2 is K, E, or R; (b) residue 14 of X2 is R, E, or K; (c) residue 18 of X2 is V, R, H, or K; (d) residue 21 of X2 is R or K; (e) residue 4 of X2 is I or W; (f) residue 7 of X2 is V, D, or E, or I; (g) residue 10 of X2 is K or R; and (h) residue 15 of X2 is A, I, or L or M; containing a residue which is and (2) Compared to X3 (SEQ ID NO: 934), (a) Residue 2 of X3 is R or K; (b) residue 6 of X3 is R or K; (c) residue 8 of X3 is R or K; (d) residue 12 of X3 is D or E; (e) residue 16 of X3 is H or Y or W; (f) residue 1 of X3 is K or R; (g) residue 5 of X3 is I or V; (h) residue 9 of X3 is Q or G; (i) residue 11 of X3 is I or L; (j) residue 13 of X3 is I or V; and (k) residue 19 of X3 is R or K; containing a residue which is and (3) Compared to X4 (SEQ ID NO: 935), (a) Residue 9 of X4 is S or T; and (b) residue 16 of X4 is G or Y; and (c) residue 8 of X4 is K or R; (d) residue 12 of X4 is Q; (e) residue 13 of X4 is K; (f) residue 15 of X4 is L or I; and (g) residue 19 of X4 is L, F, or Y; The residues include those in which:

[0037] In another embodiment, the disclosure provides a polypeptide comprising domains X2 and X3; where (a) X2 is a peptide 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO: 933); and (b) X3 is a peptide 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 KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937); wherein one, two, or all three of the following are true in comparison with SEQ ID NO:937: the residue at position 4 is Y; the residue at position 6 is F; and / or The residue at position 9 is D; wherein X2 and X3 may be in any order within the polypeptide; wherein there may be an amino acid linker between the domains; and Here, the polypeptide is capable of binding to the IL-21 receptor (IL-21Rα, CD360), but is not capable of simultaneously binding to the γc, CD132 receptor.

[0038] In this embodiment, the polypeptide can act as an IL-21 antagonist. The antagonistic IL-21 mimetic is a construct that can bind to the IL-21 receptor (IL-21Rα, CD360) but cannot simultaneously bind to the common gamma chain receptor (γc, CD132), potentially preventing heterodimerization between the IL-21 receptor (IL-21Rα, CD360) and the common gamma chain receptor (γc, CD132) and the resulting potential downstream cell signaling. Binding to IL-21R is first confirmed, followed by binding to the common gamma chain receptor (γc, CD132). c Designs can be tested for antagonist activity by confirming the lack of simultaneous binding to the receptors. c The affinity between the receptor and γ c Because receptor binding is difficult to detect by itself, γ c Receptor binding assays must be performed in the presence of IL-21R. Confirmation of IL-21R binding is important because γ c As with agonist identification, this assay was performed at the highest possible concentrations (1000 nM for IL-21R and γ c The receptor should be tested at 1000 nM.

[0039] The inventors provide a detailed description of such polypeptides that can act as IL-21 receptor antagonists. For example, at least the following polypeptides in Table 5 have been identified as having antagonistic capacity: SEQ ID NOs: 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.

[0040] The polypeptide antagonists may be used, for example, to modulate the immune response in a subject in need thereof, such as for immunosuppression in autoimmune diseases, including, but not limited to, multiple sclerosis, lupus erythematosus, and rheumatoid arthritis.

[0041] The X2 domain of this embodiment is identical to the X2 domain of the agents described in detail above, as it is the X2 domain that mediates the interaction with the IL-21 receptor (IL-21Rα, CD360) and not the γc, CD132 receptor.

[0042] In one embodiment (a) X2 is a peptide 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: 933; and (b) X3 is a peptide comprising an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:937.

[0043] In another embodiment: (a) X2 is a peptide comprising an 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 an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:937.

[0044] In a further embodiment: (a) X2 is a peptide comprising an 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 an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:937.

[0045] In one embodiment: (a) X2 is a peptide comprising an 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 an amino acid sequence that is at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:937.

[0046] In another embodiment: (a) X2 is a peptide comprising an 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 an amino acid sequence that is at least 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:937.

[0047] In order to identify various substitutions that retain receptor binding and therefore antagonist activity, the inventors performed large-scale site-saturation mutagenesis (SSM), yeast surface display assays using flow cytometry, biolayer interferometry (BLI), and other functional studies as described in the Methods section of the Examples. 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.

[0048] The inventors have identified residues 11, 14, 18, and 21 of X2 (SEQ ID NO: 933) as being in the binding interface with IL-21Rα, CD360. In some embodiments, the polypeptide has one, two, three, or all four of the following residues compared to X2 (SEQ ID NO: 933): (a) residue 11 of X2 is K, E, or R; (b) residue 14 of X2 is R, E, or K; (c) residue 18 of X2 is V, R, H, or K; and / or (d) Residue 21 of X2 is R or K.

[0049] The inventors have identified residues 4, 7, 10, and 15 of X2 (SEQ ID NO: 933) as being in the binding interface with IL-21Rα, CD360. In some embodiments, the polypeptide has one, two, three, or all four of the following residues compared to X2 (SEQ ID NO: 933): (a) residue 4 of X2 is I or W; (b) residue 7 of X2 is V, D, or E or I; (c) residue 10 of X2 is K or R; and / or (d) Residue 15 of X2 is A, I, or L or M.

[0050] In another embodiment, the polypeptide has an amino acid sequence 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; 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: 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.

[0051] In some embodiments, the polypeptide has one, two, or all three of the following residues compared to X3 (SEQ ID NO: 937): (a) residue 2 of X3 is R or K; (b) residue 12 of X3 is D or E; and / or (c) Residue 16 of X3 is H or Y / W.

[0052] In other embodiments, the polypeptide has one, two, three, four, or all five of the following residues compared to X3 (SEQ ID NO: 937): (a) residue 1 of X3 is K or R; (b) residue 5 of X3 is I or V; (c) residue 11 of X3 is I or L; (d) residue 13 of X3 is I or V; and / or (e) Residue 19 of X3 is R or K.

[0053] In a further embodiment: (i) compared to X3 (SEQ ID NO: 937): (a) All three of the following are true: residue 4 is Y; residue 6 is F; and / or residue 9 is D; (b) residue 2 of X3 is R or K; (c) residue 12 of X3 is D or E; (d) residue 16 of X3 is H, Y, or W; (e) residue 1 of X3 is K or R; (f) residue 5 of X3 is I or V; (g) residue 11 of X3 is I or L; (h) residue 13 of X3 is I or V; and (i) residue 19 of X3 is R or K; and (ii) compared to X2 (SEQ ID NO: 933): (a) residue 11 of X2 is K, E, or R; (b) residue 14 of X2 is R, E, or K; (c) residue 18 of X2 is V, R, H, or K; and (d) residue 21 of X2 is R or K; (e) residue 4 of X2 is I or W; (f) residue 7 of X2 is V, D, or E or I; (g) residue 10 of X2 is K or R; and (h) Residue 15 of X2 is A, I, or L or M.

[0054] In another embodiment, the antagonistic polypeptide further comprises an X4 domain; wherein the X4 domain comprises a helical structure; wherein the X4 domain prevents simultaneous binding of the polypeptide to the IL-21 receptor (IL-21Rα, CD360) and γc, CD132 receptors; and wherein X2, X3, and X4 can be in any order within the polypeptide, and in this embodiment, the X4 domain can be any helical domain suitable for the intended purpose, so long as the polypeptide is unable to simultaneously bind to the IL-21 receptor (IL-21Rα, CD360) and the γc, CD132 receptor.

[0055] 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.

[0056] In one embodiment of any of the Il-21-binding polypeptides of the present disclosure, the polypeptide 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 within the polypeptide. In one embodiment, X1 is a peptide comprising an amino acid sequence that is 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 amino acids selected from the following residues compared 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.

[0057] In one embodiment, the polypeptide does not have a cysteine ​​residue at position 11 compared to X1 (SEQ ID NO: 936).

[0058] The domains X2, X3, X4 (if present), and X1 (if present) may be in any order within the polypeptide of any embodiment described herein. We have provided a detailed description of "shuffled" polypeptides; see Table 2 and the columns listing domains X1, X2, X3, and X4 in domain order. In one embodiment, the polypeptide comprises the domains in the domain order X2-X3-X4, or X1-X2-X3, or X2-X3, or X1-X2-X3-X4. In other embodiments, the polypeptide comprises domains in the following 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.

[0059] Polypeptides of the present disclosure may include amino acid linkers between domains. In some embodiments, a linker is present between each of domains X2, X3, X4 (if present), and X1 (if present). In other embodiments, the polypeptide need not include a linker between any of the domains, or may include a linker between some but not all of the domains. The linker may be of any length and amino acid composition. In various non-limiting embodiments, suitable linkers include 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), GSGSGSGSG GSGGSCKKISGGSGGGSGGGGS (SEQ ID NO: 945), and (GGGGX) n (SEQ ID NO: 946); where X is Q, E, or S, and n is 2-5.

[0060] The polypeptide may be fused to one or more additional domains appropriate for the intended use. In one embodiment, the present disclosure provides a fusion protein comprising a polypeptide of any embodiment or combination of embodiments of the present disclosure and one or more functional domains. Any functional domain can be fused to a polypeptide of the present disclosure. In various non-limiting embodiments, the one or more functional domains comprise a cell-targeting domain (including, but not limited to, an antibody, an antibody fragment, a domain that extends protein half-life (e.g., albumin, albumin-binding protein, Fc fragment of an antibody), a protein that binds to a biological marker, an antigen, a ligand, a peptide, etc.), or a detectable domain (including, but not limited to, a fluorescent protein, a luminescent protein, a protein tag, etc.). In various embodiments, the one or more functional domains comprise an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 599-606. The polypeptide and the one or more functional domains may be directly adjacent to each other in the fusion protein or may be connected by a polypeptide linker suitable for the intended purpose. The one or more functional domains may be located at the N-terminus, C-terminus, or interdomain position of the X2 domain, X3 domain, X4 domain, or X1 domain (if present).

[0061] In another embodiment, the polypeptide or fusion protein is selected from the group consisting of SEQ ID NOs: 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, and an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of: 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 is selected from the group consisting of SEQ ID NOs: 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, 720-722, 723-724, 725-726, 727-738, 728-739, 730-739, 731-732, 732-733, 733-734, 735-736, 737-738, 739-740, 741-742, 742-743, 743-744, 745-746, 746-747, 747-748, 748-749, 750-751, 752-753, 754-755, 756-757, 758-759, 760-761, 761-762, 762-763, 763-764, 764-765, 765-766, 766-767, 767-768, 768-770, 769-771, 770-772, 771-77 38-739, 742-743, 746, 749-751, 771, 849-854, 856-862, 864-870, 872-874, 876-898, 900, and 902-907. In this embodiment, the polypeptide may have antagonist activity.In another embodiment, the polypeptide or fusion protein comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 polypeptide may have agonist activity.

[0062] In another aspect, the present disclosure provides a conditionally active IL-21 receptor binding protein comprising a first polypeptide element and a second polypeptide element, wherein the first polypeptide element and the second polypeptide element are not both present in the same fusion protein, and wherein the first polypeptide element and the second polypeptide element together comprise domains X2, X3, and optionally X4 as defined in any embodiment or combination of embodiments of the polypeptides of the present disclosure; where (i) the first polypeptide element comprises at least one of X2, X3, and X4 (if present), but not each of X2, X3, and X4 (if present); and (ii) the second polypeptide element comprises each of X2, X3, and X4 that is not present in the first polypeptide element; wherein said first polypeptide element and said second polypeptide element are not individually active receptor-binding proteins; and wherein the first polypeptide element and the second polypeptide element interact to form It forms an active IL-21 receptor binding protein that is capable of binding to the IL-21 receptor (IL-21Rα, CD360).

[0063] In one embodiment: (i) the first polypeptide element comprises at least one of X2, X3, and X4, but not each of X2, X3, and X4; and (ii) the second polypeptide element comprises each of X2, X3, and X4 that is not present in the first polypeptide element; wherein said first polypeptide element and said second polypeptide element are not individually active receptor-binding proteins; and wherein the first polypeptide element and the second polypeptide element interact to form It is capable of binding to the IL-21 receptor (IL-21Rα, CD360), and γc, which can also bind to the CD132 receptor, Forms active IL-21 receptor binding protein.

[0064] In another embodiment, (i) the first polypeptide element comprises at least one of X1, X2, X3, and X4, but not each of X1, X2, X3, and X4; and (ii) the second polypeptide element comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide element; wherein said first polypeptide element and said second polypeptide element are not individually active receptor-binding proteins; and wherein the first polypeptide element and the second polypeptide element interact to form It is capable of binding to the IL-21 receptor (IL-21Rα, CD360), and γc, which can also bind to the CD132 receptor, Forms active IL-21 receptor binding protein.

[0065] Surprisingly, the present disclosure demonstrates conditionally active IL-21 receptor agonists that comprise the respective first and second polypeptides listed above that are not individually receptor agonists, but that can bind to both the IL-21 receptor (IL-21Rα, CD360) and the γc, CD132 receptor, thereby forming an IL-21 active agonist through a non-covalent interaction. Thus, the conditionally active receptor agonists of the present disclosure can be used in any application in which the polypeptide agonists of the present disclosure can be used.

[0066] In another embodiment, (i) the first polypeptide element comprises X2 but not X3; and (ii) the second polypeptide element comprises X3; wherein said first polypeptide element and said second polypeptide element are not individually active receptor-binding proteins; and wherein the first polypeptide element and the second polypeptide element interact to form It forms an active IL-21 receptor-binding protein that can bind to the IL-21 receptor (IL-21Rα, CD360) but cannot simultaneously bind to the γc and CD132 receptors.

[0067] This embodiment provides a conditionally active receptor antagonist that can be used in any application where the disclosed polypeptide antagonists are available. Split IL-21 antagonists are designed to achieve targeted delivery of IL-21 antagonists 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.

[0068] The polypeptides are typically split at sites that will not interfere with the function of the protein (e.g., at the linker portion in embodiments that include a linker). Furthermore, as with the X1 domain (if present), X2 domain, X3 domain, and X4 domain (if present) in the unsplit polypeptide, the domains can form loops together in any order, and the split proteins can include any combination of the domains.

[0069] Thus, in the first and second polypeptides, X1 (if present), X2, X3, and X4 (if present) can be in any order; In a non-limiting embodiment: (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.

[0070] When the first polypeptide and / or the second polypeptide comprises two or more of the X1, X2, X3, and X4 domains, in some embodiments, the domains may be separated by an amino acid linker of any suitable length or suitable amino acid composition. There is no requirement for a linker; in one embodiment, there is no linker between any of the domains. In other embodiments, an amino acid linker may be present at 0 junction, 1 junction, or 2 junctions between the X1, X2, X3, and X4 domains of the first polypeptide and / or the second polypeptide. The amino acid linker may be of any length deemed appropriate for the intended use, including, for example, any of the linker embodiments disclosed herein. In some embodiments, the linker may be present at the N-terminus or C-terminus and may be referred to as a linker even though it does not link the two domains together.

[0071] In one embodiment, X1 (if present), X2, X3, and X4 are each a domain as recited in any embodiment or combination of embodiments of the polypeptides of the present disclosure. In another embodiment, the first and second polypeptides together comprise: SEQ ID NOs: 1 to 598, 627 to 631, 633, 635 to 641, 643 to 644, 648 to 658, 660, 662, 664 to 666, 668, 670, 672 to 675, 677 to 679, 681 to 685, 687 to 695, 698 to 702, 706 to 707, 709 to 711, 714 to 715, 717 to 735, 738 to 739, 742 to 743, 746 to 747, 749 to 798, 800 to 81 an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of 1, 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) SEQ ID NOs: 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- an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of 743, 746, 749-751, 771, 849-854, 856-862, 864-870, 872-874, 876-898, 900, and 902-907; or (c) an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 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; Includes.

[0072] In another embodiment, the first and second polypeptides are: (a) (i) a first polypeptide comprising an amino acid sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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; Includes.

[0073] In a further embodiment, the first and second polypeptides are: (a) (i) a first polypeptide comprising an amino acid sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 624; Includes.

[0074] In yet a further embodiment, the first and second polypeptides are: (a) (i) a first polypeptide comprising an amino acid sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO:624. Includes.

[0075] In yet a further embodiment, the first and second polypeptides are: (a) (i) a first polypeptide comprising an amino acid sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is at least 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 624; Includes.

[0076] In one embodiment, one or both of the first and second polypeptides are fused to a targeting domain to enable targeted delivery. In a non-limiting embodiment, the targeting domain may comprise an antibody or nanobody that can direct the split cytokine mimic to a desired cell or tissue of interest (FIG. 2C). In this example, the first and second polypeptides are fused to an anti-HER2-DARPin or anti-EGFR-DARPin (Table 4, SEQ ID NOs: 613-624) to demonstrate that the first and / or second polypeptides can be fused and reconstituted to such a targeting domain (FIG. 2D).

[0077] In another embodiment, the present disclosure provides a polypeptide comprising the amino acid sequence of any first or second polypeptide as described herein. These polypeptides may be used, for example, as conditionally active receptor agonists of the present disclosure. In one embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:607-624.

[0078] In another aspect, the present disclosure provides a non-naturally occurring polypeptide comprising domains X1, X2, X3, and X4; where (a) 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); (b) X2 is a peptide 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 DTRILKVSLKKIVRA (SEQ ID NO: 933); (c) X3 is a peptide 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 KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934); and (d) X4 is a peptide 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 KEVMERAKSAAQKILGRFL (SEQ ID NO: 935); wherein X1, X2, X3, and X4 can be in any order within the polypeptide; wherein there may be an amino acid linker anywhere between the domains; and wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21Rα, CD360).

[0079] Polypeptides according to this aspect of the disclosure can be used, for example, to treat cancer and / or modulate the immune response. In one embodiment, residues 1-16 of X1 are: 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; is selected from.

[0080] In another embodiment, residue 16 of X1 is not A. In one embodiment, residues 1-16 of X2 are: 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; is selected from.

[0081] In various embodiments, one, two, three, or all four of the following statements are true: (a) Residue 3 of X2 is not R; (b) residue 4 of X2 is not I; (c) residue 7 of X2 is not V; and / or (d) Residue 8 of X2 is not S.

[0082] In a further embodiment, residues 1-20 of X3 are: 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; Residue 10: 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; is selected from.

[0083] In various embodiments, one, two, three, four, five, six, or all seven of the following statements are true: (a) Residue 2 of X3 is not R; (b) residue 8 of X3 is not R; (c) residue 9 of X3 is not Q; (d) residue 10 of X3 is not L; (e) residue 11 of X3 is not I; (f) residue 14 of X3 is not V; and / or (g) Residue 15 of X3 is not D.

[0084] In one embodiment, residues 1-19 of X4 are: 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; is selected from.

[0085] In another embodiment, one, two, or all three of the following are true: (a) Residue 8 of X4 is not K; (b) residue 9 of X4 is not S; and / or (c) Residue 19 of X4 is not L.

[0086] In one embodiment, the polypeptide is an antagonist of the IL-21 receptor. In another embodiment, residue 11 of X1 is C. This embodiment aids in enhancing binding affinity to human common gamma chain (CD132), as well as mouse CD360 and mouse CD132.

[0087] In various embodiments, the domains are arranged N- to C-terminally 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, there is an amino acid linker between the domains.

[0088] In one embodiment, the polypeptide comprises an amino acid sequence that is 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: DEEELAEIMKEVAECARKEAEKIDNTDEDTRILKVSLKKIVRAANVIVRMREREADSKRFEIRMRQLIDIVDHVKREFASEDLKEVMERAKSAAQKILGRFL (SEQ ID NO: 938).

[0089] In this embodiment, the residues at the non-helical positions are: 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; It may be selected from the group consisting of:

[0090] In a further embodiment, the polypeptide according to this aspect of the disclosure is selected from the group consisting of SEQ ID NOs: 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, and 900-931.

[0091] In one embodiment, the polypeptide further comprises one or more functional domains. The polypeptide may be fused to any functional domain suitable for the intended use, including, but not limited to, a cell-targeting domain and a detectable domain (including, but not limited to, a fluorescent protein, a luminescent protein, etc.). In one embodiment, the one or more functional domains are translationally fusions with the polypeptide.

[0092] In another embodiment of this aspect, the disclosure provides a conditionally active non-naturally occurring IL-21 receptor binding protein, the non-naturally occurring IL-21 receptor binding protein comprising a first polypeptide element and a second polypeptide element; wherein said first polypeptide element and said second polypeptide element are not present within a fusion protein; wherein the first polypeptide element and the second polypeptide element together comprise domains X1, X2, X3, and X4 as defined in any preceding claim; where (i) the first polypeptide element comprises at least one of X1, X2, X3, and X4, but not each of X1, X2, X3, and X4; and (ii) the second polypeptide element comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide element; wherein said first polypeptide element and said second polypeptide element are not individually active receptor-binding proteins; The first polypeptide element and the second polypeptide then interact to form an active IL-21 receptor binding protein.

[0093] In various embodiments, the polypeptide is: (i) a polypeptide containing X1 but not X2, X3, and X4; (ii) a polypeptide containing X2 but not X1, X3, and X4; (iii) a polypeptide containing X3 but not X1, X2, and X4; (iv) a polypeptide containing X4 but not X1, X2, and X3; (v) a polypeptide containing X1 and X2 but not X3 and X4; (vi) a polypeptide containing X1 and X3 but not X2 and X4; (vii) a polypeptide containing X1 and X4 but not X2 and X3; (viii) a polypeptide containing X2 and X3 but not X1 and X4; (ix) a polypeptide containing X2 and X4 but not X1 and X3; (x) a polypeptide containing X3 and X4 but not X1 and X2; (xi) a polypeptide comprising X1, X2, and X3, but not X4; (xii) a polypeptide comprising X1, X2, and X4, but not X3; (xiii) a polypeptide comprising X1, X3, and X4, but not X2; and (xiv) a polypeptide comprising X2, X3, and X4, but not X1; It may be selected from the group consisting of:

[0094] In another embodiment, X1, X2, X3, and X4 are each selected from the group consisting of SEQ ID NOs: 1 to 598, 627 to 631, 633, 635 to 641, 643 to 644, 648 to 658, 660, 662, 664 to 666, 668, 670, 672 to 675, 677 to 679, 681 to 685, 687 to 695, 698 to 702, 706 to 707, 709 to 711, 714 to 715, 717 to 735, 738 to 739, 742 to 743, 746 to 747, 748 to 749, 750 to 751, 752 to 753, 754 to 755, 756 to 757, 758 to 759, 760 to 761, 762 to 763, 764 to 765, 766 to 767, 768 to 769, 770 to 771, 772 to 773, 774 to 775, 776 to 777, 778 to 779, 781 to 782, 783 to 784, 785 to 786, 787 to 788, 789 to 790, 791 to 902, 904 to 906, 908 to 909, 910 to 911, 912 to 913, 914 to 915, 916 to 91 and 900-931.

[0095] 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 sequences can comprise RNA or DNA. Such isolated nucleic acid sequences may contain additional sequences useful for facilitating expression and / or purification of the encoded proteins; such additional sequences include, but are not limited to, polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, secretion signals, nuclear localization signals, and plasma membrane localization signals. It will be clear to those skilled in the art based on the present disclosure which nucleic acid sequences encode the polypeptides of the present invention.

[0096] In another aspect, the present disclosure provides an expression vector comprising a nucleic acid of any aspect of the present disclosure operably linked to a suitable control sequence. An "expression vector" includes a vector in which a nucleic acid coding region or gene is operably linked to any control sequence capable of effectively expressing the gene product. A "control sequence" operably linked to a nucleic acid sequence of the present invention is a nucleic acid sequence capable of effecting expression of the nucleic acid molecule. A control sequence need not be contiguous with the nucleic acid sequence, so long as it functions to direct its expression. Thus, for example, a transcribed, but not translated, intervening sequence may be present between the promoter sequence and the nucleic acid sequence; in this case, 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 virus-based expression vectors. The control sequences used to express the nucleic acid sequences of the present disclosure in mammalian systems can be constitutive (expression driven by any of a variety of promoters, including, but not limited to, CMV, SV40, RSV, actin, and EF) or inducible (expression driven by any of a number of inducible promoters, including, but not limited to, tetracycline, ecdysone, and steroid-responsive promoters). The expression vector must be replicable in the host organism, either episomally or by integration into host chromosomal DNA. In various embodiments, the expression vector may comprise a plasmid, a viral-based vector (including, but not limited to, a retroviral vector or an oncolytic virus), or any other suitable expression vector. In some embodiments, the expression vector can be administered by the methods of the present disclosure to express the polypeptide in vivo for therapeutic benefit.In non-limiting embodiments, the expression vectors can be used to transform or transduce cell therapy targets (including, but not limited to, CAR-T cells or tumor cells) to achieve the therapeutic methods disclosed herein.

[0097] In a further aspect, the present disclosure provides host cells comprising an expression vector, a polypeptide, a fusion protein, a first and / or second polypeptide, a polypeptide element, a conditionally active agonist and antagonist, and / or a nucleic acid, and an expression vector disclosed herein, wherein the host cell can be either prokaryotic or eukaryotic. The cells can be transiently or stably engineered to incorporate an expression vector of the invention using techniques such as, but not limited to, bacterial transformation, calcium phosphate co-precipitation, electroporation, or liposome-, DEAE-dextran-, polycation-, or viral-mediated transformation. (See, e.g., "Molecular Cloning: A Laboratory Manual" (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press); "Culture of Animal Cells: A Manual of Basic Techniques," 2nd ed. (RI Freshney, 1987, Liss, Inc. New York, NY)). A method for producing a polypeptide according to the invention is an additional part of the invention, said method comprising the steps of: (a) culturing a host according to this aspect of the invention under conditions that promote expression of said polypeptide; and (b) optionally recovering the expressed polypeptide. Although the expressed polypeptide can be recovered from a cell-free extract, it is preferable to recover the expressed polypeptide from the culture medium.

[0098] In another aspect, the present disclosure provides a pharmaceutical composition comprising a 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 composition of the present disclosure can be used, for example, in the disclosed methods described herein. The active agent of the present disclosure can be the only active agent in the composition or can be combined with one or more other 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 agent; (e) a stabilizer; (f) a preservative, and / or (g) a buffer.

[0099] In some embodiments, the buffer in the pharmaceutical composition is Tris buffer, histidine buffer, phosphate buffer, citrate buffer, or acetate buffer. The pharmaceutical composition may also contain a lyoprotectant, such as sucrose, sorbitol, or trehalose. In certain embodiments, the pharmaceutical composition contains a preservative, such as benzalkonium chloride, benzethonium, chlorhexidine, 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 contains a bulking agent, such as glycine. In yet another embodiment, the pharmaceutical composition comprises a surfactant, such as 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 trioleate, or a combination thereof. The pharmaceutical composition may also comprise an isotonicity agent, such as a compound that renders the formulation substantially isotonic or isoosmotic with respect to human blood. Examples of isotonicity agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition further comprises a stabilizer, e.g., a molecule that substantially prevents or reduces chemical and / or physical instability of the protein of interest when combined with the protein of interest in lyophilized or liquid form. Examples of stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0100] The polypeptide, fusion protein, first polypeptide, second polypeptide, recombinant nucleic acid, expression vector and / or recombinant host cell of any embodiment herein or combination of embodiments herein may be the only active agent in the composition, or the composition may further comprise one or more other active agents suitable for the intended use.

[0101] In another aspect, the disclosure provides methods of treating cancer, comprising administering to a subject having cancer an effective tumor-treating amount of a polypeptide agonist or fusion protein, conditionally active receptor agonist, recombinant nucleic acid, expression vector, recombinant host cell, and / or pharmaceutical composition of any of the embodiments of the IL-21 agonists herein. In various embodiments, the cancer is selected from the group consisting of colon cancer, melanoma, renal cell carcinoma, head and neck squamous cell carcinoma, gastric cancer, urothelial carcinoma, Hodgkin's lymphoma, non-small cell lung cancer, small cell lung carcinoma, hepatocellular carcinoma, pancreatic cancer, Merkel cell carcinoma, colorectal cancer, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, ovarian cancer, cervical cancer; and any type of tumor selected by a diagnostic test such as microsatellite instability, tumor mutation burden, PD-L1 expression level, or an Immunoscore assay (such as those developed by the American Society for Cancer Immunotherapy).

[0102] In another aspect, the disclosure provides a method of modulating an immune response in a subject, the method comprising a polypeptide antagonist of an IL-21 antagonist embodiment disclosed herein or a fusion protein thereof, a conditionally active receptor antagonist, a recombinant nucleic acid, an expression vector, a recombinant host cell, and / or a pharmaceutical composition. In one embodiment, the immune response is an anti-cancer immune response.

[0103] By "treat" or "treating" herein is meant to accomplish one or more of the following: (a) reducing the severity of the disorder; (b) reduce or prevent the occurrence of symptoms characteristic of the disorder being treated; (c) preventing the worsening of symptoms characteristic of the disorder during treatment; (d) reducing or preventing the recurrence of the disorder in patients who have previously suffered from the disorder; and (e) reducing or preventing the recurrence of symptoms in patients who were previously symptomatic for the disorder.

[0104] The subject can be any subject suffering from the relevant disorder, hi one embodiment, the subject is a mammal (including, but not limited to, humans, dogs, cats, horses, cows, etc.).

[0105] Example overview In this invention, we have generated highly stable de novo IL-21 mimetic proteins that are ideal for therapeutic use and that mimic the interaction with the receptor and biology of native IL-21 in humans and mice. The designed IL-21 mimetic proteins include agonists, antagonists, immunocytokines, or split agonists / antagonists, as detailed in the Examples and listed in Tables 1-6. Binding to two receptors was measured in two different settings: (i) for hIL-21R binding, in the presence of hIL-21R alone; and (ii) hγ c hγ for receptor binding c and in the presence of hIL-21R. c Regarding receptor binding, the presence of hIL-21R is required in the binding assay; c The affinity for hγ alone is very weak and therefore only valid in the presence of hIL-21R. c Binding measurements are possible.

[0106] hIL-21R binding, as described in (i), is measured by flow cytometry using yeast surface display or biolayer interferometry (BLI) as described in the Methods section. For yeast surface display, SA-PE-labeled hIL-21R is used at 1-1000 nM with anti-Myc FITC antibody to label cells expressing IL-21 mimetics that bind to hIL-21R. When tested using 1000 nM hIL-21R, proteins that bind to hIL-21R are detected by a PE+FITC+ population that accounts for 0.5% or more of the cell population gated on singlets. For BLI, the assay tip is loaded with hIL-21R and binding to IL-21 mimetics ranging from 100 pM to 1000 nM is tested. Alternatively, polyhistidine-tagged IL-21 mimetics can be loaded onto the assay tip, and their binding to 100-1000 nM hIL-21R is measured. Proteins that bind to hIL-21R exhibit a shift of at least 0.1 nm upon binding in either of the two cases described above.

[0107] The resulting yeast surface display data show results with 100 nM PE-labeled hIL-21R. The resulting BLI data show the hIL-21R loading assays in the ranges described above for appropriate Kd calculations and polyhistidine-tagged protein loading assays using 100 nM hIL-21R for binding studies. In both yeast surface display and BLI, mouse cross-reactivity in binding assays against mIL-21R behaved similarly to binding assays against hIL-21R, demonstrating similar results to those for the human receptor.

[0108] (ii) c Receptor binding was measured by flow cytometry using yeast surface display or biolayer interferometry (BLI) as described in the Methods section. For yeast surface display, 1-1000 nM of unlabeled hIL-21R and 1-1000 nM of SA-PE-labeled hγ were used in combination with anti-Myc FITC antibody. c receptor, hγ in the presence of hIL-21R cLabeling of cells expressing IL-21 mimics that bind to the receptor is performed. hγ in the presence of hIL-21R. c The receptor-binding proteins were 1000 nM hIL-21R and 1000 nM hγ c In the case of receptor-based assays, 0.5% or more of the cell population gated on singlets represents a PE+FITC+ population. c The receptor is loaded and tested for binding to IL-21 mimics ranging from 100 pM to 1000 nM in the presence of 1.5-fold higher concentrations of hIL-21R compared to the IL-21 mimic. c Proteins that bind to the receptor and hIL-21R show a shift of at least 0.1 nm upon binding.

[0109] The yeast surface display data obtained were obtained using 1000 nM unlabeled hIL-21R and 200 nM PE-labeled hγ c receptor, or 1000 nM unlabeled hIL-21R and 1000 nM PE-labeled hγ c The BLI data obtained is shown for receptors within the range of hγ c Methods for analyzing receptor loading are shown. Both yeast surface display and BLI assays showed that mγ c Mouse cross-reactivity of binding assays against receptors and mIL-21R was measured using hγ c The behavior was similar to that in the binding assay for the receptor, and the results were similar to those for the human receptor.

[0110] The mimic pairs are designed so that the agonistic mimics of IL-21 have two interfaces (i.e., one for IL-21R and the other for CD360). These designed agonists can bind to both receptors simultaneously, heterodimerizing the two receptor subunits. Furthermore, the designs exhibit cross-reactivity with the human and mouse receptors. The antagonistic mimics of IL-21 only retain the ability to bind to IL-21R and γ c It is designed not to bind to γ-agonists, which occupy the same space as agonists but are involved in heterodimerization of receptor subunits.c These designs also preserve cross-reactivity between humans and mice. IL-21 mimetic immunocytokines (antibody-fused IL-21 mimetics) are examples of molecular fusions for targeted cytokine delivery. Split IL-21 mimetics were designed with the goal of achieving targeted delivery of IL-21 mimetics with minimized off-target cytokine activity.

[0111] Design and optimization of early hits Based on the human IL-21 (hIL-21) / human IL-21R (hIL-21R) complex structure, we created a scaffold mimicking the hIL-21 helix-bundle structure with improved secondary structure packing for enhanced stability. We performed helix optimization by adjusting the length of the helix-bundle scaffold, minimizing unstructured regions, and removing non-ideal long loops in native hIL-21. Wild-type hIL-21 consists of four helices; these four helices include helix A (the first helix relative to the N-terminus), helix C (the third helix relative to the N-terminus; comprising the interface with hIL-21R), and helix D (the fourth helix relative to the N-terminus; hIL-21R). cThe top two helices, B (the second helix relative to the N-terminus) and C, are not long enough to form ideal intramolecular interactions or intermolecular contacts with the receptor chain. To optimize helices B and C, we extended them to replace most of the unstructured regions, including the two long unstructured regions between the helix A / helix B and helix C / helix D helix pairs, and made adjustments aimed at improving the packing of the other two helices (i.e., helices A and D). To design the de novo scaffold interface, we used Rosetta™ to transplant the interface residues of hIL-21 into structurally compatible positions in the scaffold and then designed the remaining residues for hIL-21R. The designed proteins were evaluated and screened using the Rosetta™ score metric; their folding trajectories were assessed using fast folding (Figure 1A).

[0112] Using yeast surface display as a screening platform, we identified five hits capable of binding to hIL-21R; however, none of them were able to bind to hγ c Our best hit (21d26; Table 5, SEQ ID NO: 627) did not bind to hγ c To further enhance binding to hIL-21R, we used directed evolution to identify hIL-21R-dependent hγ c We isolated mutants that exhibited binding. Among these mutations (Table 1, SEQ ID NOS: 1-8), we isolated variant 21JC15, which has four mutations relative to 21d26: W15C, W49R, Q73H, and H99G (W15C at residue 15 in the X1 domain (SEQ ID NOS: 936); W49R at residue 21 in the X2 domain (SEQ ID NOS: 933); Q73H at residue 16 in the X3 domain, agonist version (SEQ ID NOS: 934); and H99G at residue 16 in the X4 domain (SEQ ID NOS: 935)). This variant exhibited binding to hIL-21R and hIL-21R / hγ. cNot only does it bind to mIL-21R and mIL-21R / mγ c The results also showed that the antibody binds to the human and mouse receptors, demonstrating cross-reactivity between the two. Furthermore, we used site-saturation mutagenesis (SSM) to introduce mutations into the interface residues and individually evaluated their effects on affinity (Figure 5; Table 1, SEQ ID NOs: 9-36).

[0113] Agonist Through this analysis and the combination of these mutations, we isolated the optimized variant 21h10 (Table 1, SEQ ID NO: 24; Figures 1B and 1C), along with other variants (21m1-21m6 and 21h1-21h9; Table 1, SEQ ID NOs: 9-23). ​​This variant exhibited significantly higher affinity than its parent, 21JC15 (Table 1, SEQ ID NO: 8), while retaining cross-reactivity between the human and mouse receptors (Figures 1G-1J). Notably, 21h10 exhibited the best cross-reactivity and, surprisingly, shared only 44.9% sequence identity with hIL-21 and 23.5% sequence identity with mIL-21, as measured by BLASTP analysis. 21h10 was efficiently expressed in E. coli and exhibited monodispersity by size-exclusion chromatography (Figure 1D). Furthermore, circular dichroism analysis confirmed the presence of a helical secondary structure in the protein (Fig. 1E) and demonstrated its excellent thermal stability (Fig. 1F).

[0114] ProteinMPNN was used to redesign non-interface and some interface residues, allowing for possible substitutions at these positions to diversify the agonist and experimentally validate it (Table 1, SEQ ID NOS: 37-163). Furthermore, based on 21h10 (Table 1, SEQ ID NOS: 24), affinity variants were created by attenuating the affinity at both interfaces using SSM data (Table 1, SEQ ID NOS: 164-263; Figure 6) and ProteinMPNN (Table 1, SEQ ID NOS: 264-432). These affinity variants may alter the orientation and half-life of the receptor complex, which may affect EC50, Emax, signaling pathway bias, or further downstream effects that differentiate the phenotype from native IL-21 signaling. This confirms that the agonistic IL-21 mimic is stable, allowing for interface engineering to achieve various affinities at both interfaces. Native-like, high-affinity variants are useful for mimicking the full biology and activity of native IL-21, such as for the purpose of using these variants as substitutes for native IL-21, and have full cross-reactivity between humans and mice. Lower affinity variants may be less effective than native IL-21 or may have different functions than native IL-21; however, they are useful when an IL-21 mimetic construct fused to an antibody or other targeting domain requires biodistribution directed by the target binding affinity of the fused targeting domain rather than the affinity of the fused IL-21 mimetic for the IL-21 receptor. Furthermore, if the affinity of a variant is lower, such a variant may have a higher EC50 or lower Emax in cell signaling, which may be a desirable function in the application.

[0115] SEQ ID NOS: 1-8 in Table 1 are agonistic mimetics derived from 21d26 (Table 5, SEQ ID NOS: 627) and have a combination of mutations W15C, W49R, Q73H, and H99G; all four mutations were used in 21JC15 (Table 1, SEQ ID NOS: 8). SEQ ID NOS: 9-24 in Table 1 are agonistic mimetics optimized from 21JC15 (Table 1, SEQ ID NOS: 8) and have positive mutations in site-saturation mutagenesis (SSM) data. SEQ ID NOS: 25-36 in Table 1 are agonistic mimetics with combinations of mutations applied from 21JC15 (Table 1, SEQ ID NOS: 8) to 21h10 (Table 1, SEQ ID NOS: 24). SEQ ID NOS: 37-163 in Table 1 are agonistic mimetics derived from 21h10 (Table 1, SEQ ID NOS: 24) and have been shown to bind IL-21R (CD360) or γ by ProteinMPNN. c (CD132) SEQ ID NOS: 164-432 in Table 1 are agonistic affinity variant mimetics derived from 21h10 (Table 1, SEQ ID NOS: 24) that have been shown by site saturation mutagenesis (SSM) data or ProteinMPNN to have mutations introduced at residues that are not part of the interface that contribute to receptor binding. c (CD132) with mutations introduced at residues that are part of the interface residues that contribute to receptor binding.

[0116] In Table 1, Kd1 is the Kd for hIL-21R and represents the binding affinity for hIL-21R; on the other hand, Kd2 is the Kd for the IL-21 receptor complex (hIL-21R and hγ c receptor) and hγ c Both of the above Kds were measured by BLI. Q3 represents the binding affinity to the receptor; c The percentage of the PE+FITC+ population relative to the singlet-gated cell population at 1000 nM receptor; this is the percentage of hγ measured by flow cytometry in the presence of hIL-21R. cThese concentrations represent binding to the receptor. Only the data from the above concentrations, rather than the full range of 1-1000 nM, are shown in Table 5 because the concentrations used for the presented data provide the best measure of affinity compared to lower or higher concentrations within the range. Higher concentrations, up to 1000 nM, are best for identifying binder hits, while lower concentrations, down to 1 nM, are best for discovering the most affinity-optimized variants of the mimetic. The mouse receptor binding assay performed similarly to the human receptor binding assay, and the results were similar to those for the human receptor.

[0117] Retopology / Shuffle and Split Cytokines The designed agonists can shuffle their helical domains (X1, X2, X3, and X4) while maintaining their ability to heterodimerize receptor subunits. They retain most of the binding affinity at both interfaces required for agonism (Figure 2A). X2-X3-X4-X1, X3-X4-X1-X2, and X4-X1-X2-X3 demonstrate the feasibility of engineering modifications that maintain the defined molecular structure of the IL-21Rα (CD360) and γc (CD132) receptors while preserving both interfaces for agonistic function (Table 2). When shuffling and relinking helical domains, the polypeptide linkers are either computationally designed using Rosetta™ and ProteinMPNN or one of the following linkers: (GS)n or (GGS)n, with n = 2–10. The helical domain-shuffled designs bound to the IL-21 receptor, indicating that even when shuffled in any order other than the three shown, these helical domains are highly stable, retain their interface with the IL-21 receptor, and acquire new connectivity between the helices (retopology).

[0118] The sequences of the helix domain shuffled constructs are listed in Table 2. SEQ ID NOS: 433-445 in Table 2 are agonistic mimetics with helix domains shuffled in the order X2-X3-X4-X1. SEQ ID NOS: 480-569 in Table 2 are agonistic mimetics with helix domains shuffled in the order X2-X3-X4-X1, but with a mutation at X1. SEQ ID NOS: 446-457 in Table 2 are agonistic mimetics with helix domains shuffled in the order X3-X4-X1-X2. SEQ ID NOS: 458-479 in Table 2 are agonistic mimetics with helix domains shuffled in the order X4-X1-X2-X3. The linkers between the helical domains for religation were either generated using Rosetta™ or ProteinMPNN, or were one of the following types of (GS)n or (GGS)n linkers, with n=2-10.

[0119] This molecular stability correlates with the engineering potential of the molecule and can be extended to split cytokine mimetics for conditional cytokine delivery. For IL-21 mimetics, which are four-helix bundle proteins, there are three different ways to split these helices into two linear groups (fragment X and fragment Y): H1 / H234, H12 / H34, and H123 / H4 (Figure 2B; Table 4). This can be further diversified by shuffling the helical domains (Table 2). The application of these split IL-21 mimetics for targeted delivery involves fusing each of the split fragments X and Y to a targeting domain, such as a nanobody, that can direct the split cytokine mimic to desired cells or tissues (Figure 2C). Herein, we present examples of split cytokines fused to anti-HER2-DARPins or anti-EGFR-DARPins (Table 4, SEQ ID NOS: 613-624) with the goal of demonstrating that these IL-21 mimetics can be fused to such targeting domains of interest and 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 mimetic fragments can be fused to any available targeting domain.

[0120] The sequences of the split fragments of IL-21 mimetics and their fusion constructs are listed in Table 4. SEQ ID NOS: 607-612 in Table 4 are split fragments generated from 21h10 (Table 1, SEQ ID NOS: 24) as X1 (Table 4, SEQ ID NOS: 607), X1-X2 (Table 4, SEQ ID NOS: 608), X3-X4 (Table 4, SEQ ID NOS: 609), X4 (Table 4, SEQ ID NOS: 610), X1-X2-X3 (Table 4, SEQ ID NOS: 611), and X2-X3-X4 (Table 4, SEQ ID NOS: 612). SEQ ID NOS: 613-618 in Table 4 are split fragments (Table 4, SEQ ID NOS: 607-612) fused with anti-HER2-DARPin (Table 4, SEQ ID NOS: 625). SEQ ID NOs: 619-624 in Table 4 are split fragments (Table 4, SEQ ID NOs: 607-612) fused to anti-EGFR-DARPin (Table 4, SEQ ID NO: 626). The linker between the split fragment and the targeting domain of the IL-21 mimetic is one of the following linkers: (GS)n or (GGS)n, where n=2-10.

[0121] In Table 2, Kd1 is the Kd for hIL-21R and represents the binding affinity to hIL-21R; Kd1 is broken down into kon1 and kdis1; both are measured by BLI. kon1 is the rate of binding to hIL-21R, while kdis1 is the rate of dissociation from hIL-21R. In Table 4, Kd2 is the rate of binding to the IL-21 receptor complex (hIL-21R and hγR). c receptor) and hγ as measured by BLI. c It represents the binding affinity to the receptor.

[0122] antibody fusion To target specific cells or tissues of interest, we have developed various antibody-fused IL-21 mimetic constructs (Table 3). The IL-21 mimetic was fused to the antibody heavy chain. For cis-acting targeting of effector cells such as CD8+ T cells, we have developed anti-human PD-1 targeting antibody (pembrolizumab) fusions (Table 3, SEQ ID NOs: 572-576), anti-mouse PD-1 targeting antibody fusions (Table 3, SEQ ID NOs: 584-588), anti-human CD8 targeting antibody (RED8) fusions (Table 3, SEQ ID NOs: 594-598), and anti-mouse CD8 targeting antibody (G10.1) fusions (Table 3, SEQ ID NOs: 589-593). Trans-acting constructs, such as those targeting tumor tissue, include anti-PD-L1-targeting antibody (atezolizumab) fusions (Table 3, SEQ ID NOs: 577-581) and anti-TRP1-targeting antibody (TA99) fusions (Table 3, SEQ ID NOs: 582-583). One example is 21h10-fused atezolizumab, which can form either homodimers or heterodimers utilizing Knob / Hole mutations in the antibody heavy chain (Figure 3A), and exhibits the expected size on SDS-PAGE (Figure 3B). The atezolizumab-21h10 construct can bind to each of its domain targets: human IL-21R for 21h10 and human PD-L1 for atezolizumab (Figure 3C). Furthermore, this construct can simultaneously bind to both targets (Figure 3D). In addition to targeting antibodies, we have also engineered Fc fusions for the purpose of half-life extension (Table 3, SEQ ID NOs: 570-571), demonstrating that agonistic IL-21 mimetics can be fused to any available antibody or other targeting molecule to alter their biodistribution.

[0123] SEQ ID NOS: 570-571 in Table 3 are Fc fragment fusion constructs, each of which fused the agonistic mimetic 21AG-M-p3C1 (Table 1, SEQ ID NOS: 161) to either a human Fc fragment (Table 3, SEQ ID NOS: 599) or a murine Fc fragment (Table 3, SEQ ID NOS: 600). The linker between the IL-21 mimetic and the Fc fragment was one of the (GS)n or (GGS)n linkers, with n = 2-10. SEQ ID NOS: 572-576 and 601 in Table 3 are elements used to create fusion constructs of the IL-21 mimetic with pembrolizumab. Two identical copies of the pembrolizumab light chain (Table 3, SEQ ID NOS: 572) were paired with two identical or two different copies of the pembrolizumab heavy chain (Table 3, SEQ ID NOS: 573-576 and 601). SEQ ID NOS: 577-581, and 602 in Table 3 are elements used to create fusion constructs of IL-21 mimetics and atezolizumab. Two identical copies of the atezolizumab light chain (Table 3, SEQ ID NOS: 577) are paired with two identical copies or two different copies of the atezolizumab heavy chain (Table 3, SEQ ID NOS: 578-581, and 602). SEQ ID NOS: 582-583, and 603 in Table 3 are elements used to create fusion constructs of IL-21 mimetics and TA99. Two identical copies of the TA99 light chain (Table 3, SEQ ID NOS: 582) are paired with two identical copies or two different copies of the TA99 heavy chain (Table 3, SEQ ID NOS: 583 and 603). SEQ ID NOS: 584-588, and 604 in Table 3 are elements used to create fusion constructs between IL-21 mimics and anti-mouse PD-1 monoclonal antibodies. Two identical copies of the anti-mouse PD-1 monoclonal antibody light chain (Table 3, SEQ ID NOS: 584) are paired with two identical copies or two different copies of the anti-mouse PD-1 monoclonal antibody heavy chain (Table 3, SEQ ID NOS: 585-588, and 604). SEQ ID NOS: 589-593, and 605 in Table 3 are elements used to create fusion constructs between IL-21 mimics and the anti-mouse CD8 monoclonal antibody, G10.1. Two identical copies of the G10.1 light chain (Table 3, SEQ ID NOS: 589) are paired with two identical copies or two different copies of the G10.1 heavy chain (Table 3, SEQ ID NOS: 590-593, and 605).SEQ ID NOS:594-598, and 606 in Table 3 are elements used to generate fusion constructs of IL-21 mimetics and the anti-human CD8 monoclonal antibody, RED8. Two identical copies of the RED8 light chain (Table 3, SEQ ID NOS:594) are paired with two identical or two different copies of the RED8 heavy chain (Table 3, SEQ ID NOS:595-598, and 606).

[0124] In Table 3, for the atezolizumab fusion, Kd is measured for hIL-21R and the antibody target, human PD-L1. Atezolizumab-IL-21 mimetic fusion constructs are tested by BLI for hIL-21R binding and hPD-L1 binding. For hIL-21R binding assays, hIL-21R is immobilized on the SA tip, and the atezolizumab-21h10 fusion construct (Table 3, SEQ ID NOs: 577 and 578) is tested for binding and dissociation at concentrations ranging from 0.313 to 20 nM (Figure 3C). For hPD-L1 binding assays, hPD-L1 is immobilized on the SA tip, and the atezolizumab-21h10 fusion construct (Table 3, SEQ ID NOs: 577 and 578) is tested for binding and dissociation at concentrations ranging from 0.027 to 20 nM (Figure 3C). With regard to the binding ability where it simultaneously binds to both hIL-21R and hPD-L1, in the first experiment, hPD-L1 is immobilized at the assay end, followed by binding of the atezolizumab-21h10 fusion construct (Table 3, SEQ ID NOs: 577 and 578), followed by binding of hIL-21R; in the second experiment, hIL-21R is immobilized at the assay end, followed by binding of the atezolizumab-21h10 fusion construct (Table 3, SEQ ID NOs: 577 and 578), followed by binding of hPD-L1.

[0125] antagonist Antagonistic IL-21 mimetics are cIL-21R binding was assessed by flow cytometry using streptavidin R-phycoerythrin conjugate (SAPE)-labeled human or mouse IL-21R at 100-1000 nM and / or by biolayer interferometry using human or mouse IL-21R bearing an Avi tag loaded onto a streptavidin (SA) analytical tip.

[0126] Gc binding was assessed by flow cytometry using unlabeled human or mouse IL-21R at 10-1000 nM and streptavidin R-phycoerythrin conjugate (SAPE)-labeled human or mouse Gc at 10-1000 nM, and / or by biolayer interferometry using Avi-tagged human or mouse Gc loaded onto a streptavidin (SA) analytical tip. In the assay, soluble unlabeled human or mouse IL-21R was used at a 1.5:1 ratio of IL-21R:IL-21 mimic.

[0127] The creation of antagonists from agonist designs also means that IL-21 mimetics have excellent molecular stability; i.e., they can be coupled to γ-antigens without perturbing the overall molecular integrity or other interfaces (e.g., the IL-21R interface). c Disrupting the interface is often not possible for the stability of native IL-21. Based on the original prototype and design, four types of mimetics exist and are experimentally tested: (i) Initial antagonist hits (Table 5, SEQ ID NOs: 627-631, Figure 4A) and mimetics (Figure 4B) derived from agonist 21h10 (Table 1, SEQ ID NO: 24); these used: (ii) Rosetta™ (Table 5; SEQ ID NOs: 633, 635-641, 643-644, 648-658, 660, 662, 664-666, 668, 670, 672-675, 677-679, 681-685); (iii) SSM data (Table 5; 687, 688–695, 698–702, 706–707, 709–711, 714–715, 717–735, 738–739, 742–743, 746–747, 749–751); and (iv) Protein MPNN (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). The resulting antagonists can bind to IL-21R, but γ is required for IL-21 receptor complex formation. c This indicates that IL-21 mimetics are stable and can be conferred antagonistic properties through interface mutations.

[0128] SEQ ID NOS:627-631 in Table 5 are five antagonist hits obtained in the initial screen of IL-21 mimetic designs. SEQ ID NOS:632-687 in Table 5 (designated 21AT-Rp###) are mimetics derived from 21h10 (Table 1, SEQ ID NOS:24) and were screened by Rosetta™ using γ c SEQ ID NOs:688-751 in Table 5 (designated 21AT-Sp###) are mimetics derived from 21h10 (Table 1, SEQ ID NO:24) and have been shown to be γ-mutated based on site-saturation mutagenesis (SSM) data. c The mimetics in which the interface residues were mutated are shown in SEQ ID NOs: 752 to 907 in Table 5 (designated 21AT-Mp###), which are derived from 21h10 (Table 1, SEQ ID NO: 24) and are mutated by ProteinMPNN. c It is a mimic in which the interface residues are mutated.

[0129] In Table 5, Q1 is the percentage of the PE+FITC+ population relative to the singlet-gated cell population at 100 nM PE-labeled hIL-21R, and represents binding to hIL-21R measured by flow cytometry. Q2 is the percentage of the PE+FITC+ population relative to the singlet-gated cell population at 1000 nM PE-labeled hIL-21R, and represents binding to hIL-21R measured by flow cytometry. c The percentage of the PE+FITC+ population relative to the singlet-gated cell population at 200 nM receptor, as measured by flow cytometry, represents the increase in hγ in the presence of hIL-21R. c These concentrations represent binding to the receptor. Only the data from the above concentrations, rather than the full range of 1-1000 nM, are shown in Table 5 because the concentrations used for the presented data provide the best measure of affinity compared to lower or higher concentrations within the range. Higher concentrations, up to 1000 nM, are best for identifying binder hits, while lower concentrations, down to 1 nM, are best for discovering the most affinity-optimized variants of the mimetic. The mouse receptor binding assay performed similarly to the human receptor binding assay, and the results were similar to those for the human receptor.

[0130] others Genetic fusion or chemical conjugation of functional domains for protein half-life extension or other applications is often explored for protein-based therapeutics. We designed and tested albumin-binding domain fusion constructs for half-life extension (Table 6, SEQ ID NOS: 908-909). We also designed multiple Cys- and / or Lys-relocated variants (Table 6, SEQ ID NOS: 910-931) of 21h10 (Table 1, SEQ ID NOS: 24) or 21AG-M-p3C1 (Table 1, SEQ ID NOS: 161). These variants can be chemically conjugated to PEG for half-life extension, and, if chemical conjugation sites are present within the interface of the IL-21 mimetic, can be chemically conjugated to other functional domains for conditional activation. 21h10 (Table 1, SEQ ID NOS: 24) bearing Cys15 could be chemically conjugated to PEG of various sizes to increase molecular size. The above confirms that IL-21 mimetics can be fused to antibodies or other functional domains, along with the targeting domains described above, by genetic fusion or chemical conjugation in order to extend the half-life of the IL-21 mimetics or for other functional benefits.

[0131] SEQ ID NOS: 908-909 in Table 6 are albumin-binding peptide fusion constructs in which 21AG-M-p3C1 (Table 1, SEQ ID NOS: 161) is fused to the albumin-binding peptide G148ABD3 (Table 6, SEQ ID NOS: 932), which was designed for half-life extension. The G148ABD3 domain binds to albumin in vivo, increasing the overall molecular weight and size, thereby enabling the half-life extension of the construct. SEQ ID NOs:910-931 in Table 6 are agonistic mimetics derived from 21h10 (Table 1, SEQ ID NO:24) or 21AG-M-p3C1 (Table 1, SEQ ID NO:161) with Lys (K) or Cys (C) added or removed at their non-interface residues (Table 6, SEQ ID NOs:910-925) or interface residues (Table 6, SEQ ID NOs:926-931) for chemical conjugation to functional domains (including, but not limited to, PEG, peptides, and small molecules) for various purposes (such as extended half-life and conditional activation).

[0132] method Computational design of de novo IL-21 mimetics To design mimetics of native IL-21, we used the crystal structure of hIL-21, including hIL-21R (PDB:3TGX). IL-21-like scaffolds were generated using PyRosetta™ scripts that implement the PDBInfoLabel metadata. The following residues in human IL-21 were 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. Non-fixed residues were designed using Rosetta™ FastDesign and relaxed using Rosetta™ FastRelax with a scoring function of "beta_nov16". The above designs achieved a Rosetta™ score metric of: packstat>0.6, score_per_ residue <-2.3, and sspred>0.8, Rapid folding was used to screen the designs based on their folding probability.

[0133] Yeast surface display and fluorescence-activated cell sorting The amino acid sequence of the designed protein was reverse-translated into a DNA sequence based on the Saccharomyces yeast codon frequency table; pETcon3™ vector homologous sequences were added to the 5' and 3' ends, respectively. Using competent yeast cells EBY100, the designed DNA gene block was cloned into a linearized pETcon3™ vector by yeast homologous recombination. The vector was linearized by 100-fold overdigestion with NdeI and XhoI (New England Biolabs) and then purified by gel extraction (Qiagen). Yeast transformation was verified by colony sequencing. Verified colonies were grown in SDCAA medium (2.0% glucose, 0.67% yeast nitrogen base, 0.5% casamino acids, 0.54% disodium phosphate, 0.86% monosodium phosphate) and induced in SGCAA medium (2% glucose, 0.67% yeast nitrogen base, 0.5% casamino acids, 0.54% NaHPO, 0.86% NaHPO). SGCAA induction resulted in the expression of the designed proteins, which were then displayed on the yeast surface by the Aga2p membrane protein. For all flow cytometry analyses, the induced cells were resuspended in running buffer (phosphate-buffered saline (PBS) + 1% w / v bovine serum albumin, pH 7.2–7.4). Cells expressing the designed proteins on their cell surface were labeled with two fluorophores: FITC and PE. To demonstrate protein expression, the myc tag was labeled using an anti-c-myc mAb conjugated with FITC. For IL-21R labeling, either human or mouse IL-21R with a human Fc tag (R&D Systems 991-R2, R&D Systems 596-MR) was used. For receptor complex labeling, human IL-21R with a His tag (R&D Systems 9249-R2) and human γc with an Fc tag (Acro Biosystems ILG-H5256) were used, or mouse IL-21R with a His tag (Sino Biological 51184-M08H) or mouse γc with an Fc tag (R&D Systems 784-MR) were used. cWe used either the Fc domain or the ATP-binding protein (ATP-binding protein). Target receptors fused to the Fc domain were labeled with the biotinylated ZZ domain of protein A and streptavidin-PE (SA-PE), which can bind to the biotin in protein A. PE represents the level of receptor binding. For binding hits, their signal is proportional to the protein expression level. Yeast cells were analyzed using a flow cytometer (BD Accuri C6, Thermo Fisher Attune NxT) and sorted using a fluorescence-activated cell sorting (FACS) cell sorter (Sony SH800).

[0134] Directed Evolution and Design Optimization 21d26 DNA was amplified using a random mutagenesis library kit (Agilent 200550), and the resulting library was partially sequenced using BL21(DE3) (New England Biolabs). This sequencing revealed that approximately 2-4 mutations had been introduced into the design by error-prone PCR amplification of DNA. The library was then transformed into yeast using electroporation (approximate diversity: 3.4E7). For directed evolution, the yeast library was sorted using FACS with different combinations and concentrations of the tagged receptor subunits described above.

[0135] To analyze and enhance the interfacial affinity at the 21JC15 interface, we used site-saturation mutagenesis (SSM) to dissect the two interfaces of the protein. The protein sequence of interest was divided into halves with corresponding single mutations and synthesized as two fragments for assembly (Integrated DNA Technologies). Assembly was performed by PCR using 5' and 3' primers specific to pETcon3™. Sorting four different receptor conditions ((1) tagged human IL-21R, (2) untagged human IL-21R in combination with tagged human γc, (3) tagged mouse IL-21R, and (4) untagged mouse IL-21R in combination with tagged mouse γc) allowed us to saturate variants with favorable mutations and deplete variants with unfavorable mutations. DNA for the selected SSM library was prepared using Zymoprep™ (Zymo Research), qPCR, and gel extraction (Qiagen) prior to MiSeq™ (Illumina). The selected SSM library contained 1000 nucleotides of each receptor (human IL-21R, human γc, mouse IL-21R, and mouse γ c Several mutations were identified that could improve binding affinity to ATP.

[0136] We constructed a combinatorial library of mutations by mixing positive mutations using PCR assembly; this PCR assembly used a pool of eight sets of primers (Integrated DNA Technologies) that introduced mutations with degenerate codons. Then, we sorted against either human or mouse receptors. This yielded 16 candidates with significantly improved affinity for the receptors: 10 candidates (21h1–21h10) for human receptor optimization and 6 candidates (21m1–21m6) for mouse receptor optimization. We measured the Kd of each of the 16 candidates for each receptor using biolayer interferometry (ForteBio, see Methods).

[0137] Protein expression of cytokine mimetics and split cytokine mimetics (E. coli expression) The DNA fragments encoding the designed proteins (Integrated DNA Technologies) were cloned into the pET-29b(+) plasmid with an N-terminal polyhistidine tag. The cloned plasmids were transformed into BL21 competent cells (New England Biolabs), and the cultures were grown in Terrific Broth II and induced with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG). The induced cultures were placed in baffled flasks and incubated at approximately OD . A600 The cultures were grown at 37°C on a 225 rpm shaker until a pH of 0.8 was reached. The cultures were induced at 18°C ​​for 14 hours. Each harvested culture was lysed by sonication (Qsonica™ Q500). The lysed cultures were ultracentrifuged at 18,000 g for 30 minutes and purified using immobilized metal affinity chromatography (IMAC). The eluted proteins were separated by HPLC size exclusion chromatography using a Superdex™ 75 10 / 300 GL column (GE Healthcare) on an Äkta™ (GE Healthcare Äkta™ Pure).

[0138] Immunocytokine (antibody-cytokine fusion) protein expression (mammalian expression) Antibody-cytokine fusion constructs were synthesized in the CMVR plasmid (Genscript). The cytokine mimic was fused to the C-terminus of the antibody heavy chain via a linker. The fusion constructs were expressed in HEK293 cells and purified by protein A capture (Genscript). Purification was performed using SDS-PAGE and SEC-HPLC.

[0139] Biolayer Interferometry Binding data were collected using OctetRED96 and OctetR8 (Sartorius). All biolayer interferometry analyses were performed using octet binding buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) + 1% w / v bovine serum albumin, pH 7.2–7.4). For binding affinity measurements to hIL-21R or mIL-21R, streptavidin-coated biosensors (SAForteBio) were immobilized with 5 μg / ml biotinylated hIL-21R (R&D Systems AVI9249) or biotinylated mIL-21R (Acro Biosystems ILR-M82E3) in octet binding buffer (0.8–1.0 nm shift depending on loading). Serial two-fold dilutions of ligand starting from 1 μM were used; a 300-second binding step and a 300-second dissociation step were used. Similarly, hIL-21Ra / hγ c or mIL-21Ra / mγ c To measure the binding affinity to each of the 5 μg / ml biotinylated hγ c (Acro Biosystems ILG-H85E8) or biotinylated myoglobin c Immobilization was performed in octet binding buffer containing IL-21Ra (Acro Biosystems ILA-M82E3) and in the presence of a 1.5-fold molar excess of the corresponding hIL-21Ra (R&D Systems 9249-R2) or mIL-21Ra (Sino Biological 51184-M08H) for 250 seconds of binding and 250 seconds of dissociation. For loading of polyhistidine-tagged IL-21 mimics, an anti-pentaHis (His1K) analytical tip was used for immobilization (1.0–1.5 nm shift depending on loading). Ligands used were hIL-21 (R&D Systems 8879-IL), mIL-21 (R&D Systems 594-ML), or IL-21 mimics (see Methods). Data were processed using ForteBio data analysis software (version 9.0.0.10), and parameters are displayed with standard errors.

[0140] Circular dichroism and thermal stability assays Far-UV circular dichroism measurements were performed using a spectropolarimeter (JASCO J-1500 spectropolarimeter). Protein was dissolved at 0.5 mg / mL in PBS (pH 7.4) in a 1 mm pathlength cuvette. Wavelengths were scanned from 195 nm to 260 nm. Thermal melting experiments were performed by heating the sample from 25 °C to 95 °C and then cooling it to 25 °C while monitoring the absorbance at 222 nm.

[0141] References 1. Hashmi, MH & Van Veldhuizen, PJ 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,TMet 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).

[0142] [Table 1] JPEG2025535705000002.jpg 214159 JPEG2025535705000003.jpg 214159 JPEG2025535705000004.jpg 215159 JPEG2025535705000005.jpg 213159 JPEG2025535705000006.jpg 215159 JPEG2025535705000007.jpg 215159 JPEG2025535705000008.jpg 215159 JPEG2025535705000009.jpg 216159 JPEG2025535705000010.jpg 215159 JPEG2025535705000011.jpg 215159 JPEG2025535705000012.jpg 215159 JPEG2025535705000013.jpg 214159 JPEG2025535705000014.jpg 215159 JPEG2025535705000015.jpg 215159 JPEG2025535705000016.jpg 214159 JPEG2025535705000017.jpg 214159 JPEG2025535705000018.jpg 215159 JPEG2025535705000019.jpg 214159 JPEG2025535705000020.jpg 214159 JPEG2025535705000021.jpg 214159 JPEG2025535705000022.jpg 188159

[0143]

Table 2

[0144]

Table 3

[0145]

Table 4

[0146]

Table 5

[0147]

Table 6

Claims

1. A polypeptide comprising domain X2, domain X3, and optionally domain X4; where (a) X2 is a peptide 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO:933); (b) X3 is: (i) KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934), or (ii) KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937); a peptide comprising an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of Here, in comparison with SEQ ID NO:937: The residue at position 4 is Y; The residue at position 6 is F; and / or The residue at position 9 is D; One, two, or all three of the following are true: and (c) X4 is optional and, when present, includes a helix structure, but is: wherein X2, X3, and X4, if present, may be in any order within the polypeptide; wherein there may be an amino acid linker anywhere between the domains; and wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21Rα, CD360). Polypeptide.

2. 10. The polypeptide of claim 1 ; wherein the polypeptide comprises domain X2, domain X3, and domain X4; where (a) X2 is a peptide 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO:933); (b) X3 is a peptide 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 KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934); and (c) X4 is a peptide 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 KEVMERAKSAAQKILGRFL (SEQ ID NO: 935); wherein X2, X3, and X4 may be in any order within the polypeptide; wherein there may be an amino acid linker anywhere between the domains; and wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21Rα, CD360) and also to the γc and CD132 receptors; Polypeptide.

3. 3. The polypeptide of claim 1 or 2, wherein the polypeptide has, 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, A polypeptide having amino acids selected from residues which are:

4. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide has, relative to X2 (SEQ ID NO:933): (a) residue 11 of X2 is K, E, or R; (b) residue 14 of X2 is R, E, or K; (c) residue 18 of X2 is V, R, H, or K; and / or (d) residue 21 of X2 is R or K; having one, two, three, or all four of the residues Polypeptide.

5. A polypeptide according to any one of claims 1 to 4, wherein the polypeptide has, relative to X2 (SEQ ID NO:933): (a) residue 4 of X2 is I or W; (b) residue 7 of X2 is V, D, or E or I; (c) residue 10 of X2 is K or R; and / or (d) residue 15 of X2 is A, I, or L or M; having one, two, three, or all four of the residues Polypeptide.

6. A polypeptide according to any one of claims 2 to 5, wherein the polypeptide has, as compared 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, and having amino acids selected from residues Polypeptide.

7. A polypeptide according to any one of claims 2 to 6, wherein the polypeptide has, as compared to X3 (SEQ ID NO:934): (a) residue 2 of X3 is R or K; (b) residue 6 of X3 is R or K; (c) residue 8 of X3 is R or K; (d) residue 12 of X3 is D or E; and / or (e) residue 16 of X3 is H or Y or W; containing one, two, three, or all four of the residues Polypeptide.

8. A polypeptide according to any one of claims 2 to 7, wherein the polypeptide has, as compared to X3 (SEQ ID NO:934): (a) residue 1 of X3 is K or R; (b) residue 5 of X3 is I or V; (c) residue 9 of X3 is Q or G; (d) residue 11 of X3 is I or L; (e) residue 13 of X3 is I or V; and / or (f) residue 19 of X3 is R or K; and wherein the amino acid sequence is: Polypeptide.

9. A polypeptide according to any one of claims 2 to 8, wherein the polypeptide has, as compared 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, and having amino acids selected from residues Polypeptide.

10. A polypeptide according to any one of claims 2 to 9, wherein the polypeptide has, as compared to X4 (SEQ ID NO:935): (a) residue 9 of X4 is S or T; and / or (b) residue 16 of X4 is G or Y; and wherein the residues are Polypeptide.

11. A polypeptide according to any one of claims 2 to 10, wherein the polypeptide has, as compared to X4 (SEQ ID NO:935): (a) residue 8 of X4 is K or R; (b) residue 12 of X4 is Q; (c) residue 13 of X4 is K; (d) residue 15 of X4 is L or I; and / or (e) residue 19 of X4 is L or F / Y; and wherein the amino acid sequence is: Polypeptide.

12. A polypeptide according to any one of claims 2 to 11, wherein the polypeptide is: (i) compared to X2 (SEQ ID NO:933): (a) residue 11 of X2 is K, E, or R; (b) residue 14 of X2 is R, E, or K; (c) residue 18 of X2 is V, R, H, or K; and (d) residue 21 of X2 is R or K; (e) residue 4 of X2 is I or W; (f) residue 7 of X2 is V, D or E or I; (g) residue 10 of X2 is K or R; and (h) residue 15 of X2 is A, I, or L or M; comprising a residue which is (ii) compared to X3 (SEQ ID NO:934): (a) residue 2 of X3 is R or K; (b) residue 6 of X3 is R or K; (c) residue 8 of X3 is R or K; (d) residue 12 of X3 is D or E; and (e) residue 16 of X3 is H or Y or W; (f) residue 1 of X3 is K or R; (g) residue 5 of X3 is I or V; (h) residue 9 of X3 is Q or G; (i) residue 11 of X3 is I or L; (j) residue 13 of X3 is I or V; and (k) residue 19 of X3 is R or K; comprising a residue which is and (iii) compared to X4 (SEQ ID NO: 935): (a) residue 9 of X4 is S or T; and (b) residue 16 of X4 is G or Y; and (c) residue 8 of X4 is K or R; (d) residue 12 of X4 is Q; (e) residue 13 of X4 is K; (f) residue 15 of X4 is L or I; and (g) residue 19 of X4 is L, F, or Y; containing a residue which is Polypeptide.

13. 10. The polypeptide of claim 1 ; where (a) X2 is a peptide 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 DTRILKVSLKKIVRAANVIVRM (SEQ ID NO:933); and (b) X3 is a peptide 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 KRFYIFMQDLIDIVTHVKRE (SEQ ID NO: 937); Here, in comparison with SEQ ID NO:937: The residue at position 4 is Y; The residue at position 6 is F; and / or The residue at position 9 is D; one, two, or all three of the items in are true; wherein X2 and X3 may be in any order within the polypeptide; wherein there may be an amino acid linker between the domains; and wherein the polypeptide is capable of binding to IL-21 receptor (IL-21Rα, CD360) but is not capable of simultaneously binding to γc, CD132 receptor; Polypeptide.

14. 14. The polypeptide of claim 13, wherein compared to SEQ ID NO: 937: Residue 4 is Y; residue 6 is F; and / or Residue 9 is D, two or all three of the following are true: Polypeptide.

15. 14. The polypeptide of claim 13, wherein compared to SEQ ID NO: 937: Residue 4 is Y; residue 6 is F; and / or Residue 9 is D, All three of the statements are true. Polypeptide.

16. A polypeptide according to any one of claims 13 to 15, wherein the polypeptide has, 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, and having amino acids selected from residues Polypeptide.

17. A polypeptide according to any one of claims 13 to 16, wherein the polypeptide has, relative to X2 (SEQ ID NO:933): (a) residue 11 of X2 is K, E, or R; (b) residue 14 of X2 is R, E, or K; (c) residue 18 of X2 is V, R, H, or K; and / or (d) residue 21 of X2 is R or K; A polypeptide having one, two, three, or all four of the residues:

18. A polypeptide according to any one of claims 13 to 17, 1. The polypeptide, as compared to X2 (SEQ ID NO: 933): (a) residue 4 of X2 is I or W; (b) residue 7 of X2 is V, D, or E or I; (c) residue 10 of X2 is K or R; and / or (d) residue 15 of X2 is A, I, or L or M; A polypeptide having one, two, three, or all four of the residues:

19. A polypeptide according to any one of claims 13 to 18, wherein the polypeptide has, as compared 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; 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: 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, and having amino acids selected from residues Polypeptide.

20. 20. The polypeptide according to any one of claims 13 to 19, wherein the polypeptide has, as compared to X3 (SEQ ID NO:937): (a) residue 2 of X3 is R or K; (b) residue 12 of X3 is D or E; and / or (c) residue 16 of X3 is H, Y, or W; and having one, two, or all three of the residues Polypeptide.

21. A polypeptide according to any one of claims 13 to 20, wherein the polypeptide has, as compared to X3 (SEQ ID NO:937): (a) residue 1 of X3 is K or R; (b) residue 5 of X3 is I or V; (c) residue 11 of X3 is I or L; (d) residue 13 of X3 is I or V; and / or (e) residue 19 of X3 is R or K; having one, two, three, four, or all five of the residues Polypeptide.

22. A polypeptide according to any one of claims 13 to 21, where (i) compared to X3 (SEQ ID NO:937): (a) all three of the following are true: residue 4 is Y; residue 6 is F; and / or residue 9 is D; (b) residue 2 of X3 is R or K; (c) residue 12 of X3 is D or E; (d) residue 16 of X3 is H, Y, or W; (e) residue 1 of X3 is K or R; (f) residue 5 of X3 is I or V; (g) residue 11 of X3 is I or L; (h) residue 13 of X3 is I or V; and (i) residue 19 of X3 is R or K; and and (ii) compared to X2 (SEQ ID NO:933): (a) residue 11 of X2 is K, E, or R; (b) residue 14 of X2 is R, E, or K; (c) residue 18 of X2 is V, R, H, or K; and (d) residue 21 of X2 is R or K; (e) residue 4 of X2 is I or W; (f) residue 7 of X2 is V, D, or E or I; (g) residue 10 of X2 is K or R; and (h) residue 15 of X2 is A, I, or L, or M; That is, Polypeptide.

23. A polypeptide according to any one of claims 13 to 22, further comprising an X4 domain; wherein the presence of the X4 domain precludes simultaneous binding of the polypeptide to the IL-21 receptor (IL-21Rα, CD360) and the γc, CD132 receptor; and wherein X2, X3, and X4 can be in any order within the polypeptide; Polypeptide.

24. 24. The polypeptide of claim 23, wherein X4 comprises a 19 residue peptide; and wherein the peptide is: 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, A polypeptide having an amino acid at each position selected from the amino acid residues:

25. A polypeptide according to any one of claims 1 to 24, further comprising an X1 domain; wherein the X1 domain comprises a helical structure; and wherein X1, X2, X3, and X4, if present, can be in any order within the polypeptide; Polypeptide.

26. 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). Polypeptide.

27. 27. The polypeptide of claim 26, wherein the polypeptide has, 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, and having amino acids selected from residues Polypeptide.

28. 28. The polypeptide of claim 26 or 27, wherein the polypeptide does not have a cysteine ​​residue at position 11 compared to X1 (SEQ ID NO: 936). Polypeptide.

29. A polypeptide according to any one of claims 1 to 28, wherein the polypeptide comprises in the domain order X2-X3-X4, or X1-X2-X3, or X2-X3, or X1-X2-X3-X4, Polypeptide.

30. A polypeptide according to any one of claims 1 to 15, wherein the polypeptide comprises the domains in the 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, Polypeptide.

31. A fusion protein comprising: (a) a polypeptide according to any one of claims 1 to 30; and (b) one or more functional domains; A fusion protein comprising:

32. 19. The fusion protein of claim 18, wherein the one or more functional domains comprise a cell targeting domain or a detectable domain. fusion protein.

33. 33. The fusion protein of claim 31 or 32, wherein the one or more functional domains comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 599-606. fusion protein.

34. A polypeptide or fusion protein according to any one of claims 1 to 33, comprising: (a) SEQ ID NOs: 1 to 598, 627 to 631, 633, 635 to 641, 643 to 644, 648 to 658, 660, 662, 664 to 666, 668, 670, 672 to 675, 677 to 679, 681 to 685, 687 to 695, 698 to 702, 706 to 707, 709 to 711, 714 to 715, 717 to 735, 738 to 739, 742 to 743, 746 to 747, 749 to 798, 800 to 8 an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of: 11, 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) SEQ ID NOs: 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- an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of: 743, 746, 749-751, 771, 849-854, 856-862, 864-870, 872-874, 876-898, 900, and 902-907; or (c) an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 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; A polypeptide or fusion protein comprising:

35. a conditionally active IL-21 receptor binding protein, said IL-21 receptor binding protein comprising a first polypeptide element and a second polypeptide element; wherein both said first polypeptide element and said second polypeptide element are not both present in the same fusion protein; wherein said first polypeptide element and said second polypeptide element together comprise domains X2, X3, and optionally X4, as defined in any one of claims 1 to 34; where (i) the first polypeptide element comprises at least one of X2, X3, and X4 (if present), but not each of X2, X3, and X4 (if present); and (ii) the second polypeptide element comprises each of X2, X3, and X4 (if present) that is not present in the first polypeptide element; wherein said first polypeptide element and said second polypeptide element are not individually active receptor-binding proteins; wherein the first polypeptide element and the second polypeptide interact to form an active IL-21 receptor binding protein capable of binding to the IL-21 receptor (IL-21Rα, CD360). IL-21 receptor binding protein.

36. 36. The conditionally active IL-21 receptor binding protein of claim 35, comprising: where (i) the first polypeptide element comprises at least one of X2, X3, and X4, but not each of X2, X3, and X4; (ii) the second polypeptide element comprises each of X2, X3, and X4 that is not present in the first polypeptide element; wherein the first polypeptide element and the second polypeptide interact to form an active IL-21 receptor binding protein capable of binding to the IL-21 receptor (IL-21Rα, CD360) and to the γc, CD132 receptor. IL-21 receptor binding protein.

37. 36. The conditionally active IL-21 receptor binding protein of claim 35, wherein the IL-21 receptor binding protein comprises a first polypeptide element and a second polypeptide element; wherein said first polypeptide element and said second polypeptide element are not both present within the same fusion protein; wherein the first polypeptide element and the second polypeptide element together comprise domains X1, X2, X3, and X4 as defined in any one of claims 1 to 34; where (i) the first polypeptide element comprises at least one of X1, X2, X3, and X4, but not each of X1, X2, X3, and X4; and (ii) the second polypeptide element comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide element; wherein the first polypeptide element and the second polypeptide interact to form an active IL-21 receptor binding protein capable of binding to the IL-21 receptor (IL-21Rα, CD360) and to the γc, CD132 receptor. IL-21 receptor binding protein.

38. 38. The conditionally active IL-21 receptor binding protein of any one of claims 35 to 37, wherein the first and second polypeptides together collectively comprise: (a) SEQ ID NOs: 1 to 598, 627 to 631, 633, 635 to 641, 643 to 644, 648 to 658, 660, 662, 664 to 666, 668, 670, 672 to 675, 677 to 679, 681 to 685, 687 to 695, 698 to 702, 706 to 707, 709 to 711, 714 to 715, 717 to 735, 738 to 739, 742 to 743, 746 to 747, 749 to 798, 800 to 8 an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of: 11, 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) SEQ ID NOs: 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- an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of: 743, 746, 749-751, 771, 849-854, 856-862, 864-870, 872-874, 876-898, 900, and 902-907; or (c) an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 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; Including, IL-21 receptor binding protein.

39. 39. The conditionally active IL-21 receptor binding protein of any one of claims 35 to 38, wherein said first and second polypeptides are: (a) (i) a first polypeptide comprising an amino acid sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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; Including, IL-21 receptor binding protein.

40. 40. The conditionally active IL-21 receptor binding protein of any one of claims 35 to 39, wherein one or both of said first polypeptide and said second polypeptide are fused to a targeting domain, including but not limited to, an antibody or a nanobody; IL-21 receptor binding protein.

41. A non-naturally occurring polypeptide comprising domains X1, X2, X3, and X4; where (a) 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); (b) X2 is a peptide 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 DTRILKVSLKKIVRA (SEQ ID NO:933); (c) X3 is a peptide 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 KRFEIRMRQLIDIVDHVKRE (SEQ ID NO: 934); and (d) X4 is a peptide 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 KEVMERAKSAAQKILGRFL (SEQ ID NO: 935); wherein X1, X2, X3, and X4 may be in any order within the polypeptide; wherein there may be an amino acid linker anywhere between the domains; and wherein the polypeptide is capable of binding to the IL-21 receptor (IL-21Rα, CD360). Non-naturally occurring polypeptides.

42. 42. The polypeptide of claim 41 ; where residues 1-16 of X1 are: 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, Selected from: Polypeptide.

43. 43. The polypeptide of claim 41 or 42, wherein residue 16 of X1 is not A. Polypeptide.

44. 44. The polypeptide according to any one of claims 41 to 43, where residues 1-15 of X2 are: 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, Selected from: Polypeptide.

45. 45. A polypeptide according to any one of claims 41 to 44, where (a) Residue 3 of X2 is not R; (b) residue 4 of X2 is not I; (c) residue 7 of X2 is not V; and / or (d) residue 8 of X2 is not S; One, two, three, or all four of the following are true: Polypeptide.

46. 46. ​​A polypeptide according to any one of claims 41 to 45, where residues 1-20 of X3 are: 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; Residue 10: 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, A polypeptide selected from:

47. 47. A polypeptide according to any one of claims 41 to 46, where (a) residue 2 of X3 is not R; (b) residue 8 of X3 is not R; (c) residue 9 of X3 is not Q; (d) residue 10 of X3 is not L; (e) residue 11 of X3 is not I; (f) residue 14 of X3 is not V; and / or (g) residue 15 of X3 is not D; One, two, three, four, five, six, or all seven of the following are true: Polypeptide.

48. 48. A polypeptide according to any one of claims 41 to 47, where residues 1-19 of X4 are: 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, A polypeptide selected from:

49. 49. A polypeptide according to any one of claims 41 to 48, where (a) residue 8 of X4 is not K; (b) residue 9 of X4 is not S; and / or (c) residue 19 of X4 is not L; One, two, three, four, five, six, or all seven of the following are true: Polypeptide.

50. 50. The polypeptide of any one of claims 41 to 49, wherein the polypeptide is an antagonist of the IL-21 receptor. Polypeptide.

51. 51. The polypeptide of any one of claims 41 to 50, wherein residue 16 of X4 is G.

52. 52. The polypeptide of claim 51, wherein residue 11 of X1 is C.

53. 53. A polypeptide according to any one of claims 41 to 52, wherein the domains are arranged from N-terminus to C-terminus 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. Polypeptide.

54. 54. A polypeptide according to any one of claims 41 to 53, A polypeptide wherein there is an amino acid linker between the domains.

55. 55. A polypeptide according to any one of claims 41 to 54, wherein the polypeptide comprises an amino acid sequence that is 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. Polypeptide.

56. 56. The polypeptide of claim 55, where the residues at the non-helical positions are: 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, The polypeptide may be selected from the group consisting of:

57. 57. A polypeptide according to any one of claims 41 to 56, comprising: SEQ ID NOs: 1 to 598, 627 to 631, 633, 635 to 641, 643 to 644, 648 to 658, 660, 662, 664 to 666, 668, 670, 672 to 675, 677 to 679, 681 to 685, 687 to 695, 698 to 702, 706 to 707, 709 to 711, 714 to 715, 717 to 735, 738 to 739, 742 to 743, 746 to 747, 749 to 798, 800 to 811, comprising an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of: 813-821, 823, 826, 829, 831-832, 834-835, 843, 845-847, 849-854, 856-862, 864-874, 876-898, and 900-931; Polypeptide.

58. 58. A polypeptide according to any one of claims 41 to 57, Further comprising one or more functional domains, Polypeptide.

59. 59. The polypeptide of claim 58, wherein said one or more functional domains are translationally fused with said polypeptide. Polypeptide.

60. A conditionally active non-naturally occurring IL-21 receptor binding protein, the IL-21 receptor binding protein comprising a first polypeptide element and a second polypeptide element; wherein said first polypeptide element and said second polypeptide element are not present in a fusion protein; wherein the first polypeptide element and the second polypeptide element together comprise domains X1, X2, X3, and X4 as defined in any one of claims 41 to 59; where (i) the first polypeptide element comprises at least one of X1, X2, X3, and X4, but not each of X1, X2, X3, and X4; and (ii) the second polypeptide element comprises each of X1, X2, X3, and X4 that is not present in the first polypeptide element; wherein said first polypeptide element and said second polypeptide element are not individually active receptor-binding proteins; wherein said first polypeptide element and said second polypeptide element interact to form an active IL-21 receptor binding protein. Non-natural IL-21 receptor binding proteins.

61. 61. The conditionally active IL-21 receptor binding protein of claim 60, comprising: wherein X1, X2, X3, and X4 each comprise an amino acid sequence as set forth in one of claims 52 to 70; IL-21 receptor binding protein.

62. A polypeptide comprising an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 607-624.

63. A recombinant nucleic acid encoding a polypeptide, a first polypeptide, or a second polypeptide according to any preceding claim.

64. 64. An expression vector comprising the recombinant nucleic acid of claim 63 operably 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 a polypeptide, fusion protein, first polypeptide, second polypeptide, recombinant nucleic acid, expression vector, and / or recombinant host cell according to any preceding claim, and a pharmaceutically acceptable carrier.

67. 1. A method of treating cancer comprising: administering to a subject having cancer an effective amount of a polypeptide, conditionally active IL-21 receptor agonist, first polypeptide, second polypeptide, recombinant nucleic acid, expression vector, recombinant host cell, and / or pharmaceutical composition of any preceding claim for the purpose of treating said tumor. method.

68. 68. The method of claim 67, wherein the cancer is: Colon cancer, melanoma, renal cell carcinoma, head and neck squamous cell carcinoma, gastric cancer, urothelial carcinoma, Hodgkin's 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's lymphoma, multiple myeloma, ovarian cancer, cervical cancer; and any type of tumor selected by a diagnostic test such as microsatellite instability, tumor mutation burden, PD-L1 expression level, or an Immunoscore assay (such as those developed by the American Society for Immunotherapy of Cancer), The method is selected from the group consisting of:

69. 1. A method of modulating an immune response in a subject, comprising: a polypeptide antagonist, a conditionally active IL-21 receptor antagonist, a first polypeptide, a second polypeptide, a recombinant nucleic acid, an expression vector, a recombinant host cell, and / or a pharmaceutical composition according to any preceding claim; method.

70. 71. The method of claim 70, wherein the immune response is an anti-cancer immune response, multiple sclerosis, lupus erythematosus, or rheumatoid arthritis. method.

Images